Dio oper domestication
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Transcript of Dio oper domestication
1
Welcome
2
Presented by Varsha Gayatonde
SupervisorProf J P Shahi
Co-SupervisorProf K Srivastava
3
Flow of presentation History of Agriculture
Crop domestication
Centers of domestication
Domestication genes in crops
Super-domestication
Polyploidy
Genome sequencing
NGS
GWAS
Finding adoptive genes
Re-wild the plants
Genome editing
Gene sharing
Conclusion
4
bull Story of agriculture dates back to almost 10000 BC It wasinitiated by people who depended on diets composed of wildplants and animals
bull By 4000 BC ancient peoples had completed thedomestication of all major crop species upon which humansurvival is dependent including rice wheat and maize
bull Recent research has begun to reveal the genes responsible forthis agricultural revolution boosts ldquoGene tinkeringrdquo
History of Agriculture
5
Domestication
Human influence change in genetics of plant population
leads to ldquoAdaptive syndrome of domesticationrdquo
bull May be deliberate or not
(ldquounconsciousrdquo or ldquoincidentalrdquo)
bull Due to change in selective environment and control
over reproduction (eg harvesting grains with
sickle sowing saved seeds)
6
Domesticated- refers more generally to plants that are
morphologically and genetically distinct from their wild ancestors
as a result of artificial selection or are no longer known to occur
outside of cultivation
Semi-domesticated- as a crop that is under cultivation and
subjected to conscious artificial selection pressures
Undomesticated refers to uncultivated plants that continue to be
wild-harvested with no conscious artificial selection pressures
and no discernible morphological and frasl or genetic differentiations
that could be used to distinguish them as a domesticate (eg
Brazil nut)
Nivara
Rufipogon
Glaberrima
7
Domesticated Rice
8
Wild wheat
9
Domesticated Wheat
10
Maize
11
Domesticated maize
12
Barley Domestication
Prognt H spontanium
13
14
Domesticated Pearl MilletPennisetum glaucum
better seed recovery and yield but less able to survive in natural conditions compared to wild progenitor
bull more apical dominance (less
branching)
bull compact growth habit
bull flowering at same time (rather
than spread over long period)
bull larger spikes
bull non-shattering spikelets
bull loss of bristles amp glumes around
grains
bull larger seeds
bull non-dormant seeds
bull germinate at same time
15
Ppolystachian
Oats domestication
Wild- A sterilis Modern
sativa
16
Domestication of Sorghum
S helepense
S bicolor17
JR Harlan 1976 Sci American
Divergent
selection for
different
purposes
httpenwikipediaorgwikiImageBrassica_oleracea0jpg
httpwwwcehacukimagesclip_image002_001jpg
Brassica oleracea cabbage
broccoli cauliflower kale
romanesco collards kohrabi
brussels sprouts18
Solanum pimpinellifolium
Solanum lycopersicum19
Domestication of carrot
Queen Annes Lace
Daucus carota
20
Domestication of peanut
A villosulicarpa Hoehne andA Stenosperma (Brazil)
AABB
21
Domestication of Chilli
22
StrawberryBanana
Musa acuminata Musa balbisiana (2n=3x=33) AB genomeFRAGARIA X ANANASSA
Wood berries (F vesca) and Musky strawberries (F moschata)23
Wild SoybeanGlycin soja2n=40
Cultivated soybeanGlycin max 2n=40
24
Increased in domesticate
seed germination
determinate growth
longer pods
bigger (heavier) seeds
earliness
harvest index (seed yieldbiomass)
Decreased in domesticate
seed dispersal
seed dormancy
twining
number of nodes
length of internode
number of pods
photoperiod sensitivity
Common Bean Phaseolus vulgarishttpwwwplantsciencesucdavisedugeptspb143pb143htm
copy Paul Gepts
25
Wild pigeon peaCajanus cajanifolius
Cultivated pigeon pea
26
27
28
WHY OTHERS ARE NOT DOMESTICATED
bull 350000 plants
bull 4629 mammals
bull 9200 birds
bull 10000000 insects
bull 500000 fungi
bull But only 200 plants
bull 15 mammals 5 birds and
bull 2 insects are domesticated
bull Spread of these few species
bull Little change since early
agriculture
bull Repeated domestication of these
species (sometimes)
bull Lack of new species even with
attempts with species known to be
valuable Some groups are good
candidates with no domestication
eg ferns sub-Saharan mammals
29
WHY OTHERS HAVE TO BE DOMESTICATED
New uses and demands ndash biofuels animal feed
medicinalneutraceutical waterclimate food changes
Knowledge why species arenrsquot suitable for domestication or
were not useful
Better understanding of genetics and selection
Sustainability of production
Reliability of production
To meet the food demand in an alternative way
WHY OTHERS HAVE TO BE DOMESTICATED
30
Reallocate biomass for human use
More efficient metabolism and photosynthesis increase
leaf surface decrease root system increase leaf
longevity
Grains more fertile florets larger inflorescence OR
number of ears different ways to get more seed
Larger seeds (automatic vs deliberate selection)
Oil plants increased oil content or more seed
Fiber plants long strong fibers
Domestication syndrome
31
Centers of Domestication
Fuller 2011
32
Centers of Domestication in India
Fuller 2011
33
Indian centers of domestication South India (Deccan centre) Vigna radiate Vigna mungo
Macrotyloma uniflorum (Horse gram) Sataria verticillata S plumila
wheat (macaroni) and two rowed barleybrachiaria ramosa
Orissa (Mahanadi river) Pegion pea Horsegram mung small millets
like Echinichloa Paspalum and Sataria
The Middle Ganges (Harappan civilization) Oryza nivara O rufipogon
and wild sativa Sateria pumia Cannabis sativa even the diffusion of
japonica rice
Saurashtra (Harappan civilization) Eleucine coracana Sateria italica
Paicum spp Brachypodia pearl millet and sorghum Dollicos lablab
The Himalayan foothills of the Punjab region Was a centre of
diversity for Japonica type rice and many temperate fruits and
vegetables
34
DOMESTICATION RELATED TRAITS CONTROLLED BY ONLY FEW GENES
Trait Crops
Plant architecturegrowth habit Ricemaizemilletsbeantomato
Flowering timephotoperiod sensitivity Ricemaizesorghumbeantomato
Fruit size Tomato egg plant
Grain size Ricemaizesorghumbean
Seed dispersal Brassica rice
Inflorescence modification Brassica
Dormancy Bean
35
Genes Crop Moleular and phenotypic function Causative change
Genes identified as controlling domestication traits
tb1 Maize Transcriptional regulator (TCP) plant and
inflorescence structure
regulatory change
tga1 Maize Transcriptional regulator (SBP) seed casing amino acid change
qSH1 Rice Transcriptional regulator (homeodomain)
abscission layer formation shattering
regulatory change
Rc Rice Transcriptional regulator (bHLH) seed color disrupted coding
sequence
sh4 Rice Transcriptional regulator (Myb3) abscission
layer formation shattering
regulatoryamino
acid change
fw22 Tomato Cell signaling fruit weight regulatory change
Q Wheat Transcriptional regulator (AP2) inflorescence
structure
regulatoryamino
acid change
Vrs1 Barley Inflorescence structure Premature stop Amino acid change
Genes of interest in crop domestication and improvement
36
Genes Crop Moleular and phenotypic function Causative change
c1
r1
Maize Transcriptional regulator (MYB) kernel color and
Transcriptional regulator (bHLH) kernel color
regulatory change
sh2 Maize pyrophosphorylase supersweet sweet corn transposon
insertion
su1 Maize isoamylase sweet corn gene amino acid change
brix9-2-5 Tomato Invertase fruit soluble solid content amino acid change
ovate Tomato Unknown fruit shap early stop codon
R Pea Starch branching enzyme seed sugar content transposon
insertion
ehd1 Rice B-type response regulator flowering time amino acid change
hd1 Rice Transcriptional regulator (zinc finger) flowering
time
disrupted coding
sequence
waxy Rice Starch synthase sticky grains intron splicing
defect
rht Wheat Transcriptional regulator (SH2) plant height early stop codon
vrn1 Wheat Transcriptional regulator (MADS) vernalization regulatory change
vrn2 Wheat Transcriptional regulator (ZCCT) vernalization amino acid change
Genes Identified as Controlling Varietal Differences
37
Process of domestication over the yearshellip
Rachel S Meyer 2012
38
Genetic bottleneck
Cultivated crops undergone with narrowing of diversity problem
But this is not the case in weeds
Weeds are the major threats from the beginning of domestication till
today
Problem arose due to
1 Crop mimicry
2 Genetic assimilation
3 Genetic evolution
39
Super- Domestication Vaughn et al (2007) first used the term super-domestication
The processes that lead to a domesticate with dramatically
increased yield that could not be selected in natural environments
from naturally occurring variation without recourse to new
technologies
Super-domesticates can be constructed with knowledge led
approaches based on current needs using the range of new
technologies now available
Plants exploited for continuous selection introduction hybridization
etc which boosted the process of domestication but now the plant
genetic engineering approach is exploiting plants towards synthetic
biology
40
How to use diversitybull Cross two varieties
bull Genome manipulations
Cell fusion hybrids
bull Chromosome manipulation Backcross a new species
bull Generate recombinants Chromosome recombinations
bull Use a new species wild germplasm
Transgenic approach Modern mutagenesis synthetic gene
construction by utilizing green florescent protein genome editing
NGS GWAS sequencing etchelliphelliphelliphelliphellip
41
Reduce genetic bottlenecks through polyplodization
More vigour
SC
Buffering capacity
Heterotic advantage
Enhaced vegetative
charachers
Enhanced oil
Meiotic stability
42
Approach 1 Use one tetraploid and one diploid as parents (4X ndash 2X) followed by the
chromosome doubling of triploid hybrids (Chrom doubling)
a) Cross between B juncea (AjAjBjBj ) and B oleracea (CoCo) to produce hexaploids
(AjAjBjBjCoCo)
(b) Cross between B napus (AnAnCnCn) and B nigra (BniBni) to produce hexaploids
(AnAnBniBniCnCn)
(c) Cross between B carinata (BcaBcaCcaCca) and B rapa (ArAr) to produce hexaploids
(ArArBcaBcaCcaCca)
Approach 2 Use three tetraploids as parents
(a) Cross between B napus (AnAnCnCn) and B carinata (BcaBcaCcaCca) to produce unbalanced
allotetraploids (AnBcaCnCca ndash unreduced gametes gametes with the somatic chromosome
number and cross with B juncea (AjAjBjBj) to obtain allohexaploids (AnAjBcaBjCnCca
(b) Cross between B napus (AnAnCnCn) and B juncea (AjAjBjBj) to produce unbalanced
allotetraploids (AnAjBjCn ndash unreduced gametes) and cross with B carinata (BcaBcaCcaCca) to
obtain allohexaploids (AnAjBcaBjCnCca)
(c) Cross between B carinata (BcaBcaCcaCca) and B juncea (AjAjBjBj) to produce unbalanced
allotetraploids (AjBcaBjCca - ndash unreduced gametes) and cross with B napus (AnAnCnCn) to obtain
allohexaploids (AnAjBcaBjCnCca)
Approach 3 Use three diploids as parents (2X ndash 2X ndash 2X) Cross between B rapa (ArAr) B
nigra (BniBni) and B oleracea (CoCo) sequentially to obtain hexaploid hybrids (ArArBniBniCoCo)
43
Continuehellip
Guijun Yan 2012
44
Applications of Genomic
tools in Super-Domestication
45
Genome Sequencing
Potential methods of sequencing
1 Clone by clone approach
2 Whole genome shotgun approach
3 Combination of the two methods
Till today no of cultivated plants completely sequenced -85
2016- Arachis duranensis
2015- Solanum cumersonii (Wild potato)
(Ref- NCBI)
46
GWAS
It is a study design in which many markers spread across a genome
are genotyped and test a statistical association with a phenotype are
performed locally along the genome
It is also an examination of many common genetic variants in different
individuals to see if any variant is associated with a trait
Used in successfully studying maize sorghum and barley
Method is efficient for large scale low cost genotyping (even with the
minimum number of SNPs)
Cannot be utilized generally because it needs large population size
GWAS identify rare alleles more precisely
If small population we can opt NAM
47
GWAS and whole genome prediction
Xuehui Huang and Bin Han 2014
48
Five high throuput genotyping methods
Xuehui Huang and Bin Han 2014
49
Role of NGS in domestication Capture of novel genes from wild species will be made easier by
understanding the molecular events associated with crop domestication
Re-sequencing of domesticated species can identify low diversity regions
resulting from selection during domestication
To identify gene-specific sequences to aid the cloning of homologues of key
domestication genes from wild relatives
Candidate genes from wild and domesticated plant populations can define
diversity of target genes in wild populations and lead to the discovery of key
genes for important traits by association analysis
NGS of amplicons of large numbers of candidate genes from wild and
domesticated plant populations can define diversity of target genes in
wild populations and lead to the discovery of key genes for important
traits by association analysis
50
NGS
supports the rapid domestication of new plant species and the efficient
identification and capture of novel genetic variation from related species
Allows whole-genome analysis to determine the genetic basis of
phenotypic differences
NGS allows rapid expansion of genomic analysis to investigation of non-
model species
Made rice study easy by related grass
cost-effective method for plant identification
useful strategy to analysis the chloroplast genome sequence from whole-
genome shot-gun sequencing
facilitates managing this diversity and any changes in crop performance
over time due to genetic drift
Patterns of gene expression have been evaluated in hybrids using NGS
51
Two Approaches to Finding Adaptive Genes
Ross-Ibarra et al2007
52
Techniques to re-wild the plants (transgene free)
1 Introgression
breeding
2 Specific
insertion of lost genes
3 Precision
mutagenesis
Palmgren et al2014
53
Process of rewilding
Palmgren et al2014
54
Synthetic biology projects(i) Modifying cereals including wheat to fix atmospheric nitrogen
(ii) Redesigning metabolic pathways to increase the yield of
secondary metabolites or to generate compounds with enhanced
properties
(iii) Transferring the C4 photosynthesis pathway to rice
(iv) Modifying the glycosylation pathway in plants to accommodate
production of therapeutic proteins
(v) Introducing synthetic signal transduction systems that respond to
external cues
55
Synthetic biology projects
Nicholas J Baltes et al 2015
56
Refactoring the N fixation gene cluster from Klebsiella
Karsten Temme et al 2012
57
Continuehellip
Karsten Temme et al 2012
58
Genome editing
Sequence specific nucleases
1 Meganucleases
2 ZFMs ndashZink finger motifs
3 TALENs- Transcription activator like effector nuclease
4 CRISPR CAS-9 ndash Clustered interspaced short palindromic
repeats
qmp4
59Khaoula Belhaj et al 2015
60
Genome modifications achieved in plants using sequence specific nucleases
Type of DNA
modification
Nuclease Delivery
methods
Plants Targets
Trait stacking Megnucleases Bombardment Cotton Intergenic sequence
ZFN Bombardment Maize Transgene
Rewriting host
DNA
Megnucleases Stable integration Maize Intergenic sequence
ZFN Stable integration Soybean Transgene
TALEN Stable integration Barley PAPhy_a
TALEN Agrobacterium
T-DNA (transient)
Oryza sativa SWEET14
TALEN No Protoplasts Arabidopsis
Tobacco
AtTT4 AtADH NbSurB
TALEN Bombardment Wheat MLO
TALEN Protoplasts
Stable integration
Maize PDS IPK1A IPK MRP4
CRISPRCas Protoplasts
Agrobacterium
T-DNA (transient)
Tobacco
Arabidopsis
Sorghum Oryza
OsSWEET14 transgene
61
Type of DNA
modification
Nuclease Delivery
methods
Plants Targets
Rewriting host
DNA large
deletion
Zinc-finger
nuclease
Stable
integration
Tobacco Transgene
CRISPRCas Protoplasts
Stable
integration
Rice Labdane-related
diterpenoid gene
clusters
on Chr 2 4 and 6
Zinc-finger
nuclease
Agrobacterium
T-DNA
(transient)
Tobacco CHN50 transgene
Zinc-finger
nuclease
Whiskers Maize IPK1
CRISPRCas Protoplasts Rice PDS
Controlling gene
expression
TALE repressor
(SRDX)
Stable
integration
Arabidopsis RD29A transgene
Zinc-finger
activator (VP16)
Stable
integration
Brassica
napus
KasII
Continue
62
Application of genome editing1 Introduction of precise and predictable modifications directly in an elite
background
2 multiple traits can be modified simultaneously
3 NHEJ enables gene knockout and targeted modifications
4 Introduction of transgenes at defined loci that promote high-level
transcription and do not interfere with the activity of endogenous genes
5 Site-specific nucleases also allow targeted molecular trait stacking -low
risk of segregation
6 CRISPR Cas is a transgene free approach ndash No regulatory burdens
7 frequency of off-target mutations is well below that caused by chemical
and physical mutagenesis techniques
8 In future can be utilized for metabolic engineering and molecular farming
63
Six-way Venn diagram showing the distribution of shared genefamilies (sequence clusters) among M acuminata P dactyliferaArabidopsis thaliana Oryza sativa Sorghum bicolor and Brachypodiumdistachyon genomes
A DrsquoHont et al Nature 2012 doi101038nature11241
64
Achieving super-DomesticationINTROGRESS-RECREATE-CREATE-DOMESTICATEREDOMESTICATE
bull Mobilizing left out genetic
variation still available in
land races and wild and
weedy species
bull Replaying the evolutionary
tape( resynthesis in
polyploids)
bull Domesticateredomesticate
65
We can admire and emulate how indigenous people still domesticateplants create biodiversity and manage it to sustain their future Thereis no equivalently dynamic or flexible crop breeding in modernagriculture to promote biodiversity we still have much to learn from
traditional knowledge Jan Salick 2012
2
Presented by Varsha Gayatonde
SupervisorProf J P Shahi
Co-SupervisorProf K Srivastava
3
Flow of presentation History of Agriculture
Crop domestication
Centers of domestication
Domestication genes in crops
Super-domestication
Polyploidy
Genome sequencing
NGS
GWAS
Finding adoptive genes
Re-wild the plants
Genome editing
Gene sharing
Conclusion
4
bull Story of agriculture dates back to almost 10000 BC It wasinitiated by people who depended on diets composed of wildplants and animals
bull By 4000 BC ancient peoples had completed thedomestication of all major crop species upon which humansurvival is dependent including rice wheat and maize
bull Recent research has begun to reveal the genes responsible forthis agricultural revolution boosts ldquoGene tinkeringrdquo
History of Agriculture
5
Domestication
Human influence change in genetics of plant population
leads to ldquoAdaptive syndrome of domesticationrdquo
bull May be deliberate or not
(ldquounconsciousrdquo or ldquoincidentalrdquo)
bull Due to change in selective environment and control
over reproduction (eg harvesting grains with
sickle sowing saved seeds)
6
Domesticated- refers more generally to plants that are
morphologically and genetically distinct from their wild ancestors
as a result of artificial selection or are no longer known to occur
outside of cultivation
Semi-domesticated- as a crop that is under cultivation and
subjected to conscious artificial selection pressures
Undomesticated refers to uncultivated plants that continue to be
wild-harvested with no conscious artificial selection pressures
and no discernible morphological and frasl or genetic differentiations
that could be used to distinguish them as a domesticate (eg
Brazil nut)
Nivara
Rufipogon
Glaberrima
7
Domesticated Rice
8
Wild wheat
9
Domesticated Wheat
10
Maize
11
Domesticated maize
12
Barley Domestication
Prognt H spontanium
13
14
Domesticated Pearl MilletPennisetum glaucum
better seed recovery and yield but less able to survive in natural conditions compared to wild progenitor
bull more apical dominance (less
branching)
bull compact growth habit
bull flowering at same time (rather
than spread over long period)
bull larger spikes
bull non-shattering spikelets
bull loss of bristles amp glumes around
grains
bull larger seeds
bull non-dormant seeds
bull germinate at same time
15
Ppolystachian
Oats domestication
Wild- A sterilis Modern
sativa
16
Domestication of Sorghum
S helepense
S bicolor17
JR Harlan 1976 Sci American
Divergent
selection for
different
purposes
httpenwikipediaorgwikiImageBrassica_oleracea0jpg
httpwwwcehacukimagesclip_image002_001jpg
Brassica oleracea cabbage
broccoli cauliflower kale
romanesco collards kohrabi
brussels sprouts18
Solanum pimpinellifolium
Solanum lycopersicum19
Domestication of carrot
Queen Annes Lace
Daucus carota
20
Domestication of peanut
A villosulicarpa Hoehne andA Stenosperma (Brazil)
AABB
21
Domestication of Chilli
22
StrawberryBanana
Musa acuminata Musa balbisiana (2n=3x=33) AB genomeFRAGARIA X ANANASSA
Wood berries (F vesca) and Musky strawberries (F moschata)23
Wild SoybeanGlycin soja2n=40
Cultivated soybeanGlycin max 2n=40
24
Increased in domesticate
seed germination
determinate growth
longer pods
bigger (heavier) seeds
earliness
harvest index (seed yieldbiomass)
Decreased in domesticate
seed dispersal
seed dormancy
twining
number of nodes
length of internode
number of pods
photoperiod sensitivity
Common Bean Phaseolus vulgarishttpwwwplantsciencesucdavisedugeptspb143pb143htm
copy Paul Gepts
25
Wild pigeon peaCajanus cajanifolius
Cultivated pigeon pea
26
27
28
WHY OTHERS ARE NOT DOMESTICATED
bull 350000 plants
bull 4629 mammals
bull 9200 birds
bull 10000000 insects
bull 500000 fungi
bull But only 200 plants
bull 15 mammals 5 birds and
bull 2 insects are domesticated
bull Spread of these few species
bull Little change since early
agriculture
bull Repeated domestication of these
species (sometimes)
bull Lack of new species even with
attempts with species known to be
valuable Some groups are good
candidates with no domestication
eg ferns sub-Saharan mammals
29
WHY OTHERS HAVE TO BE DOMESTICATED
New uses and demands ndash biofuels animal feed
medicinalneutraceutical waterclimate food changes
Knowledge why species arenrsquot suitable for domestication or
were not useful
Better understanding of genetics and selection
Sustainability of production
Reliability of production
To meet the food demand in an alternative way
WHY OTHERS HAVE TO BE DOMESTICATED
30
Reallocate biomass for human use
More efficient metabolism and photosynthesis increase
leaf surface decrease root system increase leaf
longevity
Grains more fertile florets larger inflorescence OR
number of ears different ways to get more seed
Larger seeds (automatic vs deliberate selection)
Oil plants increased oil content or more seed
Fiber plants long strong fibers
Domestication syndrome
31
Centers of Domestication
Fuller 2011
32
Centers of Domestication in India
Fuller 2011
33
Indian centers of domestication South India (Deccan centre) Vigna radiate Vigna mungo
Macrotyloma uniflorum (Horse gram) Sataria verticillata S plumila
wheat (macaroni) and two rowed barleybrachiaria ramosa
Orissa (Mahanadi river) Pegion pea Horsegram mung small millets
like Echinichloa Paspalum and Sataria
The Middle Ganges (Harappan civilization) Oryza nivara O rufipogon
and wild sativa Sateria pumia Cannabis sativa even the diffusion of
japonica rice
Saurashtra (Harappan civilization) Eleucine coracana Sateria italica
Paicum spp Brachypodia pearl millet and sorghum Dollicos lablab
The Himalayan foothills of the Punjab region Was a centre of
diversity for Japonica type rice and many temperate fruits and
vegetables
34
DOMESTICATION RELATED TRAITS CONTROLLED BY ONLY FEW GENES
Trait Crops
Plant architecturegrowth habit Ricemaizemilletsbeantomato
Flowering timephotoperiod sensitivity Ricemaizesorghumbeantomato
Fruit size Tomato egg plant
Grain size Ricemaizesorghumbean
Seed dispersal Brassica rice
Inflorescence modification Brassica
Dormancy Bean
35
Genes Crop Moleular and phenotypic function Causative change
Genes identified as controlling domestication traits
tb1 Maize Transcriptional regulator (TCP) plant and
inflorescence structure
regulatory change
tga1 Maize Transcriptional regulator (SBP) seed casing amino acid change
qSH1 Rice Transcriptional regulator (homeodomain)
abscission layer formation shattering
regulatory change
Rc Rice Transcriptional regulator (bHLH) seed color disrupted coding
sequence
sh4 Rice Transcriptional regulator (Myb3) abscission
layer formation shattering
regulatoryamino
acid change
fw22 Tomato Cell signaling fruit weight regulatory change
Q Wheat Transcriptional regulator (AP2) inflorescence
structure
regulatoryamino
acid change
Vrs1 Barley Inflorescence structure Premature stop Amino acid change
Genes of interest in crop domestication and improvement
36
Genes Crop Moleular and phenotypic function Causative change
c1
r1
Maize Transcriptional regulator (MYB) kernel color and
Transcriptional regulator (bHLH) kernel color
regulatory change
sh2 Maize pyrophosphorylase supersweet sweet corn transposon
insertion
su1 Maize isoamylase sweet corn gene amino acid change
brix9-2-5 Tomato Invertase fruit soluble solid content amino acid change
ovate Tomato Unknown fruit shap early stop codon
R Pea Starch branching enzyme seed sugar content transposon
insertion
ehd1 Rice B-type response regulator flowering time amino acid change
hd1 Rice Transcriptional regulator (zinc finger) flowering
time
disrupted coding
sequence
waxy Rice Starch synthase sticky grains intron splicing
defect
rht Wheat Transcriptional regulator (SH2) plant height early stop codon
vrn1 Wheat Transcriptional regulator (MADS) vernalization regulatory change
vrn2 Wheat Transcriptional regulator (ZCCT) vernalization amino acid change
Genes Identified as Controlling Varietal Differences
37
Process of domestication over the yearshellip
Rachel S Meyer 2012
38
Genetic bottleneck
Cultivated crops undergone with narrowing of diversity problem
But this is not the case in weeds
Weeds are the major threats from the beginning of domestication till
today
Problem arose due to
1 Crop mimicry
2 Genetic assimilation
3 Genetic evolution
39
Super- Domestication Vaughn et al (2007) first used the term super-domestication
The processes that lead to a domesticate with dramatically
increased yield that could not be selected in natural environments
from naturally occurring variation without recourse to new
technologies
Super-domesticates can be constructed with knowledge led
approaches based on current needs using the range of new
technologies now available
Plants exploited for continuous selection introduction hybridization
etc which boosted the process of domestication but now the plant
genetic engineering approach is exploiting plants towards synthetic
biology
40
How to use diversitybull Cross two varieties
bull Genome manipulations
Cell fusion hybrids
bull Chromosome manipulation Backcross a new species
bull Generate recombinants Chromosome recombinations
bull Use a new species wild germplasm
Transgenic approach Modern mutagenesis synthetic gene
construction by utilizing green florescent protein genome editing
NGS GWAS sequencing etchelliphelliphelliphelliphellip
41
Reduce genetic bottlenecks through polyplodization
More vigour
SC
Buffering capacity
Heterotic advantage
Enhaced vegetative
charachers
Enhanced oil
Meiotic stability
42
Approach 1 Use one tetraploid and one diploid as parents (4X ndash 2X) followed by the
chromosome doubling of triploid hybrids (Chrom doubling)
a) Cross between B juncea (AjAjBjBj ) and B oleracea (CoCo) to produce hexaploids
(AjAjBjBjCoCo)
(b) Cross between B napus (AnAnCnCn) and B nigra (BniBni) to produce hexaploids
(AnAnBniBniCnCn)
(c) Cross between B carinata (BcaBcaCcaCca) and B rapa (ArAr) to produce hexaploids
(ArArBcaBcaCcaCca)
Approach 2 Use three tetraploids as parents
(a) Cross between B napus (AnAnCnCn) and B carinata (BcaBcaCcaCca) to produce unbalanced
allotetraploids (AnBcaCnCca ndash unreduced gametes gametes with the somatic chromosome
number and cross with B juncea (AjAjBjBj) to obtain allohexaploids (AnAjBcaBjCnCca
(b) Cross between B napus (AnAnCnCn) and B juncea (AjAjBjBj) to produce unbalanced
allotetraploids (AnAjBjCn ndash unreduced gametes) and cross with B carinata (BcaBcaCcaCca) to
obtain allohexaploids (AnAjBcaBjCnCca)
(c) Cross between B carinata (BcaBcaCcaCca) and B juncea (AjAjBjBj) to produce unbalanced
allotetraploids (AjBcaBjCca - ndash unreduced gametes) and cross with B napus (AnAnCnCn) to obtain
allohexaploids (AnAjBcaBjCnCca)
Approach 3 Use three diploids as parents (2X ndash 2X ndash 2X) Cross between B rapa (ArAr) B
nigra (BniBni) and B oleracea (CoCo) sequentially to obtain hexaploid hybrids (ArArBniBniCoCo)
43
Continuehellip
Guijun Yan 2012
44
Applications of Genomic
tools in Super-Domestication
45
Genome Sequencing
Potential methods of sequencing
1 Clone by clone approach
2 Whole genome shotgun approach
3 Combination of the two methods
Till today no of cultivated plants completely sequenced -85
2016- Arachis duranensis
2015- Solanum cumersonii (Wild potato)
(Ref- NCBI)
46
GWAS
It is a study design in which many markers spread across a genome
are genotyped and test a statistical association with a phenotype are
performed locally along the genome
It is also an examination of many common genetic variants in different
individuals to see if any variant is associated with a trait
Used in successfully studying maize sorghum and barley
Method is efficient for large scale low cost genotyping (even with the
minimum number of SNPs)
Cannot be utilized generally because it needs large population size
GWAS identify rare alleles more precisely
If small population we can opt NAM
47
GWAS and whole genome prediction
Xuehui Huang and Bin Han 2014
48
Five high throuput genotyping methods
Xuehui Huang and Bin Han 2014
49
Role of NGS in domestication Capture of novel genes from wild species will be made easier by
understanding the molecular events associated with crop domestication
Re-sequencing of domesticated species can identify low diversity regions
resulting from selection during domestication
To identify gene-specific sequences to aid the cloning of homologues of key
domestication genes from wild relatives
Candidate genes from wild and domesticated plant populations can define
diversity of target genes in wild populations and lead to the discovery of key
genes for important traits by association analysis
NGS of amplicons of large numbers of candidate genes from wild and
domesticated plant populations can define diversity of target genes in
wild populations and lead to the discovery of key genes for important
traits by association analysis
50
NGS
supports the rapid domestication of new plant species and the efficient
identification and capture of novel genetic variation from related species
Allows whole-genome analysis to determine the genetic basis of
phenotypic differences
NGS allows rapid expansion of genomic analysis to investigation of non-
model species
Made rice study easy by related grass
cost-effective method for plant identification
useful strategy to analysis the chloroplast genome sequence from whole-
genome shot-gun sequencing
facilitates managing this diversity and any changes in crop performance
over time due to genetic drift
Patterns of gene expression have been evaluated in hybrids using NGS
51
Two Approaches to Finding Adaptive Genes
Ross-Ibarra et al2007
52
Techniques to re-wild the plants (transgene free)
1 Introgression
breeding
2 Specific
insertion of lost genes
3 Precision
mutagenesis
Palmgren et al2014
53
Process of rewilding
Palmgren et al2014
54
Synthetic biology projects(i) Modifying cereals including wheat to fix atmospheric nitrogen
(ii) Redesigning metabolic pathways to increase the yield of
secondary metabolites or to generate compounds with enhanced
properties
(iii) Transferring the C4 photosynthesis pathway to rice
(iv) Modifying the glycosylation pathway in plants to accommodate
production of therapeutic proteins
(v) Introducing synthetic signal transduction systems that respond to
external cues
55
Synthetic biology projects
Nicholas J Baltes et al 2015
56
Refactoring the N fixation gene cluster from Klebsiella
Karsten Temme et al 2012
57
Continuehellip
Karsten Temme et al 2012
58
Genome editing
Sequence specific nucleases
1 Meganucleases
2 ZFMs ndashZink finger motifs
3 TALENs- Transcription activator like effector nuclease
4 CRISPR CAS-9 ndash Clustered interspaced short palindromic
repeats
qmp4
59Khaoula Belhaj et al 2015
60
Genome modifications achieved in plants using sequence specific nucleases
Type of DNA
modification
Nuclease Delivery
methods
Plants Targets
Trait stacking Megnucleases Bombardment Cotton Intergenic sequence
ZFN Bombardment Maize Transgene
Rewriting host
DNA
Megnucleases Stable integration Maize Intergenic sequence
ZFN Stable integration Soybean Transgene
TALEN Stable integration Barley PAPhy_a
TALEN Agrobacterium
T-DNA (transient)
Oryza sativa SWEET14
TALEN No Protoplasts Arabidopsis
Tobacco
AtTT4 AtADH NbSurB
TALEN Bombardment Wheat MLO
TALEN Protoplasts
Stable integration
Maize PDS IPK1A IPK MRP4
CRISPRCas Protoplasts
Agrobacterium
T-DNA (transient)
Tobacco
Arabidopsis
Sorghum Oryza
OsSWEET14 transgene
61
Type of DNA
modification
Nuclease Delivery
methods
Plants Targets
Rewriting host
DNA large
deletion
Zinc-finger
nuclease
Stable
integration
Tobacco Transgene
CRISPRCas Protoplasts
Stable
integration
Rice Labdane-related
diterpenoid gene
clusters
on Chr 2 4 and 6
Zinc-finger
nuclease
Agrobacterium
T-DNA
(transient)
Tobacco CHN50 transgene
Zinc-finger
nuclease
Whiskers Maize IPK1
CRISPRCas Protoplasts Rice PDS
Controlling gene
expression
TALE repressor
(SRDX)
Stable
integration
Arabidopsis RD29A transgene
Zinc-finger
activator (VP16)
Stable
integration
Brassica
napus
KasII
Continue
62
Application of genome editing1 Introduction of precise and predictable modifications directly in an elite
background
2 multiple traits can be modified simultaneously
3 NHEJ enables gene knockout and targeted modifications
4 Introduction of transgenes at defined loci that promote high-level
transcription and do not interfere with the activity of endogenous genes
5 Site-specific nucleases also allow targeted molecular trait stacking -low
risk of segregation
6 CRISPR Cas is a transgene free approach ndash No regulatory burdens
7 frequency of off-target mutations is well below that caused by chemical
and physical mutagenesis techniques
8 In future can be utilized for metabolic engineering and molecular farming
63
Six-way Venn diagram showing the distribution of shared genefamilies (sequence clusters) among M acuminata P dactyliferaArabidopsis thaliana Oryza sativa Sorghum bicolor and Brachypodiumdistachyon genomes
A DrsquoHont et al Nature 2012 doi101038nature11241
64
Achieving super-DomesticationINTROGRESS-RECREATE-CREATE-DOMESTICATEREDOMESTICATE
bull Mobilizing left out genetic
variation still available in
land races and wild and
weedy species
bull Replaying the evolutionary
tape( resynthesis in
polyploids)
bull Domesticateredomesticate
65
We can admire and emulate how indigenous people still domesticateplants create biodiversity and manage it to sustain their future Thereis no equivalently dynamic or flexible crop breeding in modernagriculture to promote biodiversity we still have much to learn from
traditional knowledge Jan Salick 2012
3
Flow of presentation History of Agriculture
Crop domestication
Centers of domestication
Domestication genes in crops
Super-domestication
Polyploidy
Genome sequencing
NGS
GWAS
Finding adoptive genes
Re-wild the plants
Genome editing
Gene sharing
Conclusion
4
bull Story of agriculture dates back to almost 10000 BC It wasinitiated by people who depended on diets composed of wildplants and animals
bull By 4000 BC ancient peoples had completed thedomestication of all major crop species upon which humansurvival is dependent including rice wheat and maize
bull Recent research has begun to reveal the genes responsible forthis agricultural revolution boosts ldquoGene tinkeringrdquo
History of Agriculture
5
Domestication
Human influence change in genetics of plant population
leads to ldquoAdaptive syndrome of domesticationrdquo
bull May be deliberate or not
(ldquounconsciousrdquo or ldquoincidentalrdquo)
bull Due to change in selective environment and control
over reproduction (eg harvesting grains with
sickle sowing saved seeds)
6
Domesticated- refers more generally to plants that are
morphologically and genetically distinct from their wild ancestors
as a result of artificial selection or are no longer known to occur
outside of cultivation
Semi-domesticated- as a crop that is under cultivation and
subjected to conscious artificial selection pressures
Undomesticated refers to uncultivated plants that continue to be
wild-harvested with no conscious artificial selection pressures
and no discernible morphological and frasl or genetic differentiations
that could be used to distinguish them as a domesticate (eg
Brazil nut)
Nivara
Rufipogon
Glaberrima
7
Domesticated Rice
8
Wild wheat
9
Domesticated Wheat
10
Maize
11
Domesticated maize
12
Barley Domestication
Prognt H spontanium
13
14
Domesticated Pearl MilletPennisetum glaucum
better seed recovery and yield but less able to survive in natural conditions compared to wild progenitor
bull more apical dominance (less
branching)
bull compact growth habit
bull flowering at same time (rather
than spread over long period)
bull larger spikes
bull non-shattering spikelets
bull loss of bristles amp glumes around
grains
bull larger seeds
bull non-dormant seeds
bull germinate at same time
15
Ppolystachian
Oats domestication
Wild- A sterilis Modern
sativa
16
Domestication of Sorghum
S helepense
S bicolor17
JR Harlan 1976 Sci American
Divergent
selection for
different
purposes
httpenwikipediaorgwikiImageBrassica_oleracea0jpg
httpwwwcehacukimagesclip_image002_001jpg
Brassica oleracea cabbage
broccoli cauliflower kale
romanesco collards kohrabi
brussels sprouts18
Solanum pimpinellifolium
Solanum lycopersicum19
Domestication of carrot
Queen Annes Lace
Daucus carota
20
Domestication of peanut
A villosulicarpa Hoehne andA Stenosperma (Brazil)
AABB
21
Domestication of Chilli
22
StrawberryBanana
Musa acuminata Musa balbisiana (2n=3x=33) AB genomeFRAGARIA X ANANASSA
Wood berries (F vesca) and Musky strawberries (F moschata)23
Wild SoybeanGlycin soja2n=40
Cultivated soybeanGlycin max 2n=40
24
Increased in domesticate
seed germination
determinate growth
longer pods
bigger (heavier) seeds
earliness
harvest index (seed yieldbiomass)
Decreased in domesticate
seed dispersal
seed dormancy
twining
number of nodes
length of internode
number of pods
photoperiod sensitivity
Common Bean Phaseolus vulgarishttpwwwplantsciencesucdavisedugeptspb143pb143htm
copy Paul Gepts
25
Wild pigeon peaCajanus cajanifolius
Cultivated pigeon pea
26
27
28
WHY OTHERS ARE NOT DOMESTICATED
bull 350000 plants
bull 4629 mammals
bull 9200 birds
bull 10000000 insects
bull 500000 fungi
bull But only 200 plants
bull 15 mammals 5 birds and
bull 2 insects are domesticated
bull Spread of these few species
bull Little change since early
agriculture
bull Repeated domestication of these
species (sometimes)
bull Lack of new species even with
attempts with species known to be
valuable Some groups are good
candidates with no domestication
eg ferns sub-Saharan mammals
29
WHY OTHERS HAVE TO BE DOMESTICATED
New uses and demands ndash biofuels animal feed
medicinalneutraceutical waterclimate food changes
Knowledge why species arenrsquot suitable for domestication or
were not useful
Better understanding of genetics and selection
Sustainability of production
Reliability of production
To meet the food demand in an alternative way
WHY OTHERS HAVE TO BE DOMESTICATED
30
Reallocate biomass for human use
More efficient metabolism and photosynthesis increase
leaf surface decrease root system increase leaf
longevity
Grains more fertile florets larger inflorescence OR
number of ears different ways to get more seed
Larger seeds (automatic vs deliberate selection)
Oil plants increased oil content or more seed
Fiber plants long strong fibers
Domestication syndrome
31
Centers of Domestication
Fuller 2011
32
Centers of Domestication in India
Fuller 2011
33
Indian centers of domestication South India (Deccan centre) Vigna radiate Vigna mungo
Macrotyloma uniflorum (Horse gram) Sataria verticillata S plumila
wheat (macaroni) and two rowed barleybrachiaria ramosa
Orissa (Mahanadi river) Pegion pea Horsegram mung small millets
like Echinichloa Paspalum and Sataria
The Middle Ganges (Harappan civilization) Oryza nivara O rufipogon
and wild sativa Sateria pumia Cannabis sativa even the diffusion of
japonica rice
Saurashtra (Harappan civilization) Eleucine coracana Sateria italica
Paicum spp Brachypodia pearl millet and sorghum Dollicos lablab
The Himalayan foothills of the Punjab region Was a centre of
diversity for Japonica type rice and many temperate fruits and
vegetables
34
DOMESTICATION RELATED TRAITS CONTROLLED BY ONLY FEW GENES
Trait Crops
Plant architecturegrowth habit Ricemaizemilletsbeantomato
Flowering timephotoperiod sensitivity Ricemaizesorghumbeantomato
Fruit size Tomato egg plant
Grain size Ricemaizesorghumbean
Seed dispersal Brassica rice
Inflorescence modification Brassica
Dormancy Bean
35
Genes Crop Moleular and phenotypic function Causative change
Genes identified as controlling domestication traits
tb1 Maize Transcriptional regulator (TCP) plant and
inflorescence structure
regulatory change
tga1 Maize Transcriptional regulator (SBP) seed casing amino acid change
qSH1 Rice Transcriptional regulator (homeodomain)
abscission layer formation shattering
regulatory change
Rc Rice Transcriptional regulator (bHLH) seed color disrupted coding
sequence
sh4 Rice Transcriptional regulator (Myb3) abscission
layer formation shattering
regulatoryamino
acid change
fw22 Tomato Cell signaling fruit weight regulatory change
Q Wheat Transcriptional regulator (AP2) inflorescence
structure
regulatoryamino
acid change
Vrs1 Barley Inflorescence structure Premature stop Amino acid change
Genes of interest in crop domestication and improvement
36
Genes Crop Moleular and phenotypic function Causative change
c1
r1
Maize Transcriptional regulator (MYB) kernel color and
Transcriptional regulator (bHLH) kernel color
regulatory change
sh2 Maize pyrophosphorylase supersweet sweet corn transposon
insertion
su1 Maize isoamylase sweet corn gene amino acid change
brix9-2-5 Tomato Invertase fruit soluble solid content amino acid change
ovate Tomato Unknown fruit shap early stop codon
R Pea Starch branching enzyme seed sugar content transposon
insertion
ehd1 Rice B-type response regulator flowering time amino acid change
hd1 Rice Transcriptional regulator (zinc finger) flowering
time
disrupted coding
sequence
waxy Rice Starch synthase sticky grains intron splicing
defect
rht Wheat Transcriptional regulator (SH2) plant height early stop codon
vrn1 Wheat Transcriptional regulator (MADS) vernalization regulatory change
vrn2 Wheat Transcriptional regulator (ZCCT) vernalization amino acid change
Genes Identified as Controlling Varietal Differences
37
Process of domestication over the yearshellip
Rachel S Meyer 2012
38
Genetic bottleneck
Cultivated crops undergone with narrowing of diversity problem
But this is not the case in weeds
Weeds are the major threats from the beginning of domestication till
today
Problem arose due to
1 Crop mimicry
2 Genetic assimilation
3 Genetic evolution
39
Super- Domestication Vaughn et al (2007) first used the term super-domestication
The processes that lead to a domesticate with dramatically
increased yield that could not be selected in natural environments
from naturally occurring variation without recourse to new
technologies
Super-domesticates can be constructed with knowledge led
approaches based on current needs using the range of new
technologies now available
Plants exploited for continuous selection introduction hybridization
etc which boosted the process of domestication but now the plant
genetic engineering approach is exploiting plants towards synthetic
biology
40
How to use diversitybull Cross two varieties
bull Genome manipulations
Cell fusion hybrids
bull Chromosome manipulation Backcross a new species
bull Generate recombinants Chromosome recombinations
bull Use a new species wild germplasm
Transgenic approach Modern mutagenesis synthetic gene
construction by utilizing green florescent protein genome editing
NGS GWAS sequencing etchelliphelliphelliphelliphellip
41
Reduce genetic bottlenecks through polyplodization
More vigour
SC
Buffering capacity
Heterotic advantage
Enhaced vegetative
charachers
Enhanced oil
Meiotic stability
42
Approach 1 Use one tetraploid and one diploid as parents (4X ndash 2X) followed by the
chromosome doubling of triploid hybrids (Chrom doubling)
a) Cross between B juncea (AjAjBjBj ) and B oleracea (CoCo) to produce hexaploids
(AjAjBjBjCoCo)
(b) Cross between B napus (AnAnCnCn) and B nigra (BniBni) to produce hexaploids
(AnAnBniBniCnCn)
(c) Cross between B carinata (BcaBcaCcaCca) and B rapa (ArAr) to produce hexaploids
(ArArBcaBcaCcaCca)
Approach 2 Use three tetraploids as parents
(a) Cross between B napus (AnAnCnCn) and B carinata (BcaBcaCcaCca) to produce unbalanced
allotetraploids (AnBcaCnCca ndash unreduced gametes gametes with the somatic chromosome
number and cross with B juncea (AjAjBjBj) to obtain allohexaploids (AnAjBcaBjCnCca
(b) Cross between B napus (AnAnCnCn) and B juncea (AjAjBjBj) to produce unbalanced
allotetraploids (AnAjBjCn ndash unreduced gametes) and cross with B carinata (BcaBcaCcaCca) to
obtain allohexaploids (AnAjBcaBjCnCca)
(c) Cross between B carinata (BcaBcaCcaCca) and B juncea (AjAjBjBj) to produce unbalanced
allotetraploids (AjBcaBjCca - ndash unreduced gametes) and cross with B napus (AnAnCnCn) to obtain
allohexaploids (AnAjBcaBjCnCca)
Approach 3 Use three diploids as parents (2X ndash 2X ndash 2X) Cross between B rapa (ArAr) B
nigra (BniBni) and B oleracea (CoCo) sequentially to obtain hexaploid hybrids (ArArBniBniCoCo)
43
Continuehellip
Guijun Yan 2012
44
Applications of Genomic
tools in Super-Domestication
45
Genome Sequencing
Potential methods of sequencing
1 Clone by clone approach
2 Whole genome shotgun approach
3 Combination of the two methods
Till today no of cultivated plants completely sequenced -85
2016- Arachis duranensis
2015- Solanum cumersonii (Wild potato)
(Ref- NCBI)
46
GWAS
It is a study design in which many markers spread across a genome
are genotyped and test a statistical association with a phenotype are
performed locally along the genome
It is also an examination of many common genetic variants in different
individuals to see if any variant is associated with a trait
Used in successfully studying maize sorghum and barley
Method is efficient for large scale low cost genotyping (even with the
minimum number of SNPs)
Cannot be utilized generally because it needs large population size
GWAS identify rare alleles more precisely
If small population we can opt NAM
47
GWAS and whole genome prediction
Xuehui Huang and Bin Han 2014
48
Five high throuput genotyping methods
Xuehui Huang and Bin Han 2014
49
Role of NGS in domestication Capture of novel genes from wild species will be made easier by
understanding the molecular events associated with crop domestication
Re-sequencing of domesticated species can identify low diversity regions
resulting from selection during domestication
To identify gene-specific sequences to aid the cloning of homologues of key
domestication genes from wild relatives
Candidate genes from wild and domesticated plant populations can define
diversity of target genes in wild populations and lead to the discovery of key
genes for important traits by association analysis
NGS of amplicons of large numbers of candidate genes from wild and
domesticated plant populations can define diversity of target genes in
wild populations and lead to the discovery of key genes for important
traits by association analysis
50
NGS
supports the rapid domestication of new plant species and the efficient
identification and capture of novel genetic variation from related species
Allows whole-genome analysis to determine the genetic basis of
phenotypic differences
NGS allows rapid expansion of genomic analysis to investigation of non-
model species
Made rice study easy by related grass
cost-effective method for plant identification
useful strategy to analysis the chloroplast genome sequence from whole-
genome shot-gun sequencing
facilitates managing this diversity and any changes in crop performance
over time due to genetic drift
Patterns of gene expression have been evaluated in hybrids using NGS
51
Two Approaches to Finding Adaptive Genes
Ross-Ibarra et al2007
52
Techniques to re-wild the plants (transgene free)
1 Introgression
breeding
2 Specific
insertion of lost genes
3 Precision
mutagenesis
Palmgren et al2014
53
Process of rewilding
Palmgren et al2014
54
Synthetic biology projects(i) Modifying cereals including wheat to fix atmospheric nitrogen
(ii) Redesigning metabolic pathways to increase the yield of
secondary metabolites or to generate compounds with enhanced
properties
(iii) Transferring the C4 photosynthesis pathway to rice
(iv) Modifying the glycosylation pathway in plants to accommodate
production of therapeutic proteins
(v) Introducing synthetic signal transduction systems that respond to
external cues
55
Synthetic biology projects
Nicholas J Baltes et al 2015
56
Refactoring the N fixation gene cluster from Klebsiella
Karsten Temme et al 2012
57
Continuehellip
Karsten Temme et al 2012
58
Genome editing
Sequence specific nucleases
1 Meganucleases
2 ZFMs ndashZink finger motifs
3 TALENs- Transcription activator like effector nuclease
4 CRISPR CAS-9 ndash Clustered interspaced short palindromic
repeats
qmp4
59Khaoula Belhaj et al 2015
60
Genome modifications achieved in plants using sequence specific nucleases
Type of DNA
modification
Nuclease Delivery
methods
Plants Targets
Trait stacking Megnucleases Bombardment Cotton Intergenic sequence
ZFN Bombardment Maize Transgene
Rewriting host
DNA
Megnucleases Stable integration Maize Intergenic sequence
ZFN Stable integration Soybean Transgene
TALEN Stable integration Barley PAPhy_a
TALEN Agrobacterium
T-DNA (transient)
Oryza sativa SWEET14
TALEN No Protoplasts Arabidopsis
Tobacco
AtTT4 AtADH NbSurB
TALEN Bombardment Wheat MLO
TALEN Protoplasts
Stable integration
Maize PDS IPK1A IPK MRP4
CRISPRCas Protoplasts
Agrobacterium
T-DNA (transient)
Tobacco
Arabidopsis
Sorghum Oryza
OsSWEET14 transgene
61
Type of DNA
modification
Nuclease Delivery
methods
Plants Targets
Rewriting host
DNA large
deletion
Zinc-finger
nuclease
Stable
integration
Tobacco Transgene
CRISPRCas Protoplasts
Stable
integration
Rice Labdane-related
diterpenoid gene
clusters
on Chr 2 4 and 6
Zinc-finger
nuclease
Agrobacterium
T-DNA
(transient)
Tobacco CHN50 transgene
Zinc-finger
nuclease
Whiskers Maize IPK1
CRISPRCas Protoplasts Rice PDS
Controlling gene
expression
TALE repressor
(SRDX)
Stable
integration
Arabidopsis RD29A transgene
Zinc-finger
activator (VP16)
Stable
integration
Brassica
napus
KasII
Continue
62
Application of genome editing1 Introduction of precise and predictable modifications directly in an elite
background
2 multiple traits can be modified simultaneously
3 NHEJ enables gene knockout and targeted modifications
4 Introduction of transgenes at defined loci that promote high-level
transcription and do not interfere with the activity of endogenous genes
5 Site-specific nucleases also allow targeted molecular trait stacking -low
risk of segregation
6 CRISPR Cas is a transgene free approach ndash No regulatory burdens
7 frequency of off-target mutations is well below that caused by chemical
and physical mutagenesis techniques
8 In future can be utilized for metabolic engineering and molecular farming
63
Six-way Venn diagram showing the distribution of shared genefamilies (sequence clusters) among M acuminata P dactyliferaArabidopsis thaliana Oryza sativa Sorghum bicolor and Brachypodiumdistachyon genomes
A DrsquoHont et al Nature 2012 doi101038nature11241
64
Achieving super-DomesticationINTROGRESS-RECREATE-CREATE-DOMESTICATEREDOMESTICATE
bull Mobilizing left out genetic
variation still available in
land races and wild and
weedy species
bull Replaying the evolutionary
tape( resynthesis in
polyploids)
bull Domesticateredomesticate
65
We can admire and emulate how indigenous people still domesticateplants create biodiversity and manage it to sustain their future Thereis no equivalently dynamic or flexible crop breeding in modernagriculture to promote biodiversity we still have much to learn from
traditional knowledge Jan Salick 2012
4
bull Story of agriculture dates back to almost 10000 BC It wasinitiated by people who depended on diets composed of wildplants and animals
bull By 4000 BC ancient peoples had completed thedomestication of all major crop species upon which humansurvival is dependent including rice wheat and maize
bull Recent research has begun to reveal the genes responsible forthis agricultural revolution boosts ldquoGene tinkeringrdquo
History of Agriculture
5
Domestication
Human influence change in genetics of plant population
leads to ldquoAdaptive syndrome of domesticationrdquo
bull May be deliberate or not
(ldquounconsciousrdquo or ldquoincidentalrdquo)
bull Due to change in selective environment and control
over reproduction (eg harvesting grains with
sickle sowing saved seeds)
6
Domesticated- refers more generally to plants that are
morphologically and genetically distinct from their wild ancestors
as a result of artificial selection or are no longer known to occur
outside of cultivation
Semi-domesticated- as a crop that is under cultivation and
subjected to conscious artificial selection pressures
Undomesticated refers to uncultivated plants that continue to be
wild-harvested with no conscious artificial selection pressures
and no discernible morphological and frasl or genetic differentiations
that could be used to distinguish them as a domesticate (eg
Brazil nut)
Nivara
Rufipogon
Glaberrima
7
Domesticated Rice
8
Wild wheat
9
Domesticated Wheat
10
Maize
11
Domesticated maize
12
Barley Domestication
Prognt H spontanium
13
14
Domesticated Pearl MilletPennisetum glaucum
better seed recovery and yield but less able to survive in natural conditions compared to wild progenitor
bull more apical dominance (less
branching)
bull compact growth habit
bull flowering at same time (rather
than spread over long period)
bull larger spikes
bull non-shattering spikelets
bull loss of bristles amp glumes around
grains
bull larger seeds
bull non-dormant seeds
bull germinate at same time
15
Ppolystachian
Oats domestication
Wild- A sterilis Modern
sativa
16
Domestication of Sorghum
S helepense
S bicolor17
JR Harlan 1976 Sci American
Divergent
selection for
different
purposes
httpenwikipediaorgwikiImageBrassica_oleracea0jpg
httpwwwcehacukimagesclip_image002_001jpg
Brassica oleracea cabbage
broccoli cauliflower kale
romanesco collards kohrabi
brussels sprouts18
Solanum pimpinellifolium
Solanum lycopersicum19
Domestication of carrot
Queen Annes Lace
Daucus carota
20
Domestication of peanut
A villosulicarpa Hoehne andA Stenosperma (Brazil)
AABB
21
Domestication of Chilli
22
StrawberryBanana
Musa acuminata Musa balbisiana (2n=3x=33) AB genomeFRAGARIA X ANANASSA
Wood berries (F vesca) and Musky strawberries (F moschata)23
Wild SoybeanGlycin soja2n=40
Cultivated soybeanGlycin max 2n=40
24
Increased in domesticate
seed germination
determinate growth
longer pods
bigger (heavier) seeds
earliness
harvest index (seed yieldbiomass)
Decreased in domesticate
seed dispersal
seed dormancy
twining
number of nodes
length of internode
number of pods
photoperiod sensitivity
Common Bean Phaseolus vulgarishttpwwwplantsciencesucdavisedugeptspb143pb143htm
copy Paul Gepts
25
Wild pigeon peaCajanus cajanifolius
Cultivated pigeon pea
26
27
28
WHY OTHERS ARE NOT DOMESTICATED
bull 350000 plants
bull 4629 mammals
bull 9200 birds
bull 10000000 insects
bull 500000 fungi
bull But only 200 plants
bull 15 mammals 5 birds and
bull 2 insects are domesticated
bull Spread of these few species
bull Little change since early
agriculture
bull Repeated domestication of these
species (sometimes)
bull Lack of new species even with
attempts with species known to be
valuable Some groups are good
candidates with no domestication
eg ferns sub-Saharan mammals
29
WHY OTHERS HAVE TO BE DOMESTICATED
New uses and demands ndash biofuels animal feed
medicinalneutraceutical waterclimate food changes
Knowledge why species arenrsquot suitable for domestication or
were not useful
Better understanding of genetics and selection
Sustainability of production
Reliability of production
To meet the food demand in an alternative way
WHY OTHERS HAVE TO BE DOMESTICATED
30
Reallocate biomass for human use
More efficient metabolism and photosynthesis increase
leaf surface decrease root system increase leaf
longevity
Grains more fertile florets larger inflorescence OR
number of ears different ways to get more seed
Larger seeds (automatic vs deliberate selection)
Oil plants increased oil content or more seed
Fiber plants long strong fibers
Domestication syndrome
31
Centers of Domestication
Fuller 2011
32
Centers of Domestication in India
Fuller 2011
33
Indian centers of domestication South India (Deccan centre) Vigna radiate Vigna mungo
Macrotyloma uniflorum (Horse gram) Sataria verticillata S plumila
wheat (macaroni) and two rowed barleybrachiaria ramosa
Orissa (Mahanadi river) Pegion pea Horsegram mung small millets
like Echinichloa Paspalum and Sataria
The Middle Ganges (Harappan civilization) Oryza nivara O rufipogon
and wild sativa Sateria pumia Cannabis sativa even the diffusion of
japonica rice
Saurashtra (Harappan civilization) Eleucine coracana Sateria italica
Paicum spp Brachypodia pearl millet and sorghum Dollicos lablab
The Himalayan foothills of the Punjab region Was a centre of
diversity for Japonica type rice and many temperate fruits and
vegetables
34
DOMESTICATION RELATED TRAITS CONTROLLED BY ONLY FEW GENES
Trait Crops
Plant architecturegrowth habit Ricemaizemilletsbeantomato
Flowering timephotoperiod sensitivity Ricemaizesorghumbeantomato
Fruit size Tomato egg plant
Grain size Ricemaizesorghumbean
Seed dispersal Brassica rice
Inflorescence modification Brassica
Dormancy Bean
35
Genes Crop Moleular and phenotypic function Causative change
Genes identified as controlling domestication traits
tb1 Maize Transcriptional regulator (TCP) plant and
inflorescence structure
regulatory change
tga1 Maize Transcriptional regulator (SBP) seed casing amino acid change
qSH1 Rice Transcriptional regulator (homeodomain)
abscission layer formation shattering
regulatory change
Rc Rice Transcriptional regulator (bHLH) seed color disrupted coding
sequence
sh4 Rice Transcriptional regulator (Myb3) abscission
layer formation shattering
regulatoryamino
acid change
fw22 Tomato Cell signaling fruit weight regulatory change
Q Wheat Transcriptional regulator (AP2) inflorescence
structure
regulatoryamino
acid change
Vrs1 Barley Inflorescence structure Premature stop Amino acid change
Genes of interest in crop domestication and improvement
36
Genes Crop Moleular and phenotypic function Causative change
c1
r1
Maize Transcriptional regulator (MYB) kernel color and
Transcriptional regulator (bHLH) kernel color
regulatory change
sh2 Maize pyrophosphorylase supersweet sweet corn transposon
insertion
su1 Maize isoamylase sweet corn gene amino acid change
brix9-2-5 Tomato Invertase fruit soluble solid content amino acid change
ovate Tomato Unknown fruit shap early stop codon
R Pea Starch branching enzyme seed sugar content transposon
insertion
ehd1 Rice B-type response regulator flowering time amino acid change
hd1 Rice Transcriptional regulator (zinc finger) flowering
time
disrupted coding
sequence
waxy Rice Starch synthase sticky grains intron splicing
defect
rht Wheat Transcriptional regulator (SH2) plant height early stop codon
vrn1 Wheat Transcriptional regulator (MADS) vernalization regulatory change
vrn2 Wheat Transcriptional regulator (ZCCT) vernalization amino acid change
Genes Identified as Controlling Varietal Differences
37
Process of domestication over the yearshellip
Rachel S Meyer 2012
38
Genetic bottleneck
Cultivated crops undergone with narrowing of diversity problem
But this is not the case in weeds
Weeds are the major threats from the beginning of domestication till
today
Problem arose due to
1 Crop mimicry
2 Genetic assimilation
3 Genetic evolution
39
Super- Domestication Vaughn et al (2007) first used the term super-domestication
The processes that lead to a domesticate with dramatically
increased yield that could not be selected in natural environments
from naturally occurring variation without recourse to new
technologies
Super-domesticates can be constructed with knowledge led
approaches based on current needs using the range of new
technologies now available
Plants exploited for continuous selection introduction hybridization
etc which boosted the process of domestication but now the plant
genetic engineering approach is exploiting plants towards synthetic
biology
40
How to use diversitybull Cross two varieties
bull Genome manipulations
Cell fusion hybrids
bull Chromosome manipulation Backcross a new species
bull Generate recombinants Chromosome recombinations
bull Use a new species wild germplasm
Transgenic approach Modern mutagenesis synthetic gene
construction by utilizing green florescent protein genome editing
NGS GWAS sequencing etchelliphelliphelliphelliphellip
41
Reduce genetic bottlenecks through polyplodization
More vigour
SC
Buffering capacity
Heterotic advantage
Enhaced vegetative
charachers
Enhanced oil
Meiotic stability
42
Approach 1 Use one tetraploid and one diploid as parents (4X ndash 2X) followed by the
chromosome doubling of triploid hybrids (Chrom doubling)
a) Cross between B juncea (AjAjBjBj ) and B oleracea (CoCo) to produce hexaploids
(AjAjBjBjCoCo)
(b) Cross between B napus (AnAnCnCn) and B nigra (BniBni) to produce hexaploids
(AnAnBniBniCnCn)
(c) Cross between B carinata (BcaBcaCcaCca) and B rapa (ArAr) to produce hexaploids
(ArArBcaBcaCcaCca)
Approach 2 Use three tetraploids as parents
(a) Cross between B napus (AnAnCnCn) and B carinata (BcaBcaCcaCca) to produce unbalanced
allotetraploids (AnBcaCnCca ndash unreduced gametes gametes with the somatic chromosome
number and cross with B juncea (AjAjBjBj) to obtain allohexaploids (AnAjBcaBjCnCca
(b) Cross between B napus (AnAnCnCn) and B juncea (AjAjBjBj) to produce unbalanced
allotetraploids (AnAjBjCn ndash unreduced gametes) and cross with B carinata (BcaBcaCcaCca) to
obtain allohexaploids (AnAjBcaBjCnCca)
(c) Cross between B carinata (BcaBcaCcaCca) and B juncea (AjAjBjBj) to produce unbalanced
allotetraploids (AjBcaBjCca - ndash unreduced gametes) and cross with B napus (AnAnCnCn) to obtain
allohexaploids (AnAjBcaBjCnCca)
Approach 3 Use three diploids as parents (2X ndash 2X ndash 2X) Cross between B rapa (ArAr) B
nigra (BniBni) and B oleracea (CoCo) sequentially to obtain hexaploid hybrids (ArArBniBniCoCo)
43
Continuehellip
Guijun Yan 2012
44
Applications of Genomic
tools in Super-Domestication
45
Genome Sequencing
Potential methods of sequencing
1 Clone by clone approach
2 Whole genome shotgun approach
3 Combination of the two methods
Till today no of cultivated plants completely sequenced -85
2016- Arachis duranensis
2015- Solanum cumersonii (Wild potato)
(Ref- NCBI)
46
GWAS
It is a study design in which many markers spread across a genome
are genotyped and test a statistical association with a phenotype are
performed locally along the genome
It is also an examination of many common genetic variants in different
individuals to see if any variant is associated with a trait
Used in successfully studying maize sorghum and barley
Method is efficient for large scale low cost genotyping (even with the
minimum number of SNPs)
Cannot be utilized generally because it needs large population size
GWAS identify rare alleles more precisely
If small population we can opt NAM
47
GWAS and whole genome prediction
Xuehui Huang and Bin Han 2014
48
Five high throuput genotyping methods
Xuehui Huang and Bin Han 2014
49
Role of NGS in domestication Capture of novel genes from wild species will be made easier by
understanding the molecular events associated with crop domestication
Re-sequencing of domesticated species can identify low diversity regions
resulting from selection during domestication
To identify gene-specific sequences to aid the cloning of homologues of key
domestication genes from wild relatives
Candidate genes from wild and domesticated plant populations can define
diversity of target genes in wild populations and lead to the discovery of key
genes for important traits by association analysis
NGS of amplicons of large numbers of candidate genes from wild and
domesticated plant populations can define diversity of target genes in
wild populations and lead to the discovery of key genes for important
traits by association analysis
50
NGS
supports the rapid domestication of new plant species and the efficient
identification and capture of novel genetic variation from related species
Allows whole-genome analysis to determine the genetic basis of
phenotypic differences
NGS allows rapid expansion of genomic analysis to investigation of non-
model species
Made rice study easy by related grass
cost-effective method for plant identification
useful strategy to analysis the chloroplast genome sequence from whole-
genome shot-gun sequencing
facilitates managing this diversity and any changes in crop performance
over time due to genetic drift
Patterns of gene expression have been evaluated in hybrids using NGS
51
Two Approaches to Finding Adaptive Genes
Ross-Ibarra et al2007
52
Techniques to re-wild the plants (transgene free)
1 Introgression
breeding
2 Specific
insertion of lost genes
3 Precision
mutagenesis
Palmgren et al2014
53
Process of rewilding
Palmgren et al2014
54
Synthetic biology projects(i) Modifying cereals including wheat to fix atmospheric nitrogen
(ii) Redesigning metabolic pathways to increase the yield of
secondary metabolites or to generate compounds with enhanced
properties
(iii) Transferring the C4 photosynthesis pathway to rice
(iv) Modifying the glycosylation pathway in plants to accommodate
production of therapeutic proteins
(v) Introducing synthetic signal transduction systems that respond to
external cues
55
Synthetic biology projects
Nicholas J Baltes et al 2015
56
Refactoring the N fixation gene cluster from Klebsiella
Karsten Temme et al 2012
57
Continuehellip
Karsten Temme et al 2012
58
Genome editing
Sequence specific nucleases
1 Meganucleases
2 ZFMs ndashZink finger motifs
3 TALENs- Transcription activator like effector nuclease
4 CRISPR CAS-9 ndash Clustered interspaced short palindromic
repeats
qmp4
59Khaoula Belhaj et al 2015
60
Genome modifications achieved in plants using sequence specific nucleases
Type of DNA
modification
Nuclease Delivery
methods
Plants Targets
Trait stacking Megnucleases Bombardment Cotton Intergenic sequence
ZFN Bombardment Maize Transgene
Rewriting host
DNA
Megnucleases Stable integration Maize Intergenic sequence
ZFN Stable integration Soybean Transgene
TALEN Stable integration Barley PAPhy_a
TALEN Agrobacterium
T-DNA (transient)
Oryza sativa SWEET14
TALEN No Protoplasts Arabidopsis
Tobacco
AtTT4 AtADH NbSurB
TALEN Bombardment Wheat MLO
TALEN Protoplasts
Stable integration
Maize PDS IPK1A IPK MRP4
CRISPRCas Protoplasts
Agrobacterium
T-DNA (transient)
Tobacco
Arabidopsis
Sorghum Oryza
OsSWEET14 transgene
61
Type of DNA
modification
Nuclease Delivery
methods
Plants Targets
Rewriting host
DNA large
deletion
Zinc-finger
nuclease
Stable
integration
Tobacco Transgene
CRISPRCas Protoplasts
Stable
integration
Rice Labdane-related
diterpenoid gene
clusters
on Chr 2 4 and 6
Zinc-finger
nuclease
Agrobacterium
T-DNA
(transient)
Tobacco CHN50 transgene
Zinc-finger
nuclease
Whiskers Maize IPK1
CRISPRCas Protoplasts Rice PDS
Controlling gene
expression
TALE repressor
(SRDX)
Stable
integration
Arabidopsis RD29A transgene
Zinc-finger
activator (VP16)
Stable
integration
Brassica
napus
KasII
Continue
62
Application of genome editing1 Introduction of precise and predictable modifications directly in an elite
background
2 multiple traits can be modified simultaneously
3 NHEJ enables gene knockout and targeted modifications
4 Introduction of transgenes at defined loci that promote high-level
transcription and do not interfere with the activity of endogenous genes
5 Site-specific nucleases also allow targeted molecular trait stacking -low
risk of segregation
6 CRISPR Cas is a transgene free approach ndash No regulatory burdens
7 frequency of off-target mutations is well below that caused by chemical
and physical mutagenesis techniques
8 In future can be utilized for metabolic engineering and molecular farming
63
Six-way Venn diagram showing the distribution of shared genefamilies (sequence clusters) among M acuminata P dactyliferaArabidopsis thaliana Oryza sativa Sorghum bicolor and Brachypodiumdistachyon genomes
A DrsquoHont et al Nature 2012 doi101038nature11241
64
Achieving super-DomesticationINTROGRESS-RECREATE-CREATE-DOMESTICATEREDOMESTICATE
bull Mobilizing left out genetic
variation still available in
land races and wild and
weedy species
bull Replaying the evolutionary
tape( resynthesis in
polyploids)
bull Domesticateredomesticate
65
We can admire and emulate how indigenous people still domesticateplants create biodiversity and manage it to sustain their future Thereis no equivalently dynamic or flexible crop breeding in modernagriculture to promote biodiversity we still have much to learn from
traditional knowledge Jan Salick 2012
5
Domestication
Human influence change in genetics of plant population
leads to ldquoAdaptive syndrome of domesticationrdquo
bull May be deliberate or not
(ldquounconsciousrdquo or ldquoincidentalrdquo)
bull Due to change in selective environment and control
over reproduction (eg harvesting grains with
sickle sowing saved seeds)
6
Domesticated- refers more generally to plants that are
morphologically and genetically distinct from their wild ancestors
as a result of artificial selection or are no longer known to occur
outside of cultivation
Semi-domesticated- as a crop that is under cultivation and
subjected to conscious artificial selection pressures
Undomesticated refers to uncultivated plants that continue to be
wild-harvested with no conscious artificial selection pressures
and no discernible morphological and frasl or genetic differentiations
that could be used to distinguish them as a domesticate (eg
Brazil nut)
Nivara
Rufipogon
Glaberrima
7
Domesticated Rice
8
Wild wheat
9
Domesticated Wheat
10
Maize
11
Domesticated maize
12
Barley Domestication
Prognt H spontanium
13
14
Domesticated Pearl MilletPennisetum glaucum
better seed recovery and yield but less able to survive in natural conditions compared to wild progenitor
bull more apical dominance (less
branching)
bull compact growth habit
bull flowering at same time (rather
than spread over long period)
bull larger spikes
bull non-shattering spikelets
bull loss of bristles amp glumes around
grains
bull larger seeds
bull non-dormant seeds
bull germinate at same time
15
Ppolystachian
Oats domestication
Wild- A sterilis Modern
sativa
16
Domestication of Sorghum
S helepense
S bicolor17
JR Harlan 1976 Sci American
Divergent
selection for
different
purposes
httpenwikipediaorgwikiImageBrassica_oleracea0jpg
httpwwwcehacukimagesclip_image002_001jpg
Brassica oleracea cabbage
broccoli cauliflower kale
romanesco collards kohrabi
brussels sprouts18
Solanum pimpinellifolium
Solanum lycopersicum19
Domestication of carrot
Queen Annes Lace
Daucus carota
20
Domestication of peanut
A villosulicarpa Hoehne andA Stenosperma (Brazil)
AABB
21
Domestication of Chilli
22
StrawberryBanana
Musa acuminata Musa balbisiana (2n=3x=33) AB genomeFRAGARIA X ANANASSA
Wood berries (F vesca) and Musky strawberries (F moschata)23
Wild SoybeanGlycin soja2n=40
Cultivated soybeanGlycin max 2n=40
24
Increased in domesticate
seed germination
determinate growth
longer pods
bigger (heavier) seeds
earliness
harvest index (seed yieldbiomass)
Decreased in domesticate
seed dispersal
seed dormancy
twining
number of nodes
length of internode
number of pods
photoperiod sensitivity
Common Bean Phaseolus vulgarishttpwwwplantsciencesucdavisedugeptspb143pb143htm
copy Paul Gepts
25
Wild pigeon peaCajanus cajanifolius
Cultivated pigeon pea
26
27
28
WHY OTHERS ARE NOT DOMESTICATED
bull 350000 plants
bull 4629 mammals
bull 9200 birds
bull 10000000 insects
bull 500000 fungi
bull But only 200 plants
bull 15 mammals 5 birds and
bull 2 insects are domesticated
bull Spread of these few species
bull Little change since early
agriculture
bull Repeated domestication of these
species (sometimes)
bull Lack of new species even with
attempts with species known to be
valuable Some groups are good
candidates with no domestication
eg ferns sub-Saharan mammals
29
WHY OTHERS HAVE TO BE DOMESTICATED
New uses and demands ndash biofuels animal feed
medicinalneutraceutical waterclimate food changes
Knowledge why species arenrsquot suitable for domestication or
were not useful
Better understanding of genetics and selection
Sustainability of production
Reliability of production
To meet the food demand in an alternative way
WHY OTHERS HAVE TO BE DOMESTICATED
30
Reallocate biomass for human use
More efficient metabolism and photosynthesis increase
leaf surface decrease root system increase leaf
longevity
Grains more fertile florets larger inflorescence OR
number of ears different ways to get more seed
Larger seeds (automatic vs deliberate selection)
Oil plants increased oil content or more seed
Fiber plants long strong fibers
Domestication syndrome
31
Centers of Domestication
Fuller 2011
32
Centers of Domestication in India
Fuller 2011
33
Indian centers of domestication South India (Deccan centre) Vigna radiate Vigna mungo
Macrotyloma uniflorum (Horse gram) Sataria verticillata S plumila
wheat (macaroni) and two rowed barleybrachiaria ramosa
Orissa (Mahanadi river) Pegion pea Horsegram mung small millets
like Echinichloa Paspalum and Sataria
The Middle Ganges (Harappan civilization) Oryza nivara O rufipogon
and wild sativa Sateria pumia Cannabis sativa even the diffusion of
japonica rice
Saurashtra (Harappan civilization) Eleucine coracana Sateria italica
Paicum spp Brachypodia pearl millet and sorghum Dollicos lablab
The Himalayan foothills of the Punjab region Was a centre of
diversity for Japonica type rice and many temperate fruits and
vegetables
34
DOMESTICATION RELATED TRAITS CONTROLLED BY ONLY FEW GENES
Trait Crops
Plant architecturegrowth habit Ricemaizemilletsbeantomato
Flowering timephotoperiod sensitivity Ricemaizesorghumbeantomato
Fruit size Tomato egg plant
Grain size Ricemaizesorghumbean
Seed dispersal Brassica rice
Inflorescence modification Brassica
Dormancy Bean
35
Genes Crop Moleular and phenotypic function Causative change
Genes identified as controlling domestication traits
tb1 Maize Transcriptional regulator (TCP) plant and
inflorescence structure
regulatory change
tga1 Maize Transcriptional regulator (SBP) seed casing amino acid change
qSH1 Rice Transcriptional regulator (homeodomain)
abscission layer formation shattering
regulatory change
Rc Rice Transcriptional regulator (bHLH) seed color disrupted coding
sequence
sh4 Rice Transcriptional regulator (Myb3) abscission
layer formation shattering
regulatoryamino
acid change
fw22 Tomato Cell signaling fruit weight regulatory change
Q Wheat Transcriptional regulator (AP2) inflorescence
structure
regulatoryamino
acid change
Vrs1 Barley Inflorescence structure Premature stop Amino acid change
Genes of interest in crop domestication and improvement
36
Genes Crop Moleular and phenotypic function Causative change
c1
r1
Maize Transcriptional regulator (MYB) kernel color and
Transcriptional regulator (bHLH) kernel color
regulatory change
sh2 Maize pyrophosphorylase supersweet sweet corn transposon
insertion
su1 Maize isoamylase sweet corn gene amino acid change
brix9-2-5 Tomato Invertase fruit soluble solid content amino acid change
ovate Tomato Unknown fruit shap early stop codon
R Pea Starch branching enzyme seed sugar content transposon
insertion
ehd1 Rice B-type response regulator flowering time amino acid change
hd1 Rice Transcriptional regulator (zinc finger) flowering
time
disrupted coding
sequence
waxy Rice Starch synthase sticky grains intron splicing
defect
rht Wheat Transcriptional regulator (SH2) plant height early stop codon
vrn1 Wheat Transcriptional regulator (MADS) vernalization regulatory change
vrn2 Wheat Transcriptional regulator (ZCCT) vernalization amino acid change
Genes Identified as Controlling Varietal Differences
37
Process of domestication over the yearshellip
Rachel S Meyer 2012
38
Genetic bottleneck
Cultivated crops undergone with narrowing of diversity problem
But this is not the case in weeds
Weeds are the major threats from the beginning of domestication till
today
Problem arose due to
1 Crop mimicry
2 Genetic assimilation
3 Genetic evolution
39
Super- Domestication Vaughn et al (2007) first used the term super-domestication
The processes that lead to a domesticate with dramatically
increased yield that could not be selected in natural environments
from naturally occurring variation without recourse to new
technologies
Super-domesticates can be constructed with knowledge led
approaches based on current needs using the range of new
technologies now available
Plants exploited for continuous selection introduction hybridization
etc which boosted the process of domestication but now the plant
genetic engineering approach is exploiting plants towards synthetic
biology
40
How to use diversitybull Cross two varieties
bull Genome manipulations
Cell fusion hybrids
bull Chromosome manipulation Backcross a new species
bull Generate recombinants Chromosome recombinations
bull Use a new species wild germplasm
Transgenic approach Modern mutagenesis synthetic gene
construction by utilizing green florescent protein genome editing
NGS GWAS sequencing etchelliphelliphelliphelliphellip
41
Reduce genetic bottlenecks through polyplodization
More vigour
SC
Buffering capacity
Heterotic advantage
Enhaced vegetative
charachers
Enhanced oil
Meiotic stability
42
Approach 1 Use one tetraploid and one diploid as parents (4X ndash 2X) followed by the
chromosome doubling of triploid hybrids (Chrom doubling)
a) Cross between B juncea (AjAjBjBj ) and B oleracea (CoCo) to produce hexaploids
(AjAjBjBjCoCo)
(b) Cross between B napus (AnAnCnCn) and B nigra (BniBni) to produce hexaploids
(AnAnBniBniCnCn)
(c) Cross between B carinata (BcaBcaCcaCca) and B rapa (ArAr) to produce hexaploids
(ArArBcaBcaCcaCca)
Approach 2 Use three tetraploids as parents
(a) Cross between B napus (AnAnCnCn) and B carinata (BcaBcaCcaCca) to produce unbalanced
allotetraploids (AnBcaCnCca ndash unreduced gametes gametes with the somatic chromosome
number and cross with B juncea (AjAjBjBj) to obtain allohexaploids (AnAjBcaBjCnCca
(b) Cross between B napus (AnAnCnCn) and B juncea (AjAjBjBj) to produce unbalanced
allotetraploids (AnAjBjCn ndash unreduced gametes) and cross with B carinata (BcaBcaCcaCca) to
obtain allohexaploids (AnAjBcaBjCnCca)
(c) Cross between B carinata (BcaBcaCcaCca) and B juncea (AjAjBjBj) to produce unbalanced
allotetraploids (AjBcaBjCca - ndash unreduced gametes) and cross with B napus (AnAnCnCn) to obtain
allohexaploids (AnAjBcaBjCnCca)
Approach 3 Use three diploids as parents (2X ndash 2X ndash 2X) Cross between B rapa (ArAr) B
nigra (BniBni) and B oleracea (CoCo) sequentially to obtain hexaploid hybrids (ArArBniBniCoCo)
43
Continuehellip
Guijun Yan 2012
44
Applications of Genomic
tools in Super-Domestication
45
Genome Sequencing
Potential methods of sequencing
1 Clone by clone approach
2 Whole genome shotgun approach
3 Combination of the two methods
Till today no of cultivated plants completely sequenced -85
2016- Arachis duranensis
2015- Solanum cumersonii (Wild potato)
(Ref- NCBI)
46
GWAS
It is a study design in which many markers spread across a genome
are genotyped and test a statistical association with a phenotype are
performed locally along the genome
It is also an examination of many common genetic variants in different
individuals to see if any variant is associated with a trait
Used in successfully studying maize sorghum and barley
Method is efficient for large scale low cost genotyping (even with the
minimum number of SNPs)
Cannot be utilized generally because it needs large population size
GWAS identify rare alleles more precisely
If small population we can opt NAM
47
GWAS and whole genome prediction
Xuehui Huang and Bin Han 2014
48
Five high throuput genotyping methods
Xuehui Huang and Bin Han 2014
49
Role of NGS in domestication Capture of novel genes from wild species will be made easier by
understanding the molecular events associated with crop domestication
Re-sequencing of domesticated species can identify low diversity regions
resulting from selection during domestication
To identify gene-specific sequences to aid the cloning of homologues of key
domestication genes from wild relatives
Candidate genes from wild and domesticated plant populations can define
diversity of target genes in wild populations and lead to the discovery of key
genes for important traits by association analysis
NGS of amplicons of large numbers of candidate genes from wild and
domesticated plant populations can define diversity of target genes in
wild populations and lead to the discovery of key genes for important
traits by association analysis
50
NGS
supports the rapid domestication of new plant species and the efficient
identification and capture of novel genetic variation from related species
Allows whole-genome analysis to determine the genetic basis of
phenotypic differences
NGS allows rapid expansion of genomic analysis to investigation of non-
model species
Made rice study easy by related grass
cost-effective method for plant identification
useful strategy to analysis the chloroplast genome sequence from whole-
genome shot-gun sequencing
facilitates managing this diversity and any changes in crop performance
over time due to genetic drift
Patterns of gene expression have been evaluated in hybrids using NGS
51
Two Approaches to Finding Adaptive Genes
Ross-Ibarra et al2007
52
Techniques to re-wild the plants (transgene free)
1 Introgression
breeding
2 Specific
insertion of lost genes
3 Precision
mutagenesis
Palmgren et al2014
53
Process of rewilding
Palmgren et al2014
54
Synthetic biology projects(i) Modifying cereals including wheat to fix atmospheric nitrogen
(ii) Redesigning metabolic pathways to increase the yield of
secondary metabolites or to generate compounds with enhanced
properties
(iii) Transferring the C4 photosynthesis pathway to rice
(iv) Modifying the glycosylation pathway in plants to accommodate
production of therapeutic proteins
(v) Introducing synthetic signal transduction systems that respond to
external cues
55
Synthetic biology projects
Nicholas J Baltes et al 2015
56
Refactoring the N fixation gene cluster from Klebsiella
Karsten Temme et al 2012
57
Continuehellip
Karsten Temme et al 2012
58
Genome editing
Sequence specific nucleases
1 Meganucleases
2 ZFMs ndashZink finger motifs
3 TALENs- Transcription activator like effector nuclease
4 CRISPR CAS-9 ndash Clustered interspaced short palindromic
repeats
qmp4
59Khaoula Belhaj et al 2015
60
Genome modifications achieved in plants using sequence specific nucleases
Type of DNA
modification
Nuclease Delivery
methods
Plants Targets
Trait stacking Megnucleases Bombardment Cotton Intergenic sequence
ZFN Bombardment Maize Transgene
Rewriting host
DNA
Megnucleases Stable integration Maize Intergenic sequence
ZFN Stable integration Soybean Transgene
TALEN Stable integration Barley PAPhy_a
TALEN Agrobacterium
T-DNA (transient)
Oryza sativa SWEET14
TALEN No Protoplasts Arabidopsis
Tobacco
AtTT4 AtADH NbSurB
TALEN Bombardment Wheat MLO
TALEN Protoplasts
Stable integration
Maize PDS IPK1A IPK MRP4
CRISPRCas Protoplasts
Agrobacterium
T-DNA (transient)
Tobacco
Arabidopsis
Sorghum Oryza
OsSWEET14 transgene
61
Type of DNA
modification
Nuclease Delivery
methods
Plants Targets
Rewriting host
DNA large
deletion
Zinc-finger
nuclease
Stable
integration
Tobacco Transgene
CRISPRCas Protoplasts
Stable
integration
Rice Labdane-related
diterpenoid gene
clusters
on Chr 2 4 and 6
Zinc-finger
nuclease
Agrobacterium
T-DNA
(transient)
Tobacco CHN50 transgene
Zinc-finger
nuclease
Whiskers Maize IPK1
CRISPRCas Protoplasts Rice PDS
Controlling gene
expression
TALE repressor
(SRDX)
Stable
integration
Arabidopsis RD29A transgene
Zinc-finger
activator (VP16)
Stable
integration
Brassica
napus
KasII
Continue
62
Application of genome editing1 Introduction of precise and predictable modifications directly in an elite
background
2 multiple traits can be modified simultaneously
3 NHEJ enables gene knockout and targeted modifications
4 Introduction of transgenes at defined loci that promote high-level
transcription and do not interfere with the activity of endogenous genes
5 Site-specific nucleases also allow targeted molecular trait stacking -low
risk of segregation
6 CRISPR Cas is a transgene free approach ndash No regulatory burdens
7 frequency of off-target mutations is well below that caused by chemical
and physical mutagenesis techniques
8 In future can be utilized for metabolic engineering and molecular farming
63
Six-way Venn diagram showing the distribution of shared genefamilies (sequence clusters) among M acuminata P dactyliferaArabidopsis thaliana Oryza sativa Sorghum bicolor and Brachypodiumdistachyon genomes
A DrsquoHont et al Nature 2012 doi101038nature11241
64
Achieving super-DomesticationINTROGRESS-RECREATE-CREATE-DOMESTICATEREDOMESTICATE
bull Mobilizing left out genetic
variation still available in
land races and wild and
weedy species
bull Replaying the evolutionary
tape( resynthesis in
polyploids)
bull Domesticateredomesticate
65
We can admire and emulate how indigenous people still domesticateplants create biodiversity and manage it to sustain their future Thereis no equivalently dynamic or flexible crop breeding in modernagriculture to promote biodiversity we still have much to learn from
traditional knowledge Jan Salick 2012
6
Domesticated- refers more generally to plants that are
morphologically and genetically distinct from their wild ancestors
as a result of artificial selection or are no longer known to occur
outside of cultivation
Semi-domesticated- as a crop that is under cultivation and
subjected to conscious artificial selection pressures
Undomesticated refers to uncultivated plants that continue to be
wild-harvested with no conscious artificial selection pressures
and no discernible morphological and frasl or genetic differentiations
that could be used to distinguish them as a domesticate (eg
Brazil nut)
Nivara
Rufipogon
Glaberrima
7
Domesticated Rice
8
Wild wheat
9
Domesticated Wheat
10
Maize
11
Domesticated maize
12
Barley Domestication
Prognt H spontanium
13
14
Domesticated Pearl MilletPennisetum glaucum
better seed recovery and yield but less able to survive in natural conditions compared to wild progenitor
bull more apical dominance (less
branching)
bull compact growth habit
bull flowering at same time (rather
than spread over long period)
bull larger spikes
bull non-shattering spikelets
bull loss of bristles amp glumes around
grains
bull larger seeds
bull non-dormant seeds
bull germinate at same time
15
Ppolystachian
Oats domestication
Wild- A sterilis Modern
sativa
16
Domestication of Sorghum
S helepense
S bicolor17
JR Harlan 1976 Sci American
Divergent
selection for
different
purposes
httpenwikipediaorgwikiImageBrassica_oleracea0jpg
httpwwwcehacukimagesclip_image002_001jpg
Brassica oleracea cabbage
broccoli cauliflower kale
romanesco collards kohrabi
brussels sprouts18
Solanum pimpinellifolium
Solanum lycopersicum19
Domestication of carrot
Queen Annes Lace
Daucus carota
20
Domestication of peanut
A villosulicarpa Hoehne andA Stenosperma (Brazil)
AABB
21
Domestication of Chilli
22
StrawberryBanana
Musa acuminata Musa balbisiana (2n=3x=33) AB genomeFRAGARIA X ANANASSA
Wood berries (F vesca) and Musky strawberries (F moschata)23
Wild SoybeanGlycin soja2n=40
Cultivated soybeanGlycin max 2n=40
24
Increased in domesticate
seed germination
determinate growth
longer pods
bigger (heavier) seeds
earliness
harvest index (seed yieldbiomass)
Decreased in domesticate
seed dispersal
seed dormancy
twining
number of nodes
length of internode
number of pods
photoperiod sensitivity
Common Bean Phaseolus vulgarishttpwwwplantsciencesucdavisedugeptspb143pb143htm
copy Paul Gepts
25
Wild pigeon peaCajanus cajanifolius
Cultivated pigeon pea
26
27
28
WHY OTHERS ARE NOT DOMESTICATED
bull 350000 plants
bull 4629 mammals
bull 9200 birds
bull 10000000 insects
bull 500000 fungi
bull But only 200 plants
bull 15 mammals 5 birds and
bull 2 insects are domesticated
bull Spread of these few species
bull Little change since early
agriculture
bull Repeated domestication of these
species (sometimes)
bull Lack of new species even with
attempts with species known to be
valuable Some groups are good
candidates with no domestication
eg ferns sub-Saharan mammals
29
WHY OTHERS HAVE TO BE DOMESTICATED
New uses and demands ndash biofuels animal feed
medicinalneutraceutical waterclimate food changes
Knowledge why species arenrsquot suitable for domestication or
were not useful
Better understanding of genetics and selection
Sustainability of production
Reliability of production
To meet the food demand in an alternative way
WHY OTHERS HAVE TO BE DOMESTICATED
30
Reallocate biomass for human use
More efficient metabolism and photosynthesis increase
leaf surface decrease root system increase leaf
longevity
Grains more fertile florets larger inflorescence OR
number of ears different ways to get more seed
Larger seeds (automatic vs deliberate selection)
Oil plants increased oil content or more seed
Fiber plants long strong fibers
Domestication syndrome
31
Centers of Domestication
Fuller 2011
32
Centers of Domestication in India
Fuller 2011
33
Indian centers of domestication South India (Deccan centre) Vigna radiate Vigna mungo
Macrotyloma uniflorum (Horse gram) Sataria verticillata S plumila
wheat (macaroni) and two rowed barleybrachiaria ramosa
Orissa (Mahanadi river) Pegion pea Horsegram mung small millets
like Echinichloa Paspalum and Sataria
The Middle Ganges (Harappan civilization) Oryza nivara O rufipogon
and wild sativa Sateria pumia Cannabis sativa even the diffusion of
japonica rice
Saurashtra (Harappan civilization) Eleucine coracana Sateria italica
Paicum spp Brachypodia pearl millet and sorghum Dollicos lablab
The Himalayan foothills of the Punjab region Was a centre of
diversity for Japonica type rice and many temperate fruits and
vegetables
34
DOMESTICATION RELATED TRAITS CONTROLLED BY ONLY FEW GENES
Trait Crops
Plant architecturegrowth habit Ricemaizemilletsbeantomato
Flowering timephotoperiod sensitivity Ricemaizesorghumbeantomato
Fruit size Tomato egg plant
Grain size Ricemaizesorghumbean
Seed dispersal Brassica rice
Inflorescence modification Brassica
Dormancy Bean
35
Genes Crop Moleular and phenotypic function Causative change
Genes identified as controlling domestication traits
tb1 Maize Transcriptional regulator (TCP) plant and
inflorescence structure
regulatory change
tga1 Maize Transcriptional regulator (SBP) seed casing amino acid change
qSH1 Rice Transcriptional regulator (homeodomain)
abscission layer formation shattering
regulatory change
Rc Rice Transcriptional regulator (bHLH) seed color disrupted coding
sequence
sh4 Rice Transcriptional regulator (Myb3) abscission
layer formation shattering
regulatoryamino
acid change
fw22 Tomato Cell signaling fruit weight regulatory change
Q Wheat Transcriptional regulator (AP2) inflorescence
structure
regulatoryamino
acid change
Vrs1 Barley Inflorescence structure Premature stop Amino acid change
Genes of interest in crop domestication and improvement
36
Genes Crop Moleular and phenotypic function Causative change
c1
r1
Maize Transcriptional regulator (MYB) kernel color and
Transcriptional regulator (bHLH) kernel color
regulatory change
sh2 Maize pyrophosphorylase supersweet sweet corn transposon
insertion
su1 Maize isoamylase sweet corn gene amino acid change
brix9-2-5 Tomato Invertase fruit soluble solid content amino acid change
ovate Tomato Unknown fruit shap early stop codon
R Pea Starch branching enzyme seed sugar content transposon
insertion
ehd1 Rice B-type response regulator flowering time amino acid change
hd1 Rice Transcriptional regulator (zinc finger) flowering
time
disrupted coding
sequence
waxy Rice Starch synthase sticky grains intron splicing
defect
rht Wheat Transcriptional regulator (SH2) plant height early stop codon
vrn1 Wheat Transcriptional regulator (MADS) vernalization regulatory change
vrn2 Wheat Transcriptional regulator (ZCCT) vernalization amino acid change
Genes Identified as Controlling Varietal Differences
37
Process of domestication over the yearshellip
Rachel S Meyer 2012
38
Genetic bottleneck
Cultivated crops undergone with narrowing of diversity problem
But this is not the case in weeds
Weeds are the major threats from the beginning of domestication till
today
Problem arose due to
1 Crop mimicry
2 Genetic assimilation
3 Genetic evolution
39
Super- Domestication Vaughn et al (2007) first used the term super-domestication
The processes that lead to a domesticate with dramatically
increased yield that could not be selected in natural environments
from naturally occurring variation without recourse to new
technologies
Super-domesticates can be constructed with knowledge led
approaches based on current needs using the range of new
technologies now available
Plants exploited for continuous selection introduction hybridization
etc which boosted the process of domestication but now the plant
genetic engineering approach is exploiting plants towards synthetic
biology
40
How to use diversitybull Cross two varieties
bull Genome manipulations
Cell fusion hybrids
bull Chromosome manipulation Backcross a new species
bull Generate recombinants Chromosome recombinations
bull Use a new species wild germplasm
Transgenic approach Modern mutagenesis synthetic gene
construction by utilizing green florescent protein genome editing
NGS GWAS sequencing etchelliphelliphelliphelliphellip
41
Reduce genetic bottlenecks through polyplodization
More vigour
SC
Buffering capacity
Heterotic advantage
Enhaced vegetative
charachers
Enhanced oil
Meiotic stability
42
Approach 1 Use one tetraploid and one diploid as parents (4X ndash 2X) followed by the
chromosome doubling of triploid hybrids (Chrom doubling)
a) Cross between B juncea (AjAjBjBj ) and B oleracea (CoCo) to produce hexaploids
(AjAjBjBjCoCo)
(b) Cross between B napus (AnAnCnCn) and B nigra (BniBni) to produce hexaploids
(AnAnBniBniCnCn)
(c) Cross between B carinata (BcaBcaCcaCca) and B rapa (ArAr) to produce hexaploids
(ArArBcaBcaCcaCca)
Approach 2 Use three tetraploids as parents
(a) Cross between B napus (AnAnCnCn) and B carinata (BcaBcaCcaCca) to produce unbalanced
allotetraploids (AnBcaCnCca ndash unreduced gametes gametes with the somatic chromosome
number and cross with B juncea (AjAjBjBj) to obtain allohexaploids (AnAjBcaBjCnCca
(b) Cross between B napus (AnAnCnCn) and B juncea (AjAjBjBj) to produce unbalanced
allotetraploids (AnAjBjCn ndash unreduced gametes) and cross with B carinata (BcaBcaCcaCca) to
obtain allohexaploids (AnAjBcaBjCnCca)
(c) Cross between B carinata (BcaBcaCcaCca) and B juncea (AjAjBjBj) to produce unbalanced
allotetraploids (AjBcaBjCca - ndash unreduced gametes) and cross with B napus (AnAnCnCn) to obtain
allohexaploids (AnAjBcaBjCnCca)
Approach 3 Use three diploids as parents (2X ndash 2X ndash 2X) Cross between B rapa (ArAr) B
nigra (BniBni) and B oleracea (CoCo) sequentially to obtain hexaploid hybrids (ArArBniBniCoCo)
43
Continuehellip
Guijun Yan 2012
44
Applications of Genomic
tools in Super-Domestication
45
Genome Sequencing
Potential methods of sequencing
1 Clone by clone approach
2 Whole genome shotgun approach
3 Combination of the two methods
Till today no of cultivated plants completely sequenced -85
2016- Arachis duranensis
2015- Solanum cumersonii (Wild potato)
(Ref- NCBI)
46
GWAS
It is a study design in which many markers spread across a genome
are genotyped and test a statistical association with a phenotype are
performed locally along the genome
It is also an examination of many common genetic variants in different
individuals to see if any variant is associated with a trait
Used in successfully studying maize sorghum and barley
Method is efficient for large scale low cost genotyping (even with the
minimum number of SNPs)
Cannot be utilized generally because it needs large population size
GWAS identify rare alleles more precisely
If small population we can opt NAM
47
GWAS and whole genome prediction
Xuehui Huang and Bin Han 2014
48
Five high throuput genotyping methods
Xuehui Huang and Bin Han 2014
49
Role of NGS in domestication Capture of novel genes from wild species will be made easier by
understanding the molecular events associated with crop domestication
Re-sequencing of domesticated species can identify low diversity regions
resulting from selection during domestication
To identify gene-specific sequences to aid the cloning of homologues of key
domestication genes from wild relatives
Candidate genes from wild and domesticated plant populations can define
diversity of target genes in wild populations and lead to the discovery of key
genes for important traits by association analysis
NGS of amplicons of large numbers of candidate genes from wild and
domesticated plant populations can define diversity of target genes in
wild populations and lead to the discovery of key genes for important
traits by association analysis
50
NGS
supports the rapid domestication of new plant species and the efficient
identification and capture of novel genetic variation from related species
Allows whole-genome analysis to determine the genetic basis of
phenotypic differences
NGS allows rapid expansion of genomic analysis to investigation of non-
model species
Made rice study easy by related grass
cost-effective method for plant identification
useful strategy to analysis the chloroplast genome sequence from whole-
genome shot-gun sequencing
facilitates managing this diversity and any changes in crop performance
over time due to genetic drift
Patterns of gene expression have been evaluated in hybrids using NGS
51
Two Approaches to Finding Adaptive Genes
Ross-Ibarra et al2007
52
Techniques to re-wild the plants (transgene free)
1 Introgression
breeding
2 Specific
insertion of lost genes
3 Precision
mutagenesis
Palmgren et al2014
53
Process of rewilding
Palmgren et al2014
54
Synthetic biology projects(i) Modifying cereals including wheat to fix atmospheric nitrogen
(ii) Redesigning metabolic pathways to increase the yield of
secondary metabolites or to generate compounds with enhanced
properties
(iii) Transferring the C4 photosynthesis pathway to rice
(iv) Modifying the glycosylation pathway in plants to accommodate
production of therapeutic proteins
(v) Introducing synthetic signal transduction systems that respond to
external cues
55
Synthetic biology projects
Nicholas J Baltes et al 2015
56
Refactoring the N fixation gene cluster from Klebsiella
Karsten Temme et al 2012
57
Continuehellip
Karsten Temme et al 2012
58
Genome editing
Sequence specific nucleases
1 Meganucleases
2 ZFMs ndashZink finger motifs
3 TALENs- Transcription activator like effector nuclease
4 CRISPR CAS-9 ndash Clustered interspaced short palindromic
repeats
qmp4
59Khaoula Belhaj et al 2015
60
Genome modifications achieved in plants using sequence specific nucleases
Type of DNA
modification
Nuclease Delivery
methods
Plants Targets
Trait stacking Megnucleases Bombardment Cotton Intergenic sequence
ZFN Bombardment Maize Transgene
Rewriting host
DNA
Megnucleases Stable integration Maize Intergenic sequence
ZFN Stable integration Soybean Transgene
TALEN Stable integration Barley PAPhy_a
TALEN Agrobacterium
T-DNA (transient)
Oryza sativa SWEET14
TALEN No Protoplasts Arabidopsis
Tobacco
AtTT4 AtADH NbSurB
TALEN Bombardment Wheat MLO
TALEN Protoplasts
Stable integration
Maize PDS IPK1A IPK MRP4
CRISPRCas Protoplasts
Agrobacterium
T-DNA (transient)
Tobacco
Arabidopsis
Sorghum Oryza
OsSWEET14 transgene
61
Type of DNA
modification
Nuclease Delivery
methods
Plants Targets
Rewriting host
DNA large
deletion
Zinc-finger
nuclease
Stable
integration
Tobacco Transgene
CRISPRCas Protoplasts
Stable
integration
Rice Labdane-related
diterpenoid gene
clusters
on Chr 2 4 and 6
Zinc-finger
nuclease
Agrobacterium
T-DNA
(transient)
Tobacco CHN50 transgene
Zinc-finger
nuclease
Whiskers Maize IPK1
CRISPRCas Protoplasts Rice PDS
Controlling gene
expression
TALE repressor
(SRDX)
Stable
integration
Arabidopsis RD29A transgene
Zinc-finger
activator (VP16)
Stable
integration
Brassica
napus
KasII
Continue
62
Application of genome editing1 Introduction of precise and predictable modifications directly in an elite
background
2 multiple traits can be modified simultaneously
3 NHEJ enables gene knockout and targeted modifications
4 Introduction of transgenes at defined loci that promote high-level
transcription and do not interfere with the activity of endogenous genes
5 Site-specific nucleases also allow targeted molecular trait stacking -low
risk of segregation
6 CRISPR Cas is a transgene free approach ndash No regulatory burdens
7 frequency of off-target mutations is well below that caused by chemical
and physical mutagenesis techniques
8 In future can be utilized for metabolic engineering and molecular farming
63
Six-way Venn diagram showing the distribution of shared genefamilies (sequence clusters) among M acuminata P dactyliferaArabidopsis thaliana Oryza sativa Sorghum bicolor and Brachypodiumdistachyon genomes
A DrsquoHont et al Nature 2012 doi101038nature11241
64
Achieving super-DomesticationINTROGRESS-RECREATE-CREATE-DOMESTICATEREDOMESTICATE
bull Mobilizing left out genetic
variation still available in
land races and wild and
weedy species
bull Replaying the evolutionary
tape( resynthesis in
polyploids)
bull Domesticateredomesticate
65
We can admire and emulate how indigenous people still domesticateplants create biodiversity and manage it to sustain their future Thereis no equivalently dynamic or flexible crop breeding in modernagriculture to promote biodiversity we still have much to learn from
traditional knowledge Jan Salick 2012
Nivara
Rufipogon
Glaberrima
7
Domesticated Rice
8
Wild wheat
9
Domesticated Wheat
10
Maize
11
Domesticated maize
12
Barley Domestication
Prognt H spontanium
13
14
Domesticated Pearl MilletPennisetum glaucum
better seed recovery and yield but less able to survive in natural conditions compared to wild progenitor
bull more apical dominance (less
branching)
bull compact growth habit
bull flowering at same time (rather
than spread over long period)
bull larger spikes
bull non-shattering spikelets
bull loss of bristles amp glumes around
grains
bull larger seeds
bull non-dormant seeds
bull germinate at same time
15
Ppolystachian
Oats domestication
Wild- A sterilis Modern
sativa
16
Domestication of Sorghum
S helepense
S bicolor17
JR Harlan 1976 Sci American
Divergent
selection for
different
purposes
httpenwikipediaorgwikiImageBrassica_oleracea0jpg
httpwwwcehacukimagesclip_image002_001jpg
Brassica oleracea cabbage
broccoli cauliflower kale
romanesco collards kohrabi
brussels sprouts18
Solanum pimpinellifolium
Solanum lycopersicum19
Domestication of carrot
Queen Annes Lace
Daucus carota
20
Domestication of peanut
A villosulicarpa Hoehne andA Stenosperma (Brazil)
AABB
21
Domestication of Chilli
22
StrawberryBanana
Musa acuminata Musa balbisiana (2n=3x=33) AB genomeFRAGARIA X ANANASSA
Wood berries (F vesca) and Musky strawberries (F moschata)23
Wild SoybeanGlycin soja2n=40
Cultivated soybeanGlycin max 2n=40
24
Increased in domesticate
seed germination
determinate growth
longer pods
bigger (heavier) seeds
earliness
harvest index (seed yieldbiomass)
Decreased in domesticate
seed dispersal
seed dormancy
twining
number of nodes
length of internode
number of pods
photoperiod sensitivity
Common Bean Phaseolus vulgarishttpwwwplantsciencesucdavisedugeptspb143pb143htm
copy Paul Gepts
25
Wild pigeon peaCajanus cajanifolius
Cultivated pigeon pea
26
27
28
WHY OTHERS ARE NOT DOMESTICATED
bull 350000 plants
bull 4629 mammals
bull 9200 birds
bull 10000000 insects
bull 500000 fungi
bull But only 200 plants
bull 15 mammals 5 birds and
bull 2 insects are domesticated
bull Spread of these few species
bull Little change since early
agriculture
bull Repeated domestication of these
species (sometimes)
bull Lack of new species even with
attempts with species known to be
valuable Some groups are good
candidates with no domestication
eg ferns sub-Saharan mammals
29
WHY OTHERS HAVE TO BE DOMESTICATED
New uses and demands ndash biofuels animal feed
medicinalneutraceutical waterclimate food changes
Knowledge why species arenrsquot suitable for domestication or
were not useful
Better understanding of genetics and selection
Sustainability of production
Reliability of production
To meet the food demand in an alternative way
WHY OTHERS HAVE TO BE DOMESTICATED
30
Reallocate biomass for human use
More efficient metabolism and photosynthesis increase
leaf surface decrease root system increase leaf
longevity
Grains more fertile florets larger inflorescence OR
number of ears different ways to get more seed
Larger seeds (automatic vs deliberate selection)
Oil plants increased oil content or more seed
Fiber plants long strong fibers
Domestication syndrome
31
Centers of Domestication
Fuller 2011
32
Centers of Domestication in India
Fuller 2011
33
Indian centers of domestication South India (Deccan centre) Vigna radiate Vigna mungo
Macrotyloma uniflorum (Horse gram) Sataria verticillata S plumila
wheat (macaroni) and two rowed barleybrachiaria ramosa
Orissa (Mahanadi river) Pegion pea Horsegram mung small millets
like Echinichloa Paspalum and Sataria
The Middle Ganges (Harappan civilization) Oryza nivara O rufipogon
and wild sativa Sateria pumia Cannabis sativa even the diffusion of
japonica rice
Saurashtra (Harappan civilization) Eleucine coracana Sateria italica
Paicum spp Brachypodia pearl millet and sorghum Dollicos lablab
The Himalayan foothills of the Punjab region Was a centre of
diversity for Japonica type rice and many temperate fruits and
vegetables
34
DOMESTICATION RELATED TRAITS CONTROLLED BY ONLY FEW GENES
Trait Crops
Plant architecturegrowth habit Ricemaizemilletsbeantomato
Flowering timephotoperiod sensitivity Ricemaizesorghumbeantomato
Fruit size Tomato egg plant
Grain size Ricemaizesorghumbean
Seed dispersal Brassica rice
Inflorescence modification Brassica
Dormancy Bean
35
Genes Crop Moleular and phenotypic function Causative change
Genes identified as controlling domestication traits
tb1 Maize Transcriptional regulator (TCP) plant and
inflorescence structure
regulatory change
tga1 Maize Transcriptional regulator (SBP) seed casing amino acid change
qSH1 Rice Transcriptional regulator (homeodomain)
abscission layer formation shattering
regulatory change
Rc Rice Transcriptional regulator (bHLH) seed color disrupted coding
sequence
sh4 Rice Transcriptional regulator (Myb3) abscission
layer formation shattering
regulatoryamino
acid change
fw22 Tomato Cell signaling fruit weight regulatory change
Q Wheat Transcriptional regulator (AP2) inflorescence
structure
regulatoryamino
acid change
Vrs1 Barley Inflorescence structure Premature stop Amino acid change
Genes of interest in crop domestication and improvement
36
Genes Crop Moleular and phenotypic function Causative change
c1
r1
Maize Transcriptional regulator (MYB) kernel color and
Transcriptional regulator (bHLH) kernel color
regulatory change
sh2 Maize pyrophosphorylase supersweet sweet corn transposon
insertion
su1 Maize isoamylase sweet corn gene amino acid change
brix9-2-5 Tomato Invertase fruit soluble solid content amino acid change
ovate Tomato Unknown fruit shap early stop codon
R Pea Starch branching enzyme seed sugar content transposon
insertion
ehd1 Rice B-type response regulator flowering time amino acid change
hd1 Rice Transcriptional regulator (zinc finger) flowering
time
disrupted coding
sequence
waxy Rice Starch synthase sticky grains intron splicing
defect
rht Wheat Transcriptional regulator (SH2) plant height early stop codon
vrn1 Wheat Transcriptional regulator (MADS) vernalization regulatory change
vrn2 Wheat Transcriptional regulator (ZCCT) vernalization amino acid change
Genes Identified as Controlling Varietal Differences
37
Process of domestication over the yearshellip
Rachel S Meyer 2012
38
Genetic bottleneck
Cultivated crops undergone with narrowing of diversity problem
But this is not the case in weeds
Weeds are the major threats from the beginning of domestication till
today
Problem arose due to
1 Crop mimicry
2 Genetic assimilation
3 Genetic evolution
39
Super- Domestication Vaughn et al (2007) first used the term super-domestication
The processes that lead to a domesticate with dramatically
increased yield that could not be selected in natural environments
from naturally occurring variation without recourse to new
technologies
Super-domesticates can be constructed with knowledge led
approaches based on current needs using the range of new
technologies now available
Plants exploited for continuous selection introduction hybridization
etc which boosted the process of domestication but now the plant
genetic engineering approach is exploiting plants towards synthetic
biology
40
How to use diversitybull Cross two varieties
bull Genome manipulations
Cell fusion hybrids
bull Chromosome manipulation Backcross a new species
bull Generate recombinants Chromosome recombinations
bull Use a new species wild germplasm
Transgenic approach Modern mutagenesis synthetic gene
construction by utilizing green florescent protein genome editing
NGS GWAS sequencing etchelliphelliphelliphelliphellip
41
Reduce genetic bottlenecks through polyplodization
More vigour
SC
Buffering capacity
Heterotic advantage
Enhaced vegetative
charachers
Enhanced oil
Meiotic stability
42
Approach 1 Use one tetraploid and one diploid as parents (4X ndash 2X) followed by the
chromosome doubling of triploid hybrids (Chrom doubling)
a) Cross between B juncea (AjAjBjBj ) and B oleracea (CoCo) to produce hexaploids
(AjAjBjBjCoCo)
(b) Cross between B napus (AnAnCnCn) and B nigra (BniBni) to produce hexaploids
(AnAnBniBniCnCn)
(c) Cross between B carinata (BcaBcaCcaCca) and B rapa (ArAr) to produce hexaploids
(ArArBcaBcaCcaCca)
Approach 2 Use three tetraploids as parents
(a) Cross between B napus (AnAnCnCn) and B carinata (BcaBcaCcaCca) to produce unbalanced
allotetraploids (AnBcaCnCca ndash unreduced gametes gametes with the somatic chromosome
number and cross with B juncea (AjAjBjBj) to obtain allohexaploids (AnAjBcaBjCnCca
(b) Cross between B napus (AnAnCnCn) and B juncea (AjAjBjBj) to produce unbalanced
allotetraploids (AnAjBjCn ndash unreduced gametes) and cross with B carinata (BcaBcaCcaCca) to
obtain allohexaploids (AnAjBcaBjCnCca)
(c) Cross between B carinata (BcaBcaCcaCca) and B juncea (AjAjBjBj) to produce unbalanced
allotetraploids (AjBcaBjCca - ndash unreduced gametes) and cross with B napus (AnAnCnCn) to obtain
allohexaploids (AnAjBcaBjCnCca)
Approach 3 Use three diploids as parents (2X ndash 2X ndash 2X) Cross between B rapa (ArAr) B
nigra (BniBni) and B oleracea (CoCo) sequentially to obtain hexaploid hybrids (ArArBniBniCoCo)
43
Continuehellip
Guijun Yan 2012
44
Applications of Genomic
tools in Super-Domestication
45
Genome Sequencing
Potential methods of sequencing
1 Clone by clone approach
2 Whole genome shotgun approach
3 Combination of the two methods
Till today no of cultivated plants completely sequenced -85
2016- Arachis duranensis
2015- Solanum cumersonii (Wild potato)
(Ref- NCBI)
46
GWAS
It is a study design in which many markers spread across a genome
are genotyped and test a statistical association with a phenotype are
performed locally along the genome
It is also an examination of many common genetic variants in different
individuals to see if any variant is associated with a trait
Used in successfully studying maize sorghum and barley
Method is efficient for large scale low cost genotyping (even with the
minimum number of SNPs)
Cannot be utilized generally because it needs large population size
GWAS identify rare alleles more precisely
If small population we can opt NAM
47
GWAS and whole genome prediction
Xuehui Huang and Bin Han 2014
48
Five high throuput genotyping methods
Xuehui Huang and Bin Han 2014
49
Role of NGS in domestication Capture of novel genes from wild species will be made easier by
understanding the molecular events associated with crop domestication
Re-sequencing of domesticated species can identify low diversity regions
resulting from selection during domestication
To identify gene-specific sequences to aid the cloning of homologues of key
domestication genes from wild relatives
Candidate genes from wild and domesticated plant populations can define
diversity of target genes in wild populations and lead to the discovery of key
genes for important traits by association analysis
NGS of amplicons of large numbers of candidate genes from wild and
domesticated plant populations can define diversity of target genes in
wild populations and lead to the discovery of key genes for important
traits by association analysis
50
NGS
supports the rapid domestication of new plant species and the efficient
identification and capture of novel genetic variation from related species
Allows whole-genome analysis to determine the genetic basis of
phenotypic differences
NGS allows rapid expansion of genomic analysis to investigation of non-
model species
Made rice study easy by related grass
cost-effective method for plant identification
useful strategy to analysis the chloroplast genome sequence from whole-
genome shot-gun sequencing
facilitates managing this diversity and any changes in crop performance
over time due to genetic drift
Patterns of gene expression have been evaluated in hybrids using NGS
51
Two Approaches to Finding Adaptive Genes
Ross-Ibarra et al2007
52
Techniques to re-wild the plants (transgene free)
1 Introgression
breeding
2 Specific
insertion of lost genes
3 Precision
mutagenesis
Palmgren et al2014
53
Process of rewilding
Palmgren et al2014
54
Synthetic biology projects(i) Modifying cereals including wheat to fix atmospheric nitrogen
(ii) Redesigning metabolic pathways to increase the yield of
secondary metabolites or to generate compounds with enhanced
properties
(iii) Transferring the C4 photosynthesis pathway to rice
(iv) Modifying the glycosylation pathway in plants to accommodate
production of therapeutic proteins
(v) Introducing synthetic signal transduction systems that respond to
external cues
55
Synthetic biology projects
Nicholas J Baltes et al 2015
56
Refactoring the N fixation gene cluster from Klebsiella
Karsten Temme et al 2012
57
Continuehellip
Karsten Temme et al 2012
58
Genome editing
Sequence specific nucleases
1 Meganucleases
2 ZFMs ndashZink finger motifs
3 TALENs- Transcription activator like effector nuclease
4 CRISPR CAS-9 ndash Clustered interspaced short palindromic
repeats
qmp4
59Khaoula Belhaj et al 2015
60
Genome modifications achieved in plants using sequence specific nucleases
Type of DNA
modification
Nuclease Delivery
methods
Plants Targets
Trait stacking Megnucleases Bombardment Cotton Intergenic sequence
ZFN Bombardment Maize Transgene
Rewriting host
DNA
Megnucleases Stable integration Maize Intergenic sequence
ZFN Stable integration Soybean Transgene
TALEN Stable integration Barley PAPhy_a
TALEN Agrobacterium
T-DNA (transient)
Oryza sativa SWEET14
TALEN No Protoplasts Arabidopsis
Tobacco
AtTT4 AtADH NbSurB
TALEN Bombardment Wheat MLO
TALEN Protoplasts
Stable integration
Maize PDS IPK1A IPK MRP4
CRISPRCas Protoplasts
Agrobacterium
T-DNA (transient)
Tobacco
Arabidopsis
Sorghum Oryza
OsSWEET14 transgene
61
Type of DNA
modification
Nuclease Delivery
methods
Plants Targets
Rewriting host
DNA large
deletion
Zinc-finger
nuclease
Stable
integration
Tobacco Transgene
CRISPRCas Protoplasts
Stable
integration
Rice Labdane-related
diterpenoid gene
clusters
on Chr 2 4 and 6
Zinc-finger
nuclease
Agrobacterium
T-DNA
(transient)
Tobacco CHN50 transgene
Zinc-finger
nuclease
Whiskers Maize IPK1
CRISPRCas Protoplasts Rice PDS
Controlling gene
expression
TALE repressor
(SRDX)
Stable
integration
Arabidopsis RD29A transgene
Zinc-finger
activator (VP16)
Stable
integration
Brassica
napus
KasII
Continue
62
Application of genome editing1 Introduction of precise and predictable modifications directly in an elite
background
2 multiple traits can be modified simultaneously
3 NHEJ enables gene knockout and targeted modifications
4 Introduction of transgenes at defined loci that promote high-level
transcription and do not interfere with the activity of endogenous genes
5 Site-specific nucleases also allow targeted molecular trait stacking -low
risk of segregation
6 CRISPR Cas is a transgene free approach ndash No regulatory burdens
7 frequency of off-target mutations is well below that caused by chemical
and physical mutagenesis techniques
8 In future can be utilized for metabolic engineering and molecular farming
63
Six-way Venn diagram showing the distribution of shared genefamilies (sequence clusters) among M acuminata P dactyliferaArabidopsis thaliana Oryza sativa Sorghum bicolor and Brachypodiumdistachyon genomes
A DrsquoHont et al Nature 2012 doi101038nature11241
64
Achieving super-DomesticationINTROGRESS-RECREATE-CREATE-DOMESTICATEREDOMESTICATE
bull Mobilizing left out genetic
variation still available in
land races and wild and
weedy species
bull Replaying the evolutionary
tape( resynthesis in
polyploids)
bull Domesticateredomesticate
65
We can admire and emulate how indigenous people still domesticateplants create biodiversity and manage it to sustain their future Thereis no equivalently dynamic or flexible crop breeding in modernagriculture to promote biodiversity we still have much to learn from
traditional knowledge Jan Salick 2012
Domesticated Rice
8
Wild wheat
9
Domesticated Wheat
10
Maize
11
Domesticated maize
12
Barley Domestication
Prognt H spontanium
13
14
Domesticated Pearl MilletPennisetum glaucum
better seed recovery and yield but less able to survive in natural conditions compared to wild progenitor
bull more apical dominance (less
branching)
bull compact growth habit
bull flowering at same time (rather
than spread over long period)
bull larger spikes
bull non-shattering spikelets
bull loss of bristles amp glumes around
grains
bull larger seeds
bull non-dormant seeds
bull germinate at same time
15
Ppolystachian
Oats domestication
Wild- A sterilis Modern
sativa
16
Domestication of Sorghum
S helepense
S bicolor17
JR Harlan 1976 Sci American
Divergent
selection for
different
purposes
httpenwikipediaorgwikiImageBrassica_oleracea0jpg
httpwwwcehacukimagesclip_image002_001jpg
Brassica oleracea cabbage
broccoli cauliflower kale
romanesco collards kohrabi
brussels sprouts18
Solanum pimpinellifolium
Solanum lycopersicum19
Domestication of carrot
Queen Annes Lace
Daucus carota
20
Domestication of peanut
A villosulicarpa Hoehne andA Stenosperma (Brazil)
AABB
21
Domestication of Chilli
22
StrawberryBanana
Musa acuminata Musa balbisiana (2n=3x=33) AB genomeFRAGARIA X ANANASSA
Wood berries (F vesca) and Musky strawberries (F moschata)23
Wild SoybeanGlycin soja2n=40
Cultivated soybeanGlycin max 2n=40
24
Increased in domesticate
seed germination
determinate growth
longer pods
bigger (heavier) seeds
earliness
harvest index (seed yieldbiomass)
Decreased in domesticate
seed dispersal
seed dormancy
twining
number of nodes
length of internode
number of pods
photoperiod sensitivity
Common Bean Phaseolus vulgarishttpwwwplantsciencesucdavisedugeptspb143pb143htm
copy Paul Gepts
25
Wild pigeon peaCajanus cajanifolius
Cultivated pigeon pea
26
27
28
WHY OTHERS ARE NOT DOMESTICATED
bull 350000 plants
bull 4629 mammals
bull 9200 birds
bull 10000000 insects
bull 500000 fungi
bull But only 200 plants
bull 15 mammals 5 birds and
bull 2 insects are domesticated
bull Spread of these few species
bull Little change since early
agriculture
bull Repeated domestication of these
species (sometimes)
bull Lack of new species even with
attempts with species known to be
valuable Some groups are good
candidates with no domestication
eg ferns sub-Saharan mammals
29
WHY OTHERS HAVE TO BE DOMESTICATED
New uses and demands ndash biofuels animal feed
medicinalneutraceutical waterclimate food changes
Knowledge why species arenrsquot suitable for domestication or
were not useful
Better understanding of genetics and selection
Sustainability of production
Reliability of production
To meet the food demand in an alternative way
WHY OTHERS HAVE TO BE DOMESTICATED
30
Reallocate biomass for human use
More efficient metabolism and photosynthesis increase
leaf surface decrease root system increase leaf
longevity
Grains more fertile florets larger inflorescence OR
number of ears different ways to get more seed
Larger seeds (automatic vs deliberate selection)
Oil plants increased oil content or more seed
Fiber plants long strong fibers
Domestication syndrome
31
Centers of Domestication
Fuller 2011
32
Centers of Domestication in India
Fuller 2011
33
Indian centers of domestication South India (Deccan centre) Vigna radiate Vigna mungo
Macrotyloma uniflorum (Horse gram) Sataria verticillata S plumila
wheat (macaroni) and two rowed barleybrachiaria ramosa
Orissa (Mahanadi river) Pegion pea Horsegram mung small millets
like Echinichloa Paspalum and Sataria
The Middle Ganges (Harappan civilization) Oryza nivara O rufipogon
and wild sativa Sateria pumia Cannabis sativa even the diffusion of
japonica rice
Saurashtra (Harappan civilization) Eleucine coracana Sateria italica
Paicum spp Brachypodia pearl millet and sorghum Dollicos lablab
The Himalayan foothills of the Punjab region Was a centre of
diversity for Japonica type rice and many temperate fruits and
vegetables
34
DOMESTICATION RELATED TRAITS CONTROLLED BY ONLY FEW GENES
Trait Crops
Plant architecturegrowth habit Ricemaizemilletsbeantomato
Flowering timephotoperiod sensitivity Ricemaizesorghumbeantomato
Fruit size Tomato egg plant
Grain size Ricemaizesorghumbean
Seed dispersal Brassica rice
Inflorescence modification Brassica
Dormancy Bean
35
Genes Crop Moleular and phenotypic function Causative change
Genes identified as controlling domestication traits
tb1 Maize Transcriptional regulator (TCP) plant and
inflorescence structure
regulatory change
tga1 Maize Transcriptional regulator (SBP) seed casing amino acid change
qSH1 Rice Transcriptional regulator (homeodomain)
abscission layer formation shattering
regulatory change
Rc Rice Transcriptional regulator (bHLH) seed color disrupted coding
sequence
sh4 Rice Transcriptional regulator (Myb3) abscission
layer formation shattering
regulatoryamino
acid change
fw22 Tomato Cell signaling fruit weight regulatory change
Q Wheat Transcriptional regulator (AP2) inflorescence
structure
regulatoryamino
acid change
Vrs1 Barley Inflorescence structure Premature stop Amino acid change
Genes of interest in crop domestication and improvement
36
Genes Crop Moleular and phenotypic function Causative change
c1
r1
Maize Transcriptional regulator (MYB) kernel color and
Transcriptional regulator (bHLH) kernel color
regulatory change
sh2 Maize pyrophosphorylase supersweet sweet corn transposon
insertion
su1 Maize isoamylase sweet corn gene amino acid change
brix9-2-5 Tomato Invertase fruit soluble solid content amino acid change
ovate Tomato Unknown fruit shap early stop codon
R Pea Starch branching enzyme seed sugar content transposon
insertion
ehd1 Rice B-type response regulator flowering time amino acid change
hd1 Rice Transcriptional regulator (zinc finger) flowering
time
disrupted coding
sequence
waxy Rice Starch synthase sticky grains intron splicing
defect
rht Wheat Transcriptional regulator (SH2) plant height early stop codon
vrn1 Wheat Transcriptional regulator (MADS) vernalization regulatory change
vrn2 Wheat Transcriptional regulator (ZCCT) vernalization amino acid change
Genes Identified as Controlling Varietal Differences
37
Process of domestication over the yearshellip
Rachel S Meyer 2012
38
Genetic bottleneck
Cultivated crops undergone with narrowing of diversity problem
But this is not the case in weeds
Weeds are the major threats from the beginning of domestication till
today
Problem arose due to
1 Crop mimicry
2 Genetic assimilation
3 Genetic evolution
39
Super- Domestication Vaughn et al (2007) first used the term super-domestication
The processes that lead to a domesticate with dramatically
increased yield that could not be selected in natural environments
from naturally occurring variation without recourse to new
technologies
Super-domesticates can be constructed with knowledge led
approaches based on current needs using the range of new
technologies now available
Plants exploited for continuous selection introduction hybridization
etc which boosted the process of domestication but now the plant
genetic engineering approach is exploiting plants towards synthetic
biology
40
How to use diversitybull Cross two varieties
bull Genome manipulations
Cell fusion hybrids
bull Chromosome manipulation Backcross a new species
bull Generate recombinants Chromosome recombinations
bull Use a new species wild germplasm
Transgenic approach Modern mutagenesis synthetic gene
construction by utilizing green florescent protein genome editing
NGS GWAS sequencing etchelliphelliphelliphelliphellip
41
Reduce genetic bottlenecks through polyplodization
More vigour
SC
Buffering capacity
Heterotic advantage
Enhaced vegetative
charachers
Enhanced oil
Meiotic stability
42
Approach 1 Use one tetraploid and one diploid as parents (4X ndash 2X) followed by the
chromosome doubling of triploid hybrids (Chrom doubling)
a) Cross between B juncea (AjAjBjBj ) and B oleracea (CoCo) to produce hexaploids
(AjAjBjBjCoCo)
(b) Cross between B napus (AnAnCnCn) and B nigra (BniBni) to produce hexaploids
(AnAnBniBniCnCn)
(c) Cross between B carinata (BcaBcaCcaCca) and B rapa (ArAr) to produce hexaploids
(ArArBcaBcaCcaCca)
Approach 2 Use three tetraploids as parents
(a) Cross between B napus (AnAnCnCn) and B carinata (BcaBcaCcaCca) to produce unbalanced
allotetraploids (AnBcaCnCca ndash unreduced gametes gametes with the somatic chromosome
number and cross with B juncea (AjAjBjBj) to obtain allohexaploids (AnAjBcaBjCnCca
(b) Cross between B napus (AnAnCnCn) and B juncea (AjAjBjBj) to produce unbalanced
allotetraploids (AnAjBjCn ndash unreduced gametes) and cross with B carinata (BcaBcaCcaCca) to
obtain allohexaploids (AnAjBcaBjCnCca)
(c) Cross between B carinata (BcaBcaCcaCca) and B juncea (AjAjBjBj) to produce unbalanced
allotetraploids (AjBcaBjCca - ndash unreduced gametes) and cross with B napus (AnAnCnCn) to obtain
allohexaploids (AnAjBcaBjCnCca)
Approach 3 Use three diploids as parents (2X ndash 2X ndash 2X) Cross between B rapa (ArAr) B
nigra (BniBni) and B oleracea (CoCo) sequentially to obtain hexaploid hybrids (ArArBniBniCoCo)
43
Continuehellip
Guijun Yan 2012
44
Applications of Genomic
tools in Super-Domestication
45
Genome Sequencing
Potential methods of sequencing
1 Clone by clone approach
2 Whole genome shotgun approach
3 Combination of the two methods
Till today no of cultivated plants completely sequenced -85
2016- Arachis duranensis
2015- Solanum cumersonii (Wild potato)
(Ref- NCBI)
46
GWAS
It is a study design in which many markers spread across a genome
are genotyped and test a statistical association with a phenotype are
performed locally along the genome
It is also an examination of many common genetic variants in different
individuals to see if any variant is associated with a trait
Used in successfully studying maize sorghum and barley
Method is efficient for large scale low cost genotyping (even with the
minimum number of SNPs)
Cannot be utilized generally because it needs large population size
GWAS identify rare alleles more precisely
If small population we can opt NAM
47
GWAS and whole genome prediction
Xuehui Huang and Bin Han 2014
48
Five high throuput genotyping methods
Xuehui Huang and Bin Han 2014
49
Role of NGS in domestication Capture of novel genes from wild species will be made easier by
understanding the molecular events associated with crop domestication
Re-sequencing of domesticated species can identify low diversity regions
resulting from selection during domestication
To identify gene-specific sequences to aid the cloning of homologues of key
domestication genes from wild relatives
Candidate genes from wild and domesticated plant populations can define
diversity of target genes in wild populations and lead to the discovery of key
genes for important traits by association analysis
NGS of amplicons of large numbers of candidate genes from wild and
domesticated plant populations can define diversity of target genes in
wild populations and lead to the discovery of key genes for important
traits by association analysis
50
NGS
supports the rapid domestication of new plant species and the efficient
identification and capture of novel genetic variation from related species
Allows whole-genome analysis to determine the genetic basis of
phenotypic differences
NGS allows rapid expansion of genomic analysis to investigation of non-
model species
Made rice study easy by related grass
cost-effective method for plant identification
useful strategy to analysis the chloroplast genome sequence from whole-
genome shot-gun sequencing
facilitates managing this diversity and any changes in crop performance
over time due to genetic drift
Patterns of gene expression have been evaluated in hybrids using NGS
51
Two Approaches to Finding Adaptive Genes
Ross-Ibarra et al2007
52
Techniques to re-wild the plants (transgene free)
1 Introgression
breeding
2 Specific
insertion of lost genes
3 Precision
mutagenesis
Palmgren et al2014
53
Process of rewilding
Palmgren et al2014
54
Synthetic biology projects(i) Modifying cereals including wheat to fix atmospheric nitrogen
(ii) Redesigning metabolic pathways to increase the yield of
secondary metabolites or to generate compounds with enhanced
properties
(iii) Transferring the C4 photosynthesis pathway to rice
(iv) Modifying the glycosylation pathway in plants to accommodate
production of therapeutic proteins
(v) Introducing synthetic signal transduction systems that respond to
external cues
55
Synthetic biology projects
Nicholas J Baltes et al 2015
56
Refactoring the N fixation gene cluster from Klebsiella
Karsten Temme et al 2012
57
Continuehellip
Karsten Temme et al 2012
58
Genome editing
Sequence specific nucleases
1 Meganucleases
2 ZFMs ndashZink finger motifs
3 TALENs- Transcription activator like effector nuclease
4 CRISPR CAS-9 ndash Clustered interspaced short palindromic
repeats
qmp4
59Khaoula Belhaj et al 2015
60
Genome modifications achieved in plants using sequence specific nucleases
Type of DNA
modification
Nuclease Delivery
methods
Plants Targets
Trait stacking Megnucleases Bombardment Cotton Intergenic sequence
ZFN Bombardment Maize Transgene
Rewriting host
DNA
Megnucleases Stable integration Maize Intergenic sequence
ZFN Stable integration Soybean Transgene
TALEN Stable integration Barley PAPhy_a
TALEN Agrobacterium
T-DNA (transient)
Oryza sativa SWEET14
TALEN No Protoplasts Arabidopsis
Tobacco
AtTT4 AtADH NbSurB
TALEN Bombardment Wheat MLO
TALEN Protoplasts
Stable integration
Maize PDS IPK1A IPK MRP4
CRISPRCas Protoplasts
Agrobacterium
T-DNA (transient)
Tobacco
Arabidopsis
Sorghum Oryza
OsSWEET14 transgene
61
Type of DNA
modification
Nuclease Delivery
methods
Plants Targets
Rewriting host
DNA large
deletion
Zinc-finger
nuclease
Stable
integration
Tobacco Transgene
CRISPRCas Protoplasts
Stable
integration
Rice Labdane-related
diterpenoid gene
clusters
on Chr 2 4 and 6
Zinc-finger
nuclease
Agrobacterium
T-DNA
(transient)
Tobacco CHN50 transgene
Zinc-finger
nuclease
Whiskers Maize IPK1
CRISPRCas Protoplasts Rice PDS
Controlling gene
expression
TALE repressor
(SRDX)
Stable
integration
Arabidopsis RD29A transgene
Zinc-finger
activator (VP16)
Stable
integration
Brassica
napus
KasII
Continue
62
Application of genome editing1 Introduction of precise and predictable modifications directly in an elite
background
2 multiple traits can be modified simultaneously
3 NHEJ enables gene knockout and targeted modifications
4 Introduction of transgenes at defined loci that promote high-level
transcription and do not interfere with the activity of endogenous genes
5 Site-specific nucleases also allow targeted molecular trait stacking -low
risk of segregation
6 CRISPR Cas is a transgene free approach ndash No regulatory burdens
7 frequency of off-target mutations is well below that caused by chemical
and physical mutagenesis techniques
8 In future can be utilized for metabolic engineering and molecular farming
63
Six-way Venn diagram showing the distribution of shared genefamilies (sequence clusters) among M acuminata P dactyliferaArabidopsis thaliana Oryza sativa Sorghum bicolor and Brachypodiumdistachyon genomes
A DrsquoHont et al Nature 2012 doi101038nature11241
64
Achieving super-DomesticationINTROGRESS-RECREATE-CREATE-DOMESTICATEREDOMESTICATE
bull Mobilizing left out genetic
variation still available in
land races and wild and
weedy species
bull Replaying the evolutionary
tape( resynthesis in
polyploids)
bull Domesticateredomesticate
65
We can admire and emulate how indigenous people still domesticateplants create biodiversity and manage it to sustain their future Thereis no equivalently dynamic or flexible crop breeding in modernagriculture to promote biodiversity we still have much to learn from
traditional knowledge Jan Salick 2012
Wild wheat
9
Domesticated Wheat
10
Maize
11
Domesticated maize
12
Barley Domestication
Prognt H spontanium
13
14
Domesticated Pearl MilletPennisetum glaucum
better seed recovery and yield but less able to survive in natural conditions compared to wild progenitor
bull more apical dominance (less
branching)
bull compact growth habit
bull flowering at same time (rather
than spread over long period)
bull larger spikes
bull non-shattering spikelets
bull loss of bristles amp glumes around
grains
bull larger seeds
bull non-dormant seeds
bull germinate at same time
15
Ppolystachian
Oats domestication
Wild- A sterilis Modern
sativa
16
Domestication of Sorghum
S helepense
S bicolor17
JR Harlan 1976 Sci American
Divergent
selection for
different
purposes
httpenwikipediaorgwikiImageBrassica_oleracea0jpg
httpwwwcehacukimagesclip_image002_001jpg
Brassica oleracea cabbage
broccoli cauliflower kale
romanesco collards kohrabi
brussels sprouts18
Solanum pimpinellifolium
Solanum lycopersicum19
Domestication of carrot
Queen Annes Lace
Daucus carota
20
Domestication of peanut
A villosulicarpa Hoehne andA Stenosperma (Brazil)
AABB
21
Domestication of Chilli
22
StrawberryBanana
Musa acuminata Musa balbisiana (2n=3x=33) AB genomeFRAGARIA X ANANASSA
Wood berries (F vesca) and Musky strawberries (F moschata)23
Wild SoybeanGlycin soja2n=40
Cultivated soybeanGlycin max 2n=40
24
Increased in domesticate
seed germination
determinate growth
longer pods
bigger (heavier) seeds
earliness
harvest index (seed yieldbiomass)
Decreased in domesticate
seed dispersal
seed dormancy
twining
number of nodes
length of internode
number of pods
photoperiod sensitivity
Common Bean Phaseolus vulgarishttpwwwplantsciencesucdavisedugeptspb143pb143htm
copy Paul Gepts
25
Wild pigeon peaCajanus cajanifolius
Cultivated pigeon pea
26
27
28
WHY OTHERS ARE NOT DOMESTICATED
bull 350000 plants
bull 4629 mammals
bull 9200 birds
bull 10000000 insects
bull 500000 fungi
bull But only 200 plants
bull 15 mammals 5 birds and
bull 2 insects are domesticated
bull Spread of these few species
bull Little change since early
agriculture
bull Repeated domestication of these
species (sometimes)
bull Lack of new species even with
attempts with species known to be
valuable Some groups are good
candidates with no domestication
eg ferns sub-Saharan mammals
29
WHY OTHERS HAVE TO BE DOMESTICATED
New uses and demands ndash biofuels animal feed
medicinalneutraceutical waterclimate food changes
Knowledge why species arenrsquot suitable for domestication or
were not useful
Better understanding of genetics and selection
Sustainability of production
Reliability of production
To meet the food demand in an alternative way
WHY OTHERS HAVE TO BE DOMESTICATED
30
Reallocate biomass for human use
More efficient metabolism and photosynthesis increase
leaf surface decrease root system increase leaf
longevity
Grains more fertile florets larger inflorescence OR
number of ears different ways to get more seed
Larger seeds (automatic vs deliberate selection)
Oil plants increased oil content or more seed
Fiber plants long strong fibers
Domestication syndrome
31
Centers of Domestication
Fuller 2011
32
Centers of Domestication in India
Fuller 2011
33
Indian centers of domestication South India (Deccan centre) Vigna radiate Vigna mungo
Macrotyloma uniflorum (Horse gram) Sataria verticillata S plumila
wheat (macaroni) and two rowed barleybrachiaria ramosa
Orissa (Mahanadi river) Pegion pea Horsegram mung small millets
like Echinichloa Paspalum and Sataria
The Middle Ganges (Harappan civilization) Oryza nivara O rufipogon
and wild sativa Sateria pumia Cannabis sativa even the diffusion of
japonica rice
Saurashtra (Harappan civilization) Eleucine coracana Sateria italica
Paicum spp Brachypodia pearl millet and sorghum Dollicos lablab
The Himalayan foothills of the Punjab region Was a centre of
diversity for Japonica type rice and many temperate fruits and
vegetables
34
DOMESTICATION RELATED TRAITS CONTROLLED BY ONLY FEW GENES
Trait Crops
Plant architecturegrowth habit Ricemaizemilletsbeantomato
Flowering timephotoperiod sensitivity Ricemaizesorghumbeantomato
Fruit size Tomato egg plant
Grain size Ricemaizesorghumbean
Seed dispersal Brassica rice
Inflorescence modification Brassica
Dormancy Bean
35
Genes Crop Moleular and phenotypic function Causative change
Genes identified as controlling domestication traits
tb1 Maize Transcriptional regulator (TCP) plant and
inflorescence structure
regulatory change
tga1 Maize Transcriptional regulator (SBP) seed casing amino acid change
qSH1 Rice Transcriptional regulator (homeodomain)
abscission layer formation shattering
regulatory change
Rc Rice Transcriptional regulator (bHLH) seed color disrupted coding
sequence
sh4 Rice Transcriptional regulator (Myb3) abscission
layer formation shattering
regulatoryamino
acid change
fw22 Tomato Cell signaling fruit weight regulatory change
Q Wheat Transcriptional regulator (AP2) inflorescence
structure
regulatoryamino
acid change
Vrs1 Barley Inflorescence structure Premature stop Amino acid change
Genes of interest in crop domestication and improvement
36
Genes Crop Moleular and phenotypic function Causative change
c1
r1
Maize Transcriptional regulator (MYB) kernel color and
Transcriptional regulator (bHLH) kernel color
regulatory change
sh2 Maize pyrophosphorylase supersweet sweet corn transposon
insertion
su1 Maize isoamylase sweet corn gene amino acid change
brix9-2-5 Tomato Invertase fruit soluble solid content amino acid change
ovate Tomato Unknown fruit shap early stop codon
R Pea Starch branching enzyme seed sugar content transposon
insertion
ehd1 Rice B-type response regulator flowering time amino acid change
hd1 Rice Transcriptional regulator (zinc finger) flowering
time
disrupted coding
sequence
waxy Rice Starch synthase sticky grains intron splicing
defect
rht Wheat Transcriptional regulator (SH2) plant height early stop codon
vrn1 Wheat Transcriptional regulator (MADS) vernalization regulatory change
vrn2 Wheat Transcriptional regulator (ZCCT) vernalization amino acid change
Genes Identified as Controlling Varietal Differences
37
Process of domestication over the yearshellip
Rachel S Meyer 2012
38
Genetic bottleneck
Cultivated crops undergone with narrowing of diversity problem
But this is not the case in weeds
Weeds are the major threats from the beginning of domestication till
today
Problem arose due to
1 Crop mimicry
2 Genetic assimilation
3 Genetic evolution
39
Super- Domestication Vaughn et al (2007) first used the term super-domestication
The processes that lead to a domesticate with dramatically
increased yield that could not be selected in natural environments
from naturally occurring variation without recourse to new
technologies
Super-domesticates can be constructed with knowledge led
approaches based on current needs using the range of new
technologies now available
Plants exploited for continuous selection introduction hybridization
etc which boosted the process of domestication but now the plant
genetic engineering approach is exploiting plants towards synthetic
biology
40
How to use diversitybull Cross two varieties
bull Genome manipulations
Cell fusion hybrids
bull Chromosome manipulation Backcross a new species
bull Generate recombinants Chromosome recombinations
bull Use a new species wild germplasm
Transgenic approach Modern mutagenesis synthetic gene
construction by utilizing green florescent protein genome editing
NGS GWAS sequencing etchelliphelliphelliphelliphellip
41
Reduce genetic bottlenecks through polyplodization
More vigour
SC
Buffering capacity
Heterotic advantage
Enhaced vegetative
charachers
Enhanced oil
Meiotic stability
42
Approach 1 Use one tetraploid and one diploid as parents (4X ndash 2X) followed by the
chromosome doubling of triploid hybrids (Chrom doubling)
a) Cross between B juncea (AjAjBjBj ) and B oleracea (CoCo) to produce hexaploids
(AjAjBjBjCoCo)
(b) Cross between B napus (AnAnCnCn) and B nigra (BniBni) to produce hexaploids
(AnAnBniBniCnCn)
(c) Cross between B carinata (BcaBcaCcaCca) and B rapa (ArAr) to produce hexaploids
(ArArBcaBcaCcaCca)
Approach 2 Use three tetraploids as parents
(a) Cross between B napus (AnAnCnCn) and B carinata (BcaBcaCcaCca) to produce unbalanced
allotetraploids (AnBcaCnCca ndash unreduced gametes gametes with the somatic chromosome
number and cross with B juncea (AjAjBjBj) to obtain allohexaploids (AnAjBcaBjCnCca
(b) Cross between B napus (AnAnCnCn) and B juncea (AjAjBjBj) to produce unbalanced
allotetraploids (AnAjBjCn ndash unreduced gametes) and cross with B carinata (BcaBcaCcaCca) to
obtain allohexaploids (AnAjBcaBjCnCca)
(c) Cross between B carinata (BcaBcaCcaCca) and B juncea (AjAjBjBj) to produce unbalanced
allotetraploids (AjBcaBjCca - ndash unreduced gametes) and cross with B napus (AnAnCnCn) to obtain
allohexaploids (AnAjBcaBjCnCca)
Approach 3 Use three diploids as parents (2X ndash 2X ndash 2X) Cross between B rapa (ArAr) B
nigra (BniBni) and B oleracea (CoCo) sequentially to obtain hexaploid hybrids (ArArBniBniCoCo)
43
Continuehellip
Guijun Yan 2012
44
Applications of Genomic
tools in Super-Domestication
45
Genome Sequencing
Potential methods of sequencing
1 Clone by clone approach
2 Whole genome shotgun approach
3 Combination of the two methods
Till today no of cultivated plants completely sequenced -85
2016- Arachis duranensis
2015- Solanum cumersonii (Wild potato)
(Ref- NCBI)
46
GWAS
It is a study design in which many markers spread across a genome
are genotyped and test a statistical association with a phenotype are
performed locally along the genome
It is also an examination of many common genetic variants in different
individuals to see if any variant is associated with a trait
Used in successfully studying maize sorghum and barley
Method is efficient for large scale low cost genotyping (even with the
minimum number of SNPs)
Cannot be utilized generally because it needs large population size
GWAS identify rare alleles more precisely
If small population we can opt NAM
47
GWAS and whole genome prediction
Xuehui Huang and Bin Han 2014
48
Five high throuput genotyping methods
Xuehui Huang and Bin Han 2014
49
Role of NGS in domestication Capture of novel genes from wild species will be made easier by
understanding the molecular events associated with crop domestication
Re-sequencing of domesticated species can identify low diversity regions
resulting from selection during domestication
To identify gene-specific sequences to aid the cloning of homologues of key
domestication genes from wild relatives
Candidate genes from wild and domesticated plant populations can define
diversity of target genes in wild populations and lead to the discovery of key
genes for important traits by association analysis
NGS of amplicons of large numbers of candidate genes from wild and
domesticated plant populations can define diversity of target genes in
wild populations and lead to the discovery of key genes for important
traits by association analysis
50
NGS
supports the rapid domestication of new plant species and the efficient
identification and capture of novel genetic variation from related species
Allows whole-genome analysis to determine the genetic basis of
phenotypic differences
NGS allows rapid expansion of genomic analysis to investigation of non-
model species
Made rice study easy by related grass
cost-effective method for plant identification
useful strategy to analysis the chloroplast genome sequence from whole-
genome shot-gun sequencing
facilitates managing this diversity and any changes in crop performance
over time due to genetic drift
Patterns of gene expression have been evaluated in hybrids using NGS
51
Two Approaches to Finding Adaptive Genes
Ross-Ibarra et al2007
52
Techniques to re-wild the plants (transgene free)
1 Introgression
breeding
2 Specific
insertion of lost genes
3 Precision
mutagenesis
Palmgren et al2014
53
Process of rewilding
Palmgren et al2014
54
Synthetic biology projects(i) Modifying cereals including wheat to fix atmospheric nitrogen
(ii) Redesigning metabolic pathways to increase the yield of
secondary metabolites or to generate compounds with enhanced
properties
(iii) Transferring the C4 photosynthesis pathway to rice
(iv) Modifying the glycosylation pathway in plants to accommodate
production of therapeutic proteins
(v) Introducing synthetic signal transduction systems that respond to
external cues
55
Synthetic biology projects
Nicholas J Baltes et al 2015
56
Refactoring the N fixation gene cluster from Klebsiella
Karsten Temme et al 2012
57
Continuehellip
Karsten Temme et al 2012
58
Genome editing
Sequence specific nucleases
1 Meganucleases
2 ZFMs ndashZink finger motifs
3 TALENs- Transcription activator like effector nuclease
4 CRISPR CAS-9 ndash Clustered interspaced short palindromic
repeats
qmp4
59Khaoula Belhaj et al 2015
60
Genome modifications achieved in plants using sequence specific nucleases
Type of DNA
modification
Nuclease Delivery
methods
Plants Targets
Trait stacking Megnucleases Bombardment Cotton Intergenic sequence
ZFN Bombardment Maize Transgene
Rewriting host
DNA
Megnucleases Stable integration Maize Intergenic sequence
ZFN Stable integration Soybean Transgene
TALEN Stable integration Barley PAPhy_a
TALEN Agrobacterium
T-DNA (transient)
Oryza sativa SWEET14
TALEN No Protoplasts Arabidopsis
Tobacco
AtTT4 AtADH NbSurB
TALEN Bombardment Wheat MLO
TALEN Protoplasts
Stable integration
Maize PDS IPK1A IPK MRP4
CRISPRCas Protoplasts
Agrobacterium
T-DNA (transient)
Tobacco
Arabidopsis
Sorghum Oryza
OsSWEET14 transgene
61
Type of DNA
modification
Nuclease Delivery
methods
Plants Targets
Rewriting host
DNA large
deletion
Zinc-finger
nuclease
Stable
integration
Tobacco Transgene
CRISPRCas Protoplasts
Stable
integration
Rice Labdane-related
diterpenoid gene
clusters
on Chr 2 4 and 6
Zinc-finger
nuclease
Agrobacterium
T-DNA
(transient)
Tobacco CHN50 transgene
Zinc-finger
nuclease
Whiskers Maize IPK1
CRISPRCas Protoplasts Rice PDS
Controlling gene
expression
TALE repressor
(SRDX)
Stable
integration
Arabidopsis RD29A transgene
Zinc-finger
activator (VP16)
Stable
integration
Brassica
napus
KasII
Continue
62
Application of genome editing1 Introduction of precise and predictable modifications directly in an elite
background
2 multiple traits can be modified simultaneously
3 NHEJ enables gene knockout and targeted modifications
4 Introduction of transgenes at defined loci that promote high-level
transcription and do not interfere with the activity of endogenous genes
5 Site-specific nucleases also allow targeted molecular trait stacking -low
risk of segregation
6 CRISPR Cas is a transgene free approach ndash No regulatory burdens
7 frequency of off-target mutations is well below that caused by chemical
and physical mutagenesis techniques
8 In future can be utilized for metabolic engineering and molecular farming
63
Six-way Venn diagram showing the distribution of shared genefamilies (sequence clusters) among M acuminata P dactyliferaArabidopsis thaliana Oryza sativa Sorghum bicolor and Brachypodiumdistachyon genomes
A DrsquoHont et al Nature 2012 doi101038nature11241
64
Achieving super-DomesticationINTROGRESS-RECREATE-CREATE-DOMESTICATEREDOMESTICATE
bull Mobilizing left out genetic
variation still available in
land races and wild and
weedy species
bull Replaying the evolutionary
tape( resynthesis in
polyploids)
bull Domesticateredomesticate
65
We can admire and emulate how indigenous people still domesticateplants create biodiversity and manage it to sustain their future Thereis no equivalently dynamic or flexible crop breeding in modernagriculture to promote biodiversity we still have much to learn from
traditional knowledge Jan Salick 2012
Domesticated Wheat
10
Maize
11
Domesticated maize
12
Barley Domestication
Prognt H spontanium
13
14
Domesticated Pearl MilletPennisetum glaucum
better seed recovery and yield but less able to survive in natural conditions compared to wild progenitor
bull more apical dominance (less
branching)
bull compact growth habit
bull flowering at same time (rather
than spread over long period)
bull larger spikes
bull non-shattering spikelets
bull loss of bristles amp glumes around
grains
bull larger seeds
bull non-dormant seeds
bull germinate at same time
15
Ppolystachian
Oats domestication
Wild- A sterilis Modern
sativa
16
Domestication of Sorghum
S helepense
S bicolor17
JR Harlan 1976 Sci American
Divergent
selection for
different
purposes
httpenwikipediaorgwikiImageBrassica_oleracea0jpg
httpwwwcehacukimagesclip_image002_001jpg
Brassica oleracea cabbage
broccoli cauliflower kale
romanesco collards kohrabi
brussels sprouts18
Solanum pimpinellifolium
Solanum lycopersicum19
Domestication of carrot
Queen Annes Lace
Daucus carota
20
Domestication of peanut
A villosulicarpa Hoehne andA Stenosperma (Brazil)
AABB
21
Domestication of Chilli
22
StrawberryBanana
Musa acuminata Musa balbisiana (2n=3x=33) AB genomeFRAGARIA X ANANASSA
Wood berries (F vesca) and Musky strawberries (F moschata)23
Wild SoybeanGlycin soja2n=40
Cultivated soybeanGlycin max 2n=40
24
Increased in domesticate
seed germination
determinate growth
longer pods
bigger (heavier) seeds
earliness
harvest index (seed yieldbiomass)
Decreased in domesticate
seed dispersal
seed dormancy
twining
number of nodes
length of internode
number of pods
photoperiod sensitivity
Common Bean Phaseolus vulgarishttpwwwplantsciencesucdavisedugeptspb143pb143htm
copy Paul Gepts
25
Wild pigeon peaCajanus cajanifolius
Cultivated pigeon pea
26
27
28
WHY OTHERS ARE NOT DOMESTICATED
bull 350000 plants
bull 4629 mammals
bull 9200 birds
bull 10000000 insects
bull 500000 fungi
bull But only 200 plants
bull 15 mammals 5 birds and
bull 2 insects are domesticated
bull Spread of these few species
bull Little change since early
agriculture
bull Repeated domestication of these
species (sometimes)
bull Lack of new species even with
attempts with species known to be
valuable Some groups are good
candidates with no domestication
eg ferns sub-Saharan mammals
29
WHY OTHERS HAVE TO BE DOMESTICATED
New uses and demands ndash biofuels animal feed
medicinalneutraceutical waterclimate food changes
Knowledge why species arenrsquot suitable for domestication or
were not useful
Better understanding of genetics and selection
Sustainability of production
Reliability of production
To meet the food demand in an alternative way
WHY OTHERS HAVE TO BE DOMESTICATED
30
Reallocate biomass for human use
More efficient metabolism and photosynthesis increase
leaf surface decrease root system increase leaf
longevity
Grains more fertile florets larger inflorescence OR
number of ears different ways to get more seed
Larger seeds (automatic vs deliberate selection)
Oil plants increased oil content or more seed
Fiber plants long strong fibers
Domestication syndrome
31
Centers of Domestication
Fuller 2011
32
Centers of Domestication in India
Fuller 2011
33
Indian centers of domestication South India (Deccan centre) Vigna radiate Vigna mungo
Macrotyloma uniflorum (Horse gram) Sataria verticillata S plumila
wheat (macaroni) and two rowed barleybrachiaria ramosa
Orissa (Mahanadi river) Pegion pea Horsegram mung small millets
like Echinichloa Paspalum and Sataria
The Middle Ganges (Harappan civilization) Oryza nivara O rufipogon
and wild sativa Sateria pumia Cannabis sativa even the diffusion of
japonica rice
Saurashtra (Harappan civilization) Eleucine coracana Sateria italica
Paicum spp Brachypodia pearl millet and sorghum Dollicos lablab
The Himalayan foothills of the Punjab region Was a centre of
diversity for Japonica type rice and many temperate fruits and
vegetables
34
DOMESTICATION RELATED TRAITS CONTROLLED BY ONLY FEW GENES
Trait Crops
Plant architecturegrowth habit Ricemaizemilletsbeantomato
Flowering timephotoperiod sensitivity Ricemaizesorghumbeantomato
Fruit size Tomato egg plant
Grain size Ricemaizesorghumbean
Seed dispersal Brassica rice
Inflorescence modification Brassica
Dormancy Bean
35
Genes Crop Moleular and phenotypic function Causative change
Genes identified as controlling domestication traits
tb1 Maize Transcriptional regulator (TCP) plant and
inflorescence structure
regulatory change
tga1 Maize Transcriptional regulator (SBP) seed casing amino acid change
qSH1 Rice Transcriptional regulator (homeodomain)
abscission layer formation shattering
regulatory change
Rc Rice Transcriptional regulator (bHLH) seed color disrupted coding
sequence
sh4 Rice Transcriptional regulator (Myb3) abscission
layer formation shattering
regulatoryamino
acid change
fw22 Tomato Cell signaling fruit weight regulatory change
Q Wheat Transcriptional regulator (AP2) inflorescence
structure
regulatoryamino
acid change
Vrs1 Barley Inflorescence structure Premature stop Amino acid change
Genes of interest in crop domestication and improvement
36
Genes Crop Moleular and phenotypic function Causative change
c1
r1
Maize Transcriptional regulator (MYB) kernel color and
Transcriptional regulator (bHLH) kernel color
regulatory change
sh2 Maize pyrophosphorylase supersweet sweet corn transposon
insertion
su1 Maize isoamylase sweet corn gene amino acid change
brix9-2-5 Tomato Invertase fruit soluble solid content amino acid change
ovate Tomato Unknown fruit shap early stop codon
R Pea Starch branching enzyme seed sugar content transposon
insertion
ehd1 Rice B-type response regulator flowering time amino acid change
hd1 Rice Transcriptional regulator (zinc finger) flowering
time
disrupted coding
sequence
waxy Rice Starch synthase sticky grains intron splicing
defect
rht Wheat Transcriptional regulator (SH2) plant height early stop codon
vrn1 Wheat Transcriptional regulator (MADS) vernalization regulatory change
vrn2 Wheat Transcriptional regulator (ZCCT) vernalization amino acid change
Genes Identified as Controlling Varietal Differences
37
Process of domestication over the yearshellip
Rachel S Meyer 2012
38
Genetic bottleneck
Cultivated crops undergone with narrowing of diversity problem
But this is not the case in weeds
Weeds are the major threats from the beginning of domestication till
today
Problem arose due to
1 Crop mimicry
2 Genetic assimilation
3 Genetic evolution
39
Super- Domestication Vaughn et al (2007) first used the term super-domestication
The processes that lead to a domesticate with dramatically
increased yield that could not be selected in natural environments
from naturally occurring variation without recourse to new
technologies
Super-domesticates can be constructed with knowledge led
approaches based on current needs using the range of new
technologies now available
Plants exploited for continuous selection introduction hybridization
etc which boosted the process of domestication but now the plant
genetic engineering approach is exploiting plants towards synthetic
biology
40
How to use diversitybull Cross two varieties
bull Genome manipulations
Cell fusion hybrids
bull Chromosome manipulation Backcross a new species
bull Generate recombinants Chromosome recombinations
bull Use a new species wild germplasm
Transgenic approach Modern mutagenesis synthetic gene
construction by utilizing green florescent protein genome editing
NGS GWAS sequencing etchelliphelliphelliphelliphellip
41
Reduce genetic bottlenecks through polyplodization
More vigour
SC
Buffering capacity
Heterotic advantage
Enhaced vegetative
charachers
Enhanced oil
Meiotic stability
42
Approach 1 Use one tetraploid and one diploid as parents (4X ndash 2X) followed by the
chromosome doubling of triploid hybrids (Chrom doubling)
a) Cross between B juncea (AjAjBjBj ) and B oleracea (CoCo) to produce hexaploids
(AjAjBjBjCoCo)
(b) Cross between B napus (AnAnCnCn) and B nigra (BniBni) to produce hexaploids
(AnAnBniBniCnCn)
(c) Cross between B carinata (BcaBcaCcaCca) and B rapa (ArAr) to produce hexaploids
(ArArBcaBcaCcaCca)
Approach 2 Use three tetraploids as parents
(a) Cross between B napus (AnAnCnCn) and B carinata (BcaBcaCcaCca) to produce unbalanced
allotetraploids (AnBcaCnCca ndash unreduced gametes gametes with the somatic chromosome
number and cross with B juncea (AjAjBjBj) to obtain allohexaploids (AnAjBcaBjCnCca
(b) Cross between B napus (AnAnCnCn) and B juncea (AjAjBjBj) to produce unbalanced
allotetraploids (AnAjBjCn ndash unreduced gametes) and cross with B carinata (BcaBcaCcaCca) to
obtain allohexaploids (AnAjBcaBjCnCca)
(c) Cross between B carinata (BcaBcaCcaCca) and B juncea (AjAjBjBj) to produce unbalanced
allotetraploids (AjBcaBjCca - ndash unreduced gametes) and cross with B napus (AnAnCnCn) to obtain
allohexaploids (AnAjBcaBjCnCca)
Approach 3 Use three diploids as parents (2X ndash 2X ndash 2X) Cross between B rapa (ArAr) B
nigra (BniBni) and B oleracea (CoCo) sequentially to obtain hexaploid hybrids (ArArBniBniCoCo)
43
Continuehellip
Guijun Yan 2012
44
Applications of Genomic
tools in Super-Domestication
45
Genome Sequencing
Potential methods of sequencing
1 Clone by clone approach
2 Whole genome shotgun approach
3 Combination of the two methods
Till today no of cultivated plants completely sequenced -85
2016- Arachis duranensis
2015- Solanum cumersonii (Wild potato)
(Ref- NCBI)
46
GWAS
It is a study design in which many markers spread across a genome
are genotyped and test a statistical association with a phenotype are
performed locally along the genome
It is also an examination of many common genetic variants in different
individuals to see if any variant is associated with a trait
Used in successfully studying maize sorghum and barley
Method is efficient for large scale low cost genotyping (even with the
minimum number of SNPs)
Cannot be utilized generally because it needs large population size
GWAS identify rare alleles more precisely
If small population we can opt NAM
47
GWAS and whole genome prediction
Xuehui Huang and Bin Han 2014
48
Five high throuput genotyping methods
Xuehui Huang and Bin Han 2014
49
Role of NGS in domestication Capture of novel genes from wild species will be made easier by
understanding the molecular events associated with crop domestication
Re-sequencing of domesticated species can identify low diversity regions
resulting from selection during domestication
To identify gene-specific sequences to aid the cloning of homologues of key
domestication genes from wild relatives
Candidate genes from wild and domesticated plant populations can define
diversity of target genes in wild populations and lead to the discovery of key
genes for important traits by association analysis
NGS of amplicons of large numbers of candidate genes from wild and
domesticated plant populations can define diversity of target genes in
wild populations and lead to the discovery of key genes for important
traits by association analysis
50
NGS
supports the rapid domestication of new plant species and the efficient
identification and capture of novel genetic variation from related species
Allows whole-genome analysis to determine the genetic basis of
phenotypic differences
NGS allows rapid expansion of genomic analysis to investigation of non-
model species
Made rice study easy by related grass
cost-effective method for plant identification
useful strategy to analysis the chloroplast genome sequence from whole-
genome shot-gun sequencing
facilitates managing this diversity and any changes in crop performance
over time due to genetic drift
Patterns of gene expression have been evaluated in hybrids using NGS
51
Two Approaches to Finding Adaptive Genes
Ross-Ibarra et al2007
52
Techniques to re-wild the plants (transgene free)
1 Introgression
breeding
2 Specific
insertion of lost genes
3 Precision
mutagenesis
Palmgren et al2014
53
Process of rewilding
Palmgren et al2014
54
Synthetic biology projects(i) Modifying cereals including wheat to fix atmospheric nitrogen
(ii) Redesigning metabolic pathways to increase the yield of
secondary metabolites or to generate compounds with enhanced
properties
(iii) Transferring the C4 photosynthesis pathway to rice
(iv) Modifying the glycosylation pathway in plants to accommodate
production of therapeutic proteins
(v) Introducing synthetic signal transduction systems that respond to
external cues
55
Synthetic biology projects
Nicholas J Baltes et al 2015
56
Refactoring the N fixation gene cluster from Klebsiella
Karsten Temme et al 2012
57
Continuehellip
Karsten Temme et al 2012
58
Genome editing
Sequence specific nucleases
1 Meganucleases
2 ZFMs ndashZink finger motifs
3 TALENs- Transcription activator like effector nuclease
4 CRISPR CAS-9 ndash Clustered interspaced short palindromic
repeats
qmp4
59Khaoula Belhaj et al 2015
60
Genome modifications achieved in plants using sequence specific nucleases
Type of DNA
modification
Nuclease Delivery
methods
Plants Targets
Trait stacking Megnucleases Bombardment Cotton Intergenic sequence
ZFN Bombardment Maize Transgene
Rewriting host
DNA
Megnucleases Stable integration Maize Intergenic sequence
ZFN Stable integration Soybean Transgene
TALEN Stable integration Barley PAPhy_a
TALEN Agrobacterium
T-DNA (transient)
Oryza sativa SWEET14
TALEN No Protoplasts Arabidopsis
Tobacco
AtTT4 AtADH NbSurB
TALEN Bombardment Wheat MLO
TALEN Protoplasts
Stable integration
Maize PDS IPK1A IPK MRP4
CRISPRCas Protoplasts
Agrobacterium
T-DNA (transient)
Tobacco
Arabidopsis
Sorghum Oryza
OsSWEET14 transgene
61
Type of DNA
modification
Nuclease Delivery
methods
Plants Targets
Rewriting host
DNA large
deletion
Zinc-finger
nuclease
Stable
integration
Tobacco Transgene
CRISPRCas Protoplasts
Stable
integration
Rice Labdane-related
diterpenoid gene
clusters
on Chr 2 4 and 6
Zinc-finger
nuclease
Agrobacterium
T-DNA
(transient)
Tobacco CHN50 transgene
Zinc-finger
nuclease
Whiskers Maize IPK1
CRISPRCas Protoplasts Rice PDS
Controlling gene
expression
TALE repressor
(SRDX)
Stable
integration
Arabidopsis RD29A transgene
Zinc-finger
activator (VP16)
Stable
integration
Brassica
napus
KasII
Continue
62
Application of genome editing1 Introduction of precise and predictable modifications directly in an elite
background
2 multiple traits can be modified simultaneously
3 NHEJ enables gene knockout and targeted modifications
4 Introduction of transgenes at defined loci that promote high-level
transcription and do not interfere with the activity of endogenous genes
5 Site-specific nucleases also allow targeted molecular trait stacking -low
risk of segregation
6 CRISPR Cas is a transgene free approach ndash No regulatory burdens
7 frequency of off-target mutations is well below that caused by chemical
and physical mutagenesis techniques
8 In future can be utilized for metabolic engineering and molecular farming
63
Six-way Venn diagram showing the distribution of shared genefamilies (sequence clusters) among M acuminata P dactyliferaArabidopsis thaliana Oryza sativa Sorghum bicolor and Brachypodiumdistachyon genomes
A DrsquoHont et al Nature 2012 doi101038nature11241
64
Achieving super-DomesticationINTROGRESS-RECREATE-CREATE-DOMESTICATEREDOMESTICATE
bull Mobilizing left out genetic
variation still available in
land races and wild and
weedy species
bull Replaying the evolutionary
tape( resynthesis in
polyploids)
bull Domesticateredomesticate
65
We can admire and emulate how indigenous people still domesticateplants create biodiversity and manage it to sustain their future Thereis no equivalently dynamic or flexible crop breeding in modernagriculture to promote biodiversity we still have much to learn from
traditional knowledge Jan Salick 2012
Maize
11
Domesticated maize
12
Barley Domestication
Prognt H spontanium
13
14
Domesticated Pearl MilletPennisetum glaucum
better seed recovery and yield but less able to survive in natural conditions compared to wild progenitor
bull more apical dominance (less
branching)
bull compact growth habit
bull flowering at same time (rather
than spread over long period)
bull larger spikes
bull non-shattering spikelets
bull loss of bristles amp glumes around
grains
bull larger seeds
bull non-dormant seeds
bull germinate at same time
15
Ppolystachian
Oats domestication
Wild- A sterilis Modern
sativa
16
Domestication of Sorghum
S helepense
S bicolor17
JR Harlan 1976 Sci American
Divergent
selection for
different
purposes
httpenwikipediaorgwikiImageBrassica_oleracea0jpg
httpwwwcehacukimagesclip_image002_001jpg
Brassica oleracea cabbage
broccoli cauliflower kale
romanesco collards kohrabi
brussels sprouts18
Solanum pimpinellifolium
Solanum lycopersicum19
Domestication of carrot
Queen Annes Lace
Daucus carota
20
Domestication of peanut
A villosulicarpa Hoehne andA Stenosperma (Brazil)
AABB
21
Domestication of Chilli
22
StrawberryBanana
Musa acuminata Musa balbisiana (2n=3x=33) AB genomeFRAGARIA X ANANASSA
Wood berries (F vesca) and Musky strawberries (F moschata)23
Wild SoybeanGlycin soja2n=40
Cultivated soybeanGlycin max 2n=40
24
Increased in domesticate
seed germination
determinate growth
longer pods
bigger (heavier) seeds
earliness
harvest index (seed yieldbiomass)
Decreased in domesticate
seed dispersal
seed dormancy
twining
number of nodes
length of internode
number of pods
photoperiod sensitivity
Common Bean Phaseolus vulgarishttpwwwplantsciencesucdavisedugeptspb143pb143htm
copy Paul Gepts
25
Wild pigeon peaCajanus cajanifolius
Cultivated pigeon pea
26
27
28
WHY OTHERS ARE NOT DOMESTICATED
bull 350000 plants
bull 4629 mammals
bull 9200 birds
bull 10000000 insects
bull 500000 fungi
bull But only 200 plants
bull 15 mammals 5 birds and
bull 2 insects are domesticated
bull Spread of these few species
bull Little change since early
agriculture
bull Repeated domestication of these
species (sometimes)
bull Lack of new species even with
attempts with species known to be
valuable Some groups are good
candidates with no domestication
eg ferns sub-Saharan mammals
29
WHY OTHERS HAVE TO BE DOMESTICATED
New uses and demands ndash biofuels animal feed
medicinalneutraceutical waterclimate food changes
Knowledge why species arenrsquot suitable for domestication or
were not useful
Better understanding of genetics and selection
Sustainability of production
Reliability of production
To meet the food demand in an alternative way
WHY OTHERS HAVE TO BE DOMESTICATED
30
Reallocate biomass for human use
More efficient metabolism and photosynthesis increase
leaf surface decrease root system increase leaf
longevity
Grains more fertile florets larger inflorescence OR
number of ears different ways to get more seed
Larger seeds (automatic vs deliberate selection)
Oil plants increased oil content or more seed
Fiber plants long strong fibers
Domestication syndrome
31
Centers of Domestication
Fuller 2011
32
Centers of Domestication in India
Fuller 2011
33
Indian centers of domestication South India (Deccan centre) Vigna radiate Vigna mungo
Macrotyloma uniflorum (Horse gram) Sataria verticillata S plumila
wheat (macaroni) and two rowed barleybrachiaria ramosa
Orissa (Mahanadi river) Pegion pea Horsegram mung small millets
like Echinichloa Paspalum and Sataria
The Middle Ganges (Harappan civilization) Oryza nivara O rufipogon
and wild sativa Sateria pumia Cannabis sativa even the diffusion of
japonica rice
Saurashtra (Harappan civilization) Eleucine coracana Sateria italica
Paicum spp Brachypodia pearl millet and sorghum Dollicos lablab
The Himalayan foothills of the Punjab region Was a centre of
diversity for Japonica type rice and many temperate fruits and
vegetables
34
DOMESTICATION RELATED TRAITS CONTROLLED BY ONLY FEW GENES
Trait Crops
Plant architecturegrowth habit Ricemaizemilletsbeantomato
Flowering timephotoperiod sensitivity Ricemaizesorghumbeantomato
Fruit size Tomato egg plant
Grain size Ricemaizesorghumbean
Seed dispersal Brassica rice
Inflorescence modification Brassica
Dormancy Bean
35
Genes Crop Moleular and phenotypic function Causative change
Genes identified as controlling domestication traits
tb1 Maize Transcriptional regulator (TCP) plant and
inflorescence structure
regulatory change
tga1 Maize Transcriptional regulator (SBP) seed casing amino acid change
qSH1 Rice Transcriptional regulator (homeodomain)
abscission layer formation shattering
regulatory change
Rc Rice Transcriptional regulator (bHLH) seed color disrupted coding
sequence
sh4 Rice Transcriptional regulator (Myb3) abscission
layer formation shattering
regulatoryamino
acid change
fw22 Tomato Cell signaling fruit weight regulatory change
Q Wheat Transcriptional regulator (AP2) inflorescence
structure
regulatoryamino
acid change
Vrs1 Barley Inflorescence structure Premature stop Amino acid change
Genes of interest in crop domestication and improvement
36
Genes Crop Moleular and phenotypic function Causative change
c1
r1
Maize Transcriptional regulator (MYB) kernel color and
Transcriptional regulator (bHLH) kernel color
regulatory change
sh2 Maize pyrophosphorylase supersweet sweet corn transposon
insertion
su1 Maize isoamylase sweet corn gene amino acid change
brix9-2-5 Tomato Invertase fruit soluble solid content amino acid change
ovate Tomato Unknown fruit shap early stop codon
R Pea Starch branching enzyme seed sugar content transposon
insertion
ehd1 Rice B-type response regulator flowering time amino acid change
hd1 Rice Transcriptional regulator (zinc finger) flowering
time
disrupted coding
sequence
waxy Rice Starch synthase sticky grains intron splicing
defect
rht Wheat Transcriptional regulator (SH2) plant height early stop codon
vrn1 Wheat Transcriptional regulator (MADS) vernalization regulatory change
vrn2 Wheat Transcriptional regulator (ZCCT) vernalization amino acid change
Genes Identified as Controlling Varietal Differences
37
Process of domestication over the yearshellip
Rachel S Meyer 2012
38
Genetic bottleneck
Cultivated crops undergone with narrowing of diversity problem
But this is not the case in weeds
Weeds are the major threats from the beginning of domestication till
today
Problem arose due to
1 Crop mimicry
2 Genetic assimilation
3 Genetic evolution
39
Super- Domestication Vaughn et al (2007) first used the term super-domestication
The processes that lead to a domesticate with dramatically
increased yield that could not be selected in natural environments
from naturally occurring variation without recourse to new
technologies
Super-domesticates can be constructed with knowledge led
approaches based on current needs using the range of new
technologies now available
Plants exploited for continuous selection introduction hybridization
etc which boosted the process of domestication but now the plant
genetic engineering approach is exploiting plants towards synthetic
biology
40
How to use diversitybull Cross two varieties
bull Genome manipulations
Cell fusion hybrids
bull Chromosome manipulation Backcross a new species
bull Generate recombinants Chromosome recombinations
bull Use a new species wild germplasm
Transgenic approach Modern mutagenesis synthetic gene
construction by utilizing green florescent protein genome editing
NGS GWAS sequencing etchelliphelliphelliphelliphellip
41
Reduce genetic bottlenecks through polyplodization
More vigour
SC
Buffering capacity
Heterotic advantage
Enhaced vegetative
charachers
Enhanced oil
Meiotic stability
42
Approach 1 Use one tetraploid and one diploid as parents (4X ndash 2X) followed by the
chromosome doubling of triploid hybrids (Chrom doubling)
a) Cross between B juncea (AjAjBjBj ) and B oleracea (CoCo) to produce hexaploids
(AjAjBjBjCoCo)
(b) Cross between B napus (AnAnCnCn) and B nigra (BniBni) to produce hexaploids
(AnAnBniBniCnCn)
(c) Cross between B carinata (BcaBcaCcaCca) and B rapa (ArAr) to produce hexaploids
(ArArBcaBcaCcaCca)
Approach 2 Use three tetraploids as parents
(a) Cross between B napus (AnAnCnCn) and B carinata (BcaBcaCcaCca) to produce unbalanced
allotetraploids (AnBcaCnCca ndash unreduced gametes gametes with the somatic chromosome
number and cross with B juncea (AjAjBjBj) to obtain allohexaploids (AnAjBcaBjCnCca
(b) Cross between B napus (AnAnCnCn) and B juncea (AjAjBjBj) to produce unbalanced
allotetraploids (AnAjBjCn ndash unreduced gametes) and cross with B carinata (BcaBcaCcaCca) to
obtain allohexaploids (AnAjBcaBjCnCca)
(c) Cross between B carinata (BcaBcaCcaCca) and B juncea (AjAjBjBj) to produce unbalanced
allotetraploids (AjBcaBjCca - ndash unreduced gametes) and cross with B napus (AnAnCnCn) to obtain
allohexaploids (AnAjBcaBjCnCca)
Approach 3 Use three diploids as parents (2X ndash 2X ndash 2X) Cross between B rapa (ArAr) B
nigra (BniBni) and B oleracea (CoCo) sequentially to obtain hexaploid hybrids (ArArBniBniCoCo)
43
Continuehellip
Guijun Yan 2012
44
Applications of Genomic
tools in Super-Domestication
45
Genome Sequencing
Potential methods of sequencing
1 Clone by clone approach
2 Whole genome shotgun approach
3 Combination of the two methods
Till today no of cultivated plants completely sequenced -85
2016- Arachis duranensis
2015- Solanum cumersonii (Wild potato)
(Ref- NCBI)
46
GWAS
It is a study design in which many markers spread across a genome
are genotyped and test a statistical association with a phenotype are
performed locally along the genome
It is also an examination of many common genetic variants in different
individuals to see if any variant is associated with a trait
Used in successfully studying maize sorghum and barley
Method is efficient for large scale low cost genotyping (even with the
minimum number of SNPs)
Cannot be utilized generally because it needs large population size
GWAS identify rare alleles more precisely
If small population we can opt NAM
47
GWAS and whole genome prediction
Xuehui Huang and Bin Han 2014
48
Five high throuput genotyping methods
Xuehui Huang and Bin Han 2014
49
Role of NGS in domestication Capture of novel genes from wild species will be made easier by
understanding the molecular events associated with crop domestication
Re-sequencing of domesticated species can identify low diversity regions
resulting from selection during domestication
To identify gene-specific sequences to aid the cloning of homologues of key
domestication genes from wild relatives
Candidate genes from wild and domesticated plant populations can define
diversity of target genes in wild populations and lead to the discovery of key
genes for important traits by association analysis
NGS of amplicons of large numbers of candidate genes from wild and
domesticated plant populations can define diversity of target genes in
wild populations and lead to the discovery of key genes for important
traits by association analysis
50
NGS
supports the rapid domestication of new plant species and the efficient
identification and capture of novel genetic variation from related species
Allows whole-genome analysis to determine the genetic basis of
phenotypic differences
NGS allows rapid expansion of genomic analysis to investigation of non-
model species
Made rice study easy by related grass
cost-effective method for plant identification
useful strategy to analysis the chloroplast genome sequence from whole-
genome shot-gun sequencing
facilitates managing this diversity and any changes in crop performance
over time due to genetic drift
Patterns of gene expression have been evaluated in hybrids using NGS
51
Two Approaches to Finding Adaptive Genes
Ross-Ibarra et al2007
52
Techniques to re-wild the plants (transgene free)
1 Introgression
breeding
2 Specific
insertion of lost genes
3 Precision
mutagenesis
Palmgren et al2014
53
Process of rewilding
Palmgren et al2014
54
Synthetic biology projects(i) Modifying cereals including wheat to fix atmospheric nitrogen
(ii) Redesigning metabolic pathways to increase the yield of
secondary metabolites or to generate compounds with enhanced
properties
(iii) Transferring the C4 photosynthesis pathway to rice
(iv) Modifying the glycosylation pathway in plants to accommodate
production of therapeutic proteins
(v) Introducing synthetic signal transduction systems that respond to
external cues
55
Synthetic biology projects
Nicholas J Baltes et al 2015
56
Refactoring the N fixation gene cluster from Klebsiella
Karsten Temme et al 2012
57
Continuehellip
Karsten Temme et al 2012
58
Genome editing
Sequence specific nucleases
1 Meganucleases
2 ZFMs ndashZink finger motifs
3 TALENs- Transcription activator like effector nuclease
4 CRISPR CAS-9 ndash Clustered interspaced short palindromic
repeats
qmp4
59Khaoula Belhaj et al 2015
60
Genome modifications achieved in plants using sequence specific nucleases
Type of DNA
modification
Nuclease Delivery
methods
Plants Targets
Trait stacking Megnucleases Bombardment Cotton Intergenic sequence
ZFN Bombardment Maize Transgene
Rewriting host
DNA
Megnucleases Stable integration Maize Intergenic sequence
ZFN Stable integration Soybean Transgene
TALEN Stable integration Barley PAPhy_a
TALEN Agrobacterium
T-DNA (transient)
Oryza sativa SWEET14
TALEN No Protoplasts Arabidopsis
Tobacco
AtTT4 AtADH NbSurB
TALEN Bombardment Wheat MLO
TALEN Protoplasts
Stable integration
Maize PDS IPK1A IPK MRP4
CRISPRCas Protoplasts
Agrobacterium
T-DNA (transient)
Tobacco
Arabidopsis
Sorghum Oryza
OsSWEET14 transgene
61
Type of DNA
modification
Nuclease Delivery
methods
Plants Targets
Rewriting host
DNA large
deletion
Zinc-finger
nuclease
Stable
integration
Tobacco Transgene
CRISPRCas Protoplasts
Stable
integration
Rice Labdane-related
diterpenoid gene
clusters
on Chr 2 4 and 6
Zinc-finger
nuclease
Agrobacterium
T-DNA
(transient)
Tobacco CHN50 transgene
Zinc-finger
nuclease
Whiskers Maize IPK1
CRISPRCas Protoplasts Rice PDS
Controlling gene
expression
TALE repressor
(SRDX)
Stable
integration
Arabidopsis RD29A transgene
Zinc-finger
activator (VP16)
Stable
integration
Brassica
napus
KasII
Continue
62
Application of genome editing1 Introduction of precise and predictable modifications directly in an elite
background
2 multiple traits can be modified simultaneously
3 NHEJ enables gene knockout and targeted modifications
4 Introduction of transgenes at defined loci that promote high-level
transcription and do not interfere with the activity of endogenous genes
5 Site-specific nucleases also allow targeted molecular trait stacking -low
risk of segregation
6 CRISPR Cas is a transgene free approach ndash No regulatory burdens
7 frequency of off-target mutations is well below that caused by chemical
and physical mutagenesis techniques
8 In future can be utilized for metabolic engineering and molecular farming
63
Six-way Venn diagram showing the distribution of shared genefamilies (sequence clusters) among M acuminata P dactyliferaArabidopsis thaliana Oryza sativa Sorghum bicolor and Brachypodiumdistachyon genomes
A DrsquoHont et al Nature 2012 doi101038nature11241
64
Achieving super-DomesticationINTROGRESS-RECREATE-CREATE-DOMESTICATEREDOMESTICATE
bull Mobilizing left out genetic
variation still available in
land races and wild and
weedy species
bull Replaying the evolutionary
tape( resynthesis in
polyploids)
bull Domesticateredomesticate
65
We can admire and emulate how indigenous people still domesticateplants create biodiversity and manage it to sustain their future Thereis no equivalently dynamic or flexible crop breeding in modernagriculture to promote biodiversity we still have much to learn from
traditional knowledge Jan Salick 2012
Domesticated maize
12
Barley Domestication
Prognt H spontanium
13
14
Domesticated Pearl MilletPennisetum glaucum
better seed recovery and yield but less able to survive in natural conditions compared to wild progenitor
bull more apical dominance (less
branching)
bull compact growth habit
bull flowering at same time (rather
than spread over long period)
bull larger spikes
bull non-shattering spikelets
bull loss of bristles amp glumes around
grains
bull larger seeds
bull non-dormant seeds
bull germinate at same time
15
Ppolystachian
Oats domestication
Wild- A sterilis Modern
sativa
16
Domestication of Sorghum
S helepense
S bicolor17
JR Harlan 1976 Sci American
Divergent
selection for
different
purposes
httpenwikipediaorgwikiImageBrassica_oleracea0jpg
httpwwwcehacukimagesclip_image002_001jpg
Brassica oleracea cabbage
broccoli cauliflower kale
romanesco collards kohrabi
brussels sprouts18
Solanum pimpinellifolium
Solanum lycopersicum19
Domestication of carrot
Queen Annes Lace
Daucus carota
20
Domestication of peanut
A villosulicarpa Hoehne andA Stenosperma (Brazil)
AABB
21
Domestication of Chilli
22
StrawberryBanana
Musa acuminata Musa balbisiana (2n=3x=33) AB genomeFRAGARIA X ANANASSA
Wood berries (F vesca) and Musky strawberries (F moschata)23
Wild SoybeanGlycin soja2n=40
Cultivated soybeanGlycin max 2n=40
24
Increased in domesticate
seed germination
determinate growth
longer pods
bigger (heavier) seeds
earliness
harvest index (seed yieldbiomass)
Decreased in domesticate
seed dispersal
seed dormancy
twining
number of nodes
length of internode
number of pods
photoperiod sensitivity
Common Bean Phaseolus vulgarishttpwwwplantsciencesucdavisedugeptspb143pb143htm
copy Paul Gepts
25
Wild pigeon peaCajanus cajanifolius
Cultivated pigeon pea
26
27
28
WHY OTHERS ARE NOT DOMESTICATED
bull 350000 plants
bull 4629 mammals
bull 9200 birds
bull 10000000 insects
bull 500000 fungi
bull But only 200 plants
bull 15 mammals 5 birds and
bull 2 insects are domesticated
bull Spread of these few species
bull Little change since early
agriculture
bull Repeated domestication of these
species (sometimes)
bull Lack of new species even with
attempts with species known to be
valuable Some groups are good
candidates with no domestication
eg ferns sub-Saharan mammals
29
WHY OTHERS HAVE TO BE DOMESTICATED
New uses and demands ndash biofuels animal feed
medicinalneutraceutical waterclimate food changes
Knowledge why species arenrsquot suitable for domestication or
were not useful
Better understanding of genetics and selection
Sustainability of production
Reliability of production
To meet the food demand in an alternative way
WHY OTHERS HAVE TO BE DOMESTICATED
30
Reallocate biomass for human use
More efficient metabolism and photosynthesis increase
leaf surface decrease root system increase leaf
longevity
Grains more fertile florets larger inflorescence OR
number of ears different ways to get more seed
Larger seeds (automatic vs deliberate selection)
Oil plants increased oil content or more seed
Fiber plants long strong fibers
Domestication syndrome
31
Centers of Domestication
Fuller 2011
32
Centers of Domestication in India
Fuller 2011
33
Indian centers of domestication South India (Deccan centre) Vigna radiate Vigna mungo
Macrotyloma uniflorum (Horse gram) Sataria verticillata S plumila
wheat (macaroni) and two rowed barleybrachiaria ramosa
Orissa (Mahanadi river) Pegion pea Horsegram mung small millets
like Echinichloa Paspalum and Sataria
The Middle Ganges (Harappan civilization) Oryza nivara O rufipogon
and wild sativa Sateria pumia Cannabis sativa even the diffusion of
japonica rice
Saurashtra (Harappan civilization) Eleucine coracana Sateria italica
Paicum spp Brachypodia pearl millet and sorghum Dollicos lablab
The Himalayan foothills of the Punjab region Was a centre of
diversity for Japonica type rice and many temperate fruits and
vegetables
34
DOMESTICATION RELATED TRAITS CONTROLLED BY ONLY FEW GENES
Trait Crops
Plant architecturegrowth habit Ricemaizemilletsbeantomato
Flowering timephotoperiod sensitivity Ricemaizesorghumbeantomato
Fruit size Tomato egg plant
Grain size Ricemaizesorghumbean
Seed dispersal Brassica rice
Inflorescence modification Brassica
Dormancy Bean
35
Genes Crop Moleular and phenotypic function Causative change
Genes identified as controlling domestication traits
tb1 Maize Transcriptional regulator (TCP) plant and
inflorescence structure
regulatory change
tga1 Maize Transcriptional regulator (SBP) seed casing amino acid change
qSH1 Rice Transcriptional regulator (homeodomain)
abscission layer formation shattering
regulatory change
Rc Rice Transcriptional regulator (bHLH) seed color disrupted coding
sequence
sh4 Rice Transcriptional regulator (Myb3) abscission
layer formation shattering
regulatoryamino
acid change
fw22 Tomato Cell signaling fruit weight regulatory change
Q Wheat Transcriptional regulator (AP2) inflorescence
structure
regulatoryamino
acid change
Vrs1 Barley Inflorescence structure Premature stop Amino acid change
Genes of interest in crop domestication and improvement
36
Genes Crop Moleular and phenotypic function Causative change
c1
r1
Maize Transcriptional regulator (MYB) kernel color and
Transcriptional regulator (bHLH) kernel color
regulatory change
sh2 Maize pyrophosphorylase supersweet sweet corn transposon
insertion
su1 Maize isoamylase sweet corn gene amino acid change
brix9-2-5 Tomato Invertase fruit soluble solid content amino acid change
ovate Tomato Unknown fruit shap early stop codon
R Pea Starch branching enzyme seed sugar content transposon
insertion
ehd1 Rice B-type response regulator flowering time amino acid change
hd1 Rice Transcriptional regulator (zinc finger) flowering
time
disrupted coding
sequence
waxy Rice Starch synthase sticky grains intron splicing
defect
rht Wheat Transcriptional regulator (SH2) plant height early stop codon
vrn1 Wheat Transcriptional regulator (MADS) vernalization regulatory change
vrn2 Wheat Transcriptional regulator (ZCCT) vernalization amino acid change
Genes Identified as Controlling Varietal Differences
37
Process of domestication over the yearshellip
Rachel S Meyer 2012
38
Genetic bottleneck
Cultivated crops undergone with narrowing of diversity problem
But this is not the case in weeds
Weeds are the major threats from the beginning of domestication till
today
Problem arose due to
1 Crop mimicry
2 Genetic assimilation
3 Genetic evolution
39
Super- Domestication Vaughn et al (2007) first used the term super-domestication
The processes that lead to a domesticate with dramatically
increased yield that could not be selected in natural environments
from naturally occurring variation without recourse to new
technologies
Super-domesticates can be constructed with knowledge led
approaches based on current needs using the range of new
technologies now available
Plants exploited for continuous selection introduction hybridization
etc which boosted the process of domestication but now the plant
genetic engineering approach is exploiting plants towards synthetic
biology
40
How to use diversitybull Cross two varieties
bull Genome manipulations
Cell fusion hybrids
bull Chromosome manipulation Backcross a new species
bull Generate recombinants Chromosome recombinations
bull Use a new species wild germplasm
Transgenic approach Modern mutagenesis synthetic gene
construction by utilizing green florescent protein genome editing
NGS GWAS sequencing etchelliphelliphelliphelliphellip
41
Reduce genetic bottlenecks through polyplodization
More vigour
SC
Buffering capacity
Heterotic advantage
Enhaced vegetative
charachers
Enhanced oil
Meiotic stability
42
Approach 1 Use one tetraploid and one diploid as parents (4X ndash 2X) followed by the
chromosome doubling of triploid hybrids (Chrom doubling)
a) Cross between B juncea (AjAjBjBj ) and B oleracea (CoCo) to produce hexaploids
(AjAjBjBjCoCo)
(b) Cross between B napus (AnAnCnCn) and B nigra (BniBni) to produce hexaploids
(AnAnBniBniCnCn)
(c) Cross between B carinata (BcaBcaCcaCca) and B rapa (ArAr) to produce hexaploids
(ArArBcaBcaCcaCca)
Approach 2 Use three tetraploids as parents
(a) Cross between B napus (AnAnCnCn) and B carinata (BcaBcaCcaCca) to produce unbalanced
allotetraploids (AnBcaCnCca ndash unreduced gametes gametes with the somatic chromosome
number and cross with B juncea (AjAjBjBj) to obtain allohexaploids (AnAjBcaBjCnCca
(b) Cross between B napus (AnAnCnCn) and B juncea (AjAjBjBj) to produce unbalanced
allotetraploids (AnAjBjCn ndash unreduced gametes) and cross with B carinata (BcaBcaCcaCca) to
obtain allohexaploids (AnAjBcaBjCnCca)
(c) Cross between B carinata (BcaBcaCcaCca) and B juncea (AjAjBjBj) to produce unbalanced
allotetraploids (AjBcaBjCca - ndash unreduced gametes) and cross with B napus (AnAnCnCn) to obtain
allohexaploids (AnAjBcaBjCnCca)
Approach 3 Use three diploids as parents (2X ndash 2X ndash 2X) Cross between B rapa (ArAr) B
nigra (BniBni) and B oleracea (CoCo) sequentially to obtain hexaploid hybrids (ArArBniBniCoCo)
43
Continuehellip
Guijun Yan 2012
44
Applications of Genomic
tools in Super-Domestication
45
Genome Sequencing
Potential methods of sequencing
1 Clone by clone approach
2 Whole genome shotgun approach
3 Combination of the two methods
Till today no of cultivated plants completely sequenced -85
2016- Arachis duranensis
2015- Solanum cumersonii (Wild potato)
(Ref- NCBI)
46
GWAS
It is a study design in which many markers spread across a genome
are genotyped and test a statistical association with a phenotype are
performed locally along the genome
It is also an examination of many common genetic variants in different
individuals to see if any variant is associated with a trait
Used in successfully studying maize sorghum and barley
Method is efficient for large scale low cost genotyping (even with the
minimum number of SNPs)
Cannot be utilized generally because it needs large population size
GWAS identify rare alleles more precisely
If small population we can opt NAM
47
GWAS and whole genome prediction
Xuehui Huang and Bin Han 2014
48
Five high throuput genotyping methods
Xuehui Huang and Bin Han 2014
49
Role of NGS in domestication Capture of novel genes from wild species will be made easier by
understanding the molecular events associated with crop domestication
Re-sequencing of domesticated species can identify low diversity regions
resulting from selection during domestication
To identify gene-specific sequences to aid the cloning of homologues of key
domestication genes from wild relatives
Candidate genes from wild and domesticated plant populations can define
diversity of target genes in wild populations and lead to the discovery of key
genes for important traits by association analysis
NGS of amplicons of large numbers of candidate genes from wild and
domesticated plant populations can define diversity of target genes in
wild populations and lead to the discovery of key genes for important
traits by association analysis
50
NGS
supports the rapid domestication of new plant species and the efficient
identification and capture of novel genetic variation from related species
Allows whole-genome analysis to determine the genetic basis of
phenotypic differences
NGS allows rapid expansion of genomic analysis to investigation of non-
model species
Made rice study easy by related grass
cost-effective method for plant identification
useful strategy to analysis the chloroplast genome sequence from whole-
genome shot-gun sequencing
facilitates managing this diversity and any changes in crop performance
over time due to genetic drift
Patterns of gene expression have been evaluated in hybrids using NGS
51
Two Approaches to Finding Adaptive Genes
Ross-Ibarra et al2007
52
Techniques to re-wild the plants (transgene free)
1 Introgression
breeding
2 Specific
insertion of lost genes
3 Precision
mutagenesis
Palmgren et al2014
53
Process of rewilding
Palmgren et al2014
54
Synthetic biology projects(i) Modifying cereals including wheat to fix atmospheric nitrogen
(ii) Redesigning metabolic pathways to increase the yield of
secondary metabolites or to generate compounds with enhanced
properties
(iii) Transferring the C4 photosynthesis pathway to rice
(iv) Modifying the glycosylation pathway in plants to accommodate
production of therapeutic proteins
(v) Introducing synthetic signal transduction systems that respond to
external cues
55
Synthetic biology projects
Nicholas J Baltes et al 2015
56
Refactoring the N fixation gene cluster from Klebsiella
Karsten Temme et al 2012
57
Continuehellip
Karsten Temme et al 2012
58
Genome editing
Sequence specific nucleases
1 Meganucleases
2 ZFMs ndashZink finger motifs
3 TALENs- Transcription activator like effector nuclease
4 CRISPR CAS-9 ndash Clustered interspaced short palindromic
repeats
qmp4
59Khaoula Belhaj et al 2015
60
Genome modifications achieved in plants using sequence specific nucleases
Type of DNA
modification
Nuclease Delivery
methods
Plants Targets
Trait stacking Megnucleases Bombardment Cotton Intergenic sequence
ZFN Bombardment Maize Transgene
Rewriting host
DNA
Megnucleases Stable integration Maize Intergenic sequence
ZFN Stable integration Soybean Transgene
TALEN Stable integration Barley PAPhy_a
TALEN Agrobacterium
T-DNA (transient)
Oryza sativa SWEET14
TALEN No Protoplasts Arabidopsis
Tobacco
AtTT4 AtADH NbSurB
TALEN Bombardment Wheat MLO
TALEN Protoplasts
Stable integration
Maize PDS IPK1A IPK MRP4
CRISPRCas Protoplasts
Agrobacterium
T-DNA (transient)
Tobacco
Arabidopsis
Sorghum Oryza
OsSWEET14 transgene
61
Type of DNA
modification
Nuclease Delivery
methods
Plants Targets
Rewriting host
DNA large
deletion
Zinc-finger
nuclease
Stable
integration
Tobacco Transgene
CRISPRCas Protoplasts
Stable
integration
Rice Labdane-related
diterpenoid gene
clusters
on Chr 2 4 and 6
Zinc-finger
nuclease
Agrobacterium
T-DNA
(transient)
Tobacco CHN50 transgene
Zinc-finger
nuclease
Whiskers Maize IPK1
CRISPRCas Protoplasts Rice PDS
Controlling gene
expression
TALE repressor
(SRDX)
Stable
integration
Arabidopsis RD29A transgene
Zinc-finger
activator (VP16)
Stable
integration
Brassica
napus
KasII
Continue
62
Application of genome editing1 Introduction of precise and predictable modifications directly in an elite
background
2 multiple traits can be modified simultaneously
3 NHEJ enables gene knockout and targeted modifications
4 Introduction of transgenes at defined loci that promote high-level
transcription and do not interfere with the activity of endogenous genes
5 Site-specific nucleases also allow targeted molecular trait stacking -low
risk of segregation
6 CRISPR Cas is a transgene free approach ndash No regulatory burdens
7 frequency of off-target mutations is well below that caused by chemical
and physical mutagenesis techniques
8 In future can be utilized for metabolic engineering and molecular farming
63
Six-way Venn diagram showing the distribution of shared genefamilies (sequence clusters) among M acuminata P dactyliferaArabidopsis thaliana Oryza sativa Sorghum bicolor and Brachypodiumdistachyon genomes
A DrsquoHont et al Nature 2012 doi101038nature11241
64
Achieving super-DomesticationINTROGRESS-RECREATE-CREATE-DOMESTICATEREDOMESTICATE
bull Mobilizing left out genetic
variation still available in
land races and wild and
weedy species
bull Replaying the evolutionary
tape( resynthesis in
polyploids)
bull Domesticateredomesticate
65
We can admire and emulate how indigenous people still domesticateplants create biodiversity and manage it to sustain their future Thereis no equivalently dynamic or flexible crop breeding in modernagriculture to promote biodiversity we still have much to learn from
traditional knowledge Jan Salick 2012
Barley Domestication
Prognt H spontanium
13
14
Domesticated Pearl MilletPennisetum glaucum
better seed recovery and yield but less able to survive in natural conditions compared to wild progenitor
bull more apical dominance (less
branching)
bull compact growth habit
bull flowering at same time (rather
than spread over long period)
bull larger spikes
bull non-shattering spikelets
bull loss of bristles amp glumes around
grains
bull larger seeds
bull non-dormant seeds
bull germinate at same time
15
Ppolystachian
Oats domestication
Wild- A sterilis Modern
sativa
16
Domestication of Sorghum
S helepense
S bicolor17
JR Harlan 1976 Sci American
Divergent
selection for
different
purposes
httpenwikipediaorgwikiImageBrassica_oleracea0jpg
httpwwwcehacukimagesclip_image002_001jpg
Brassica oleracea cabbage
broccoli cauliflower kale
romanesco collards kohrabi
brussels sprouts18
Solanum pimpinellifolium
Solanum lycopersicum19
Domestication of carrot
Queen Annes Lace
Daucus carota
20
Domestication of peanut
A villosulicarpa Hoehne andA Stenosperma (Brazil)
AABB
21
Domestication of Chilli
22
StrawberryBanana
Musa acuminata Musa balbisiana (2n=3x=33) AB genomeFRAGARIA X ANANASSA
Wood berries (F vesca) and Musky strawberries (F moschata)23
Wild SoybeanGlycin soja2n=40
Cultivated soybeanGlycin max 2n=40
24
Increased in domesticate
seed germination
determinate growth
longer pods
bigger (heavier) seeds
earliness
harvest index (seed yieldbiomass)
Decreased in domesticate
seed dispersal
seed dormancy
twining
number of nodes
length of internode
number of pods
photoperiod sensitivity
Common Bean Phaseolus vulgarishttpwwwplantsciencesucdavisedugeptspb143pb143htm
copy Paul Gepts
25
Wild pigeon peaCajanus cajanifolius
Cultivated pigeon pea
26
27
28
WHY OTHERS ARE NOT DOMESTICATED
bull 350000 plants
bull 4629 mammals
bull 9200 birds
bull 10000000 insects
bull 500000 fungi
bull But only 200 plants
bull 15 mammals 5 birds and
bull 2 insects are domesticated
bull Spread of these few species
bull Little change since early
agriculture
bull Repeated domestication of these
species (sometimes)
bull Lack of new species even with
attempts with species known to be
valuable Some groups are good
candidates with no domestication
eg ferns sub-Saharan mammals
29
WHY OTHERS HAVE TO BE DOMESTICATED
New uses and demands ndash biofuels animal feed
medicinalneutraceutical waterclimate food changes
Knowledge why species arenrsquot suitable for domestication or
were not useful
Better understanding of genetics and selection
Sustainability of production
Reliability of production
To meet the food demand in an alternative way
WHY OTHERS HAVE TO BE DOMESTICATED
30
Reallocate biomass for human use
More efficient metabolism and photosynthesis increase
leaf surface decrease root system increase leaf
longevity
Grains more fertile florets larger inflorescence OR
number of ears different ways to get more seed
Larger seeds (automatic vs deliberate selection)
Oil plants increased oil content or more seed
Fiber plants long strong fibers
Domestication syndrome
31
Centers of Domestication
Fuller 2011
32
Centers of Domestication in India
Fuller 2011
33
Indian centers of domestication South India (Deccan centre) Vigna radiate Vigna mungo
Macrotyloma uniflorum (Horse gram) Sataria verticillata S plumila
wheat (macaroni) and two rowed barleybrachiaria ramosa
Orissa (Mahanadi river) Pegion pea Horsegram mung small millets
like Echinichloa Paspalum and Sataria
The Middle Ganges (Harappan civilization) Oryza nivara O rufipogon
and wild sativa Sateria pumia Cannabis sativa even the diffusion of
japonica rice
Saurashtra (Harappan civilization) Eleucine coracana Sateria italica
Paicum spp Brachypodia pearl millet and sorghum Dollicos lablab
The Himalayan foothills of the Punjab region Was a centre of
diversity for Japonica type rice and many temperate fruits and
vegetables
34
DOMESTICATION RELATED TRAITS CONTROLLED BY ONLY FEW GENES
Trait Crops
Plant architecturegrowth habit Ricemaizemilletsbeantomato
Flowering timephotoperiod sensitivity Ricemaizesorghumbeantomato
Fruit size Tomato egg plant
Grain size Ricemaizesorghumbean
Seed dispersal Brassica rice
Inflorescence modification Brassica
Dormancy Bean
35
Genes Crop Moleular and phenotypic function Causative change
Genes identified as controlling domestication traits
tb1 Maize Transcriptional regulator (TCP) plant and
inflorescence structure
regulatory change
tga1 Maize Transcriptional regulator (SBP) seed casing amino acid change
qSH1 Rice Transcriptional regulator (homeodomain)
abscission layer formation shattering
regulatory change
Rc Rice Transcriptional regulator (bHLH) seed color disrupted coding
sequence
sh4 Rice Transcriptional regulator (Myb3) abscission
layer formation shattering
regulatoryamino
acid change
fw22 Tomato Cell signaling fruit weight regulatory change
Q Wheat Transcriptional regulator (AP2) inflorescence
structure
regulatoryamino
acid change
Vrs1 Barley Inflorescence structure Premature stop Amino acid change
Genes of interest in crop domestication and improvement
36
Genes Crop Moleular and phenotypic function Causative change
c1
r1
Maize Transcriptional regulator (MYB) kernel color and
Transcriptional regulator (bHLH) kernel color
regulatory change
sh2 Maize pyrophosphorylase supersweet sweet corn transposon
insertion
su1 Maize isoamylase sweet corn gene amino acid change
brix9-2-5 Tomato Invertase fruit soluble solid content amino acid change
ovate Tomato Unknown fruit shap early stop codon
R Pea Starch branching enzyme seed sugar content transposon
insertion
ehd1 Rice B-type response regulator flowering time amino acid change
hd1 Rice Transcriptional regulator (zinc finger) flowering
time
disrupted coding
sequence
waxy Rice Starch synthase sticky grains intron splicing
defect
rht Wheat Transcriptional regulator (SH2) plant height early stop codon
vrn1 Wheat Transcriptional regulator (MADS) vernalization regulatory change
vrn2 Wheat Transcriptional regulator (ZCCT) vernalization amino acid change
Genes Identified as Controlling Varietal Differences
37
Process of domestication over the yearshellip
Rachel S Meyer 2012
38
Genetic bottleneck
Cultivated crops undergone with narrowing of diversity problem
But this is not the case in weeds
Weeds are the major threats from the beginning of domestication till
today
Problem arose due to
1 Crop mimicry
2 Genetic assimilation
3 Genetic evolution
39
Super- Domestication Vaughn et al (2007) first used the term super-domestication
The processes that lead to a domesticate with dramatically
increased yield that could not be selected in natural environments
from naturally occurring variation without recourse to new
technologies
Super-domesticates can be constructed with knowledge led
approaches based on current needs using the range of new
technologies now available
Plants exploited for continuous selection introduction hybridization
etc which boosted the process of domestication but now the plant
genetic engineering approach is exploiting plants towards synthetic
biology
40
How to use diversitybull Cross two varieties
bull Genome manipulations
Cell fusion hybrids
bull Chromosome manipulation Backcross a new species
bull Generate recombinants Chromosome recombinations
bull Use a new species wild germplasm
Transgenic approach Modern mutagenesis synthetic gene
construction by utilizing green florescent protein genome editing
NGS GWAS sequencing etchelliphelliphelliphelliphellip
41
Reduce genetic bottlenecks through polyplodization
More vigour
SC
Buffering capacity
Heterotic advantage
Enhaced vegetative
charachers
Enhanced oil
Meiotic stability
42
Approach 1 Use one tetraploid and one diploid as parents (4X ndash 2X) followed by the
chromosome doubling of triploid hybrids (Chrom doubling)
a) Cross between B juncea (AjAjBjBj ) and B oleracea (CoCo) to produce hexaploids
(AjAjBjBjCoCo)
(b) Cross between B napus (AnAnCnCn) and B nigra (BniBni) to produce hexaploids
(AnAnBniBniCnCn)
(c) Cross between B carinata (BcaBcaCcaCca) and B rapa (ArAr) to produce hexaploids
(ArArBcaBcaCcaCca)
Approach 2 Use three tetraploids as parents
(a) Cross between B napus (AnAnCnCn) and B carinata (BcaBcaCcaCca) to produce unbalanced
allotetraploids (AnBcaCnCca ndash unreduced gametes gametes with the somatic chromosome
number and cross with B juncea (AjAjBjBj) to obtain allohexaploids (AnAjBcaBjCnCca
(b) Cross between B napus (AnAnCnCn) and B juncea (AjAjBjBj) to produce unbalanced
allotetraploids (AnAjBjCn ndash unreduced gametes) and cross with B carinata (BcaBcaCcaCca) to
obtain allohexaploids (AnAjBcaBjCnCca)
(c) Cross between B carinata (BcaBcaCcaCca) and B juncea (AjAjBjBj) to produce unbalanced
allotetraploids (AjBcaBjCca - ndash unreduced gametes) and cross with B napus (AnAnCnCn) to obtain
allohexaploids (AnAjBcaBjCnCca)
Approach 3 Use three diploids as parents (2X ndash 2X ndash 2X) Cross between B rapa (ArAr) B
nigra (BniBni) and B oleracea (CoCo) sequentially to obtain hexaploid hybrids (ArArBniBniCoCo)
43
Continuehellip
Guijun Yan 2012
44
Applications of Genomic
tools in Super-Domestication
45
Genome Sequencing
Potential methods of sequencing
1 Clone by clone approach
2 Whole genome shotgun approach
3 Combination of the two methods
Till today no of cultivated plants completely sequenced -85
2016- Arachis duranensis
2015- Solanum cumersonii (Wild potato)
(Ref- NCBI)
46
GWAS
It is a study design in which many markers spread across a genome
are genotyped and test a statistical association with a phenotype are
performed locally along the genome
It is also an examination of many common genetic variants in different
individuals to see if any variant is associated with a trait
Used in successfully studying maize sorghum and barley
Method is efficient for large scale low cost genotyping (even with the
minimum number of SNPs)
Cannot be utilized generally because it needs large population size
GWAS identify rare alleles more precisely
If small population we can opt NAM
47
GWAS and whole genome prediction
Xuehui Huang and Bin Han 2014
48
Five high throuput genotyping methods
Xuehui Huang and Bin Han 2014
49
Role of NGS in domestication Capture of novel genes from wild species will be made easier by
understanding the molecular events associated with crop domestication
Re-sequencing of domesticated species can identify low diversity regions
resulting from selection during domestication
To identify gene-specific sequences to aid the cloning of homologues of key
domestication genes from wild relatives
Candidate genes from wild and domesticated plant populations can define
diversity of target genes in wild populations and lead to the discovery of key
genes for important traits by association analysis
NGS of amplicons of large numbers of candidate genes from wild and
domesticated plant populations can define diversity of target genes in
wild populations and lead to the discovery of key genes for important
traits by association analysis
50
NGS
supports the rapid domestication of new plant species and the efficient
identification and capture of novel genetic variation from related species
Allows whole-genome analysis to determine the genetic basis of
phenotypic differences
NGS allows rapid expansion of genomic analysis to investigation of non-
model species
Made rice study easy by related grass
cost-effective method for plant identification
useful strategy to analysis the chloroplast genome sequence from whole-
genome shot-gun sequencing
facilitates managing this diversity and any changes in crop performance
over time due to genetic drift
Patterns of gene expression have been evaluated in hybrids using NGS
51
Two Approaches to Finding Adaptive Genes
Ross-Ibarra et al2007
52
Techniques to re-wild the plants (transgene free)
1 Introgression
breeding
2 Specific
insertion of lost genes
3 Precision
mutagenesis
Palmgren et al2014
53
Process of rewilding
Palmgren et al2014
54
Synthetic biology projects(i) Modifying cereals including wheat to fix atmospheric nitrogen
(ii) Redesigning metabolic pathways to increase the yield of
secondary metabolites or to generate compounds with enhanced
properties
(iii) Transferring the C4 photosynthesis pathway to rice
(iv) Modifying the glycosylation pathway in plants to accommodate
production of therapeutic proteins
(v) Introducing synthetic signal transduction systems that respond to
external cues
55
Synthetic biology projects
Nicholas J Baltes et al 2015
56
Refactoring the N fixation gene cluster from Klebsiella
Karsten Temme et al 2012
57
Continuehellip
Karsten Temme et al 2012
58
Genome editing
Sequence specific nucleases
1 Meganucleases
2 ZFMs ndashZink finger motifs
3 TALENs- Transcription activator like effector nuclease
4 CRISPR CAS-9 ndash Clustered interspaced short palindromic
repeats
qmp4
59Khaoula Belhaj et al 2015
60
Genome modifications achieved in plants using sequence specific nucleases
Type of DNA
modification
Nuclease Delivery
methods
Plants Targets
Trait stacking Megnucleases Bombardment Cotton Intergenic sequence
ZFN Bombardment Maize Transgene
Rewriting host
DNA
Megnucleases Stable integration Maize Intergenic sequence
ZFN Stable integration Soybean Transgene
TALEN Stable integration Barley PAPhy_a
TALEN Agrobacterium
T-DNA (transient)
Oryza sativa SWEET14
TALEN No Protoplasts Arabidopsis
Tobacco
AtTT4 AtADH NbSurB
TALEN Bombardment Wheat MLO
TALEN Protoplasts
Stable integration
Maize PDS IPK1A IPK MRP4
CRISPRCas Protoplasts
Agrobacterium
T-DNA (transient)
Tobacco
Arabidopsis
Sorghum Oryza
OsSWEET14 transgene
61
Type of DNA
modification
Nuclease Delivery
methods
Plants Targets
Rewriting host
DNA large
deletion
Zinc-finger
nuclease
Stable
integration
Tobacco Transgene
CRISPRCas Protoplasts
Stable
integration
Rice Labdane-related
diterpenoid gene
clusters
on Chr 2 4 and 6
Zinc-finger
nuclease
Agrobacterium
T-DNA
(transient)
Tobacco CHN50 transgene
Zinc-finger
nuclease
Whiskers Maize IPK1
CRISPRCas Protoplasts Rice PDS
Controlling gene
expression
TALE repressor
(SRDX)
Stable
integration
Arabidopsis RD29A transgene
Zinc-finger
activator (VP16)
Stable
integration
Brassica
napus
KasII
Continue
62
Application of genome editing1 Introduction of precise and predictable modifications directly in an elite
background
2 multiple traits can be modified simultaneously
3 NHEJ enables gene knockout and targeted modifications
4 Introduction of transgenes at defined loci that promote high-level
transcription and do not interfere with the activity of endogenous genes
5 Site-specific nucleases also allow targeted molecular trait stacking -low
risk of segregation
6 CRISPR Cas is a transgene free approach ndash No regulatory burdens
7 frequency of off-target mutations is well below that caused by chemical
and physical mutagenesis techniques
8 In future can be utilized for metabolic engineering and molecular farming
63
Six-way Venn diagram showing the distribution of shared genefamilies (sequence clusters) among M acuminata P dactyliferaArabidopsis thaliana Oryza sativa Sorghum bicolor and Brachypodiumdistachyon genomes
A DrsquoHont et al Nature 2012 doi101038nature11241
64
Achieving super-DomesticationINTROGRESS-RECREATE-CREATE-DOMESTICATEREDOMESTICATE
bull Mobilizing left out genetic
variation still available in
land races and wild and
weedy species
bull Replaying the evolutionary
tape( resynthesis in
polyploids)
bull Domesticateredomesticate
65
We can admire and emulate how indigenous people still domesticateplants create biodiversity and manage it to sustain their future Thereis no equivalently dynamic or flexible crop breeding in modernagriculture to promote biodiversity we still have much to learn from
traditional knowledge Jan Salick 2012
14
Domesticated Pearl MilletPennisetum glaucum
better seed recovery and yield but less able to survive in natural conditions compared to wild progenitor
bull more apical dominance (less
branching)
bull compact growth habit
bull flowering at same time (rather
than spread over long period)
bull larger spikes
bull non-shattering spikelets
bull loss of bristles amp glumes around
grains
bull larger seeds
bull non-dormant seeds
bull germinate at same time
15
Ppolystachian
Oats domestication
Wild- A sterilis Modern
sativa
16
Domestication of Sorghum
S helepense
S bicolor17
JR Harlan 1976 Sci American
Divergent
selection for
different
purposes
httpenwikipediaorgwikiImageBrassica_oleracea0jpg
httpwwwcehacukimagesclip_image002_001jpg
Brassica oleracea cabbage
broccoli cauliflower kale
romanesco collards kohrabi
brussels sprouts18
Solanum pimpinellifolium
Solanum lycopersicum19
Domestication of carrot
Queen Annes Lace
Daucus carota
20
Domestication of peanut
A villosulicarpa Hoehne andA Stenosperma (Brazil)
AABB
21
Domestication of Chilli
22
StrawberryBanana
Musa acuminata Musa balbisiana (2n=3x=33) AB genomeFRAGARIA X ANANASSA
Wood berries (F vesca) and Musky strawberries (F moschata)23
Wild SoybeanGlycin soja2n=40
Cultivated soybeanGlycin max 2n=40
24
Increased in domesticate
seed germination
determinate growth
longer pods
bigger (heavier) seeds
earliness
harvest index (seed yieldbiomass)
Decreased in domesticate
seed dispersal
seed dormancy
twining
number of nodes
length of internode
number of pods
photoperiod sensitivity
Common Bean Phaseolus vulgarishttpwwwplantsciencesucdavisedugeptspb143pb143htm
copy Paul Gepts
25
Wild pigeon peaCajanus cajanifolius
Cultivated pigeon pea
26
27
28
WHY OTHERS ARE NOT DOMESTICATED
bull 350000 plants
bull 4629 mammals
bull 9200 birds
bull 10000000 insects
bull 500000 fungi
bull But only 200 plants
bull 15 mammals 5 birds and
bull 2 insects are domesticated
bull Spread of these few species
bull Little change since early
agriculture
bull Repeated domestication of these
species (sometimes)
bull Lack of new species even with
attempts with species known to be
valuable Some groups are good
candidates with no domestication
eg ferns sub-Saharan mammals
29
WHY OTHERS HAVE TO BE DOMESTICATED
New uses and demands ndash biofuels animal feed
medicinalneutraceutical waterclimate food changes
Knowledge why species arenrsquot suitable for domestication or
were not useful
Better understanding of genetics and selection
Sustainability of production
Reliability of production
To meet the food demand in an alternative way
WHY OTHERS HAVE TO BE DOMESTICATED
30
Reallocate biomass for human use
More efficient metabolism and photosynthesis increase
leaf surface decrease root system increase leaf
longevity
Grains more fertile florets larger inflorescence OR
number of ears different ways to get more seed
Larger seeds (automatic vs deliberate selection)
Oil plants increased oil content or more seed
Fiber plants long strong fibers
Domestication syndrome
31
Centers of Domestication
Fuller 2011
32
Centers of Domestication in India
Fuller 2011
33
Indian centers of domestication South India (Deccan centre) Vigna radiate Vigna mungo
Macrotyloma uniflorum (Horse gram) Sataria verticillata S plumila
wheat (macaroni) and two rowed barleybrachiaria ramosa
Orissa (Mahanadi river) Pegion pea Horsegram mung small millets
like Echinichloa Paspalum and Sataria
The Middle Ganges (Harappan civilization) Oryza nivara O rufipogon
and wild sativa Sateria pumia Cannabis sativa even the diffusion of
japonica rice
Saurashtra (Harappan civilization) Eleucine coracana Sateria italica
Paicum spp Brachypodia pearl millet and sorghum Dollicos lablab
The Himalayan foothills of the Punjab region Was a centre of
diversity for Japonica type rice and many temperate fruits and
vegetables
34
DOMESTICATION RELATED TRAITS CONTROLLED BY ONLY FEW GENES
Trait Crops
Plant architecturegrowth habit Ricemaizemilletsbeantomato
Flowering timephotoperiod sensitivity Ricemaizesorghumbeantomato
Fruit size Tomato egg plant
Grain size Ricemaizesorghumbean
Seed dispersal Brassica rice
Inflorescence modification Brassica
Dormancy Bean
35
Genes Crop Moleular and phenotypic function Causative change
Genes identified as controlling domestication traits
tb1 Maize Transcriptional regulator (TCP) plant and
inflorescence structure
regulatory change
tga1 Maize Transcriptional regulator (SBP) seed casing amino acid change
qSH1 Rice Transcriptional regulator (homeodomain)
abscission layer formation shattering
regulatory change
Rc Rice Transcriptional regulator (bHLH) seed color disrupted coding
sequence
sh4 Rice Transcriptional regulator (Myb3) abscission
layer formation shattering
regulatoryamino
acid change
fw22 Tomato Cell signaling fruit weight regulatory change
Q Wheat Transcriptional regulator (AP2) inflorescence
structure
regulatoryamino
acid change
Vrs1 Barley Inflorescence structure Premature stop Amino acid change
Genes of interest in crop domestication and improvement
36
Genes Crop Moleular and phenotypic function Causative change
c1
r1
Maize Transcriptional regulator (MYB) kernel color and
Transcriptional regulator (bHLH) kernel color
regulatory change
sh2 Maize pyrophosphorylase supersweet sweet corn transposon
insertion
su1 Maize isoamylase sweet corn gene amino acid change
brix9-2-5 Tomato Invertase fruit soluble solid content amino acid change
ovate Tomato Unknown fruit shap early stop codon
R Pea Starch branching enzyme seed sugar content transposon
insertion
ehd1 Rice B-type response regulator flowering time amino acid change
hd1 Rice Transcriptional regulator (zinc finger) flowering
time
disrupted coding
sequence
waxy Rice Starch synthase sticky grains intron splicing
defect
rht Wheat Transcriptional regulator (SH2) plant height early stop codon
vrn1 Wheat Transcriptional regulator (MADS) vernalization regulatory change
vrn2 Wheat Transcriptional regulator (ZCCT) vernalization amino acid change
Genes Identified as Controlling Varietal Differences
37
Process of domestication over the yearshellip
Rachel S Meyer 2012
38
Genetic bottleneck
Cultivated crops undergone with narrowing of diversity problem
But this is not the case in weeds
Weeds are the major threats from the beginning of domestication till
today
Problem arose due to
1 Crop mimicry
2 Genetic assimilation
3 Genetic evolution
39
Super- Domestication Vaughn et al (2007) first used the term super-domestication
The processes that lead to a domesticate with dramatically
increased yield that could not be selected in natural environments
from naturally occurring variation without recourse to new
technologies
Super-domesticates can be constructed with knowledge led
approaches based on current needs using the range of new
technologies now available
Plants exploited for continuous selection introduction hybridization
etc which boosted the process of domestication but now the plant
genetic engineering approach is exploiting plants towards synthetic
biology
40
How to use diversitybull Cross two varieties
bull Genome manipulations
Cell fusion hybrids
bull Chromosome manipulation Backcross a new species
bull Generate recombinants Chromosome recombinations
bull Use a new species wild germplasm
Transgenic approach Modern mutagenesis synthetic gene
construction by utilizing green florescent protein genome editing
NGS GWAS sequencing etchelliphelliphelliphelliphellip
41
Reduce genetic bottlenecks through polyplodization
More vigour
SC
Buffering capacity
Heterotic advantage
Enhaced vegetative
charachers
Enhanced oil
Meiotic stability
42
Approach 1 Use one tetraploid and one diploid as parents (4X ndash 2X) followed by the
chromosome doubling of triploid hybrids (Chrom doubling)
a) Cross between B juncea (AjAjBjBj ) and B oleracea (CoCo) to produce hexaploids
(AjAjBjBjCoCo)
(b) Cross between B napus (AnAnCnCn) and B nigra (BniBni) to produce hexaploids
(AnAnBniBniCnCn)
(c) Cross between B carinata (BcaBcaCcaCca) and B rapa (ArAr) to produce hexaploids
(ArArBcaBcaCcaCca)
Approach 2 Use three tetraploids as parents
(a) Cross between B napus (AnAnCnCn) and B carinata (BcaBcaCcaCca) to produce unbalanced
allotetraploids (AnBcaCnCca ndash unreduced gametes gametes with the somatic chromosome
number and cross with B juncea (AjAjBjBj) to obtain allohexaploids (AnAjBcaBjCnCca
(b) Cross between B napus (AnAnCnCn) and B juncea (AjAjBjBj) to produce unbalanced
allotetraploids (AnAjBjCn ndash unreduced gametes) and cross with B carinata (BcaBcaCcaCca) to
obtain allohexaploids (AnAjBcaBjCnCca)
(c) Cross between B carinata (BcaBcaCcaCca) and B juncea (AjAjBjBj) to produce unbalanced
allotetraploids (AjBcaBjCca - ndash unreduced gametes) and cross with B napus (AnAnCnCn) to obtain
allohexaploids (AnAjBcaBjCnCca)
Approach 3 Use three diploids as parents (2X ndash 2X ndash 2X) Cross between B rapa (ArAr) B
nigra (BniBni) and B oleracea (CoCo) sequentially to obtain hexaploid hybrids (ArArBniBniCoCo)
43
Continuehellip
Guijun Yan 2012
44
Applications of Genomic
tools in Super-Domestication
45
Genome Sequencing
Potential methods of sequencing
1 Clone by clone approach
2 Whole genome shotgun approach
3 Combination of the two methods
Till today no of cultivated plants completely sequenced -85
2016- Arachis duranensis
2015- Solanum cumersonii (Wild potato)
(Ref- NCBI)
46
GWAS
It is a study design in which many markers spread across a genome
are genotyped and test a statistical association with a phenotype are
performed locally along the genome
It is also an examination of many common genetic variants in different
individuals to see if any variant is associated with a trait
Used in successfully studying maize sorghum and barley
Method is efficient for large scale low cost genotyping (even with the
minimum number of SNPs)
Cannot be utilized generally because it needs large population size
GWAS identify rare alleles more precisely
If small population we can opt NAM
47
GWAS and whole genome prediction
Xuehui Huang and Bin Han 2014
48
Five high throuput genotyping methods
Xuehui Huang and Bin Han 2014
49
Role of NGS in domestication Capture of novel genes from wild species will be made easier by
understanding the molecular events associated with crop domestication
Re-sequencing of domesticated species can identify low diversity regions
resulting from selection during domestication
To identify gene-specific sequences to aid the cloning of homologues of key
domestication genes from wild relatives
Candidate genes from wild and domesticated plant populations can define
diversity of target genes in wild populations and lead to the discovery of key
genes for important traits by association analysis
NGS of amplicons of large numbers of candidate genes from wild and
domesticated plant populations can define diversity of target genes in
wild populations and lead to the discovery of key genes for important
traits by association analysis
50
NGS
supports the rapid domestication of new plant species and the efficient
identification and capture of novel genetic variation from related species
Allows whole-genome analysis to determine the genetic basis of
phenotypic differences
NGS allows rapid expansion of genomic analysis to investigation of non-
model species
Made rice study easy by related grass
cost-effective method for plant identification
useful strategy to analysis the chloroplast genome sequence from whole-
genome shot-gun sequencing
facilitates managing this diversity and any changes in crop performance
over time due to genetic drift
Patterns of gene expression have been evaluated in hybrids using NGS
51
Two Approaches to Finding Adaptive Genes
Ross-Ibarra et al2007
52
Techniques to re-wild the plants (transgene free)
1 Introgression
breeding
2 Specific
insertion of lost genes
3 Precision
mutagenesis
Palmgren et al2014
53
Process of rewilding
Palmgren et al2014
54
Synthetic biology projects(i) Modifying cereals including wheat to fix atmospheric nitrogen
(ii) Redesigning metabolic pathways to increase the yield of
secondary metabolites or to generate compounds with enhanced
properties
(iii) Transferring the C4 photosynthesis pathway to rice
(iv) Modifying the glycosylation pathway in plants to accommodate
production of therapeutic proteins
(v) Introducing synthetic signal transduction systems that respond to
external cues
55
Synthetic biology projects
Nicholas J Baltes et al 2015
56
Refactoring the N fixation gene cluster from Klebsiella
Karsten Temme et al 2012
57
Continuehellip
Karsten Temme et al 2012
58
Genome editing
Sequence specific nucleases
1 Meganucleases
2 ZFMs ndashZink finger motifs
3 TALENs- Transcription activator like effector nuclease
4 CRISPR CAS-9 ndash Clustered interspaced short palindromic
repeats
qmp4
59Khaoula Belhaj et al 2015
60
Genome modifications achieved in plants using sequence specific nucleases
Type of DNA
modification
Nuclease Delivery
methods
Plants Targets
Trait stacking Megnucleases Bombardment Cotton Intergenic sequence
ZFN Bombardment Maize Transgene
Rewriting host
DNA
Megnucleases Stable integration Maize Intergenic sequence
ZFN Stable integration Soybean Transgene
TALEN Stable integration Barley PAPhy_a
TALEN Agrobacterium
T-DNA (transient)
Oryza sativa SWEET14
TALEN No Protoplasts Arabidopsis
Tobacco
AtTT4 AtADH NbSurB
TALEN Bombardment Wheat MLO
TALEN Protoplasts
Stable integration
Maize PDS IPK1A IPK MRP4
CRISPRCas Protoplasts
Agrobacterium
T-DNA (transient)
Tobacco
Arabidopsis
Sorghum Oryza
OsSWEET14 transgene
61
Type of DNA
modification
Nuclease Delivery
methods
Plants Targets
Rewriting host
DNA large
deletion
Zinc-finger
nuclease
Stable
integration
Tobacco Transgene
CRISPRCas Protoplasts
Stable
integration
Rice Labdane-related
diterpenoid gene
clusters
on Chr 2 4 and 6
Zinc-finger
nuclease
Agrobacterium
T-DNA
(transient)
Tobacco CHN50 transgene
Zinc-finger
nuclease
Whiskers Maize IPK1
CRISPRCas Protoplasts Rice PDS
Controlling gene
expression
TALE repressor
(SRDX)
Stable
integration
Arabidopsis RD29A transgene
Zinc-finger
activator (VP16)
Stable
integration
Brassica
napus
KasII
Continue
62
Application of genome editing1 Introduction of precise and predictable modifications directly in an elite
background
2 multiple traits can be modified simultaneously
3 NHEJ enables gene knockout and targeted modifications
4 Introduction of transgenes at defined loci that promote high-level
transcription and do not interfere with the activity of endogenous genes
5 Site-specific nucleases also allow targeted molecular trait stacking -low
risk of segregation
6 CRISPR Cas is a transgene free approach ndash No regulatory burdens
7 frequency of off-target mutations is well below that caused by chemical
and physical mutagenesis techniques
8 In future can be utilized for metabolic engineering and molecular farming
63
Six-way Venn diagram showing the distribution of shared genefamilies (sequence clusters) among M acuminata P dactyliferaArabidopsis thaliana Oryza sativa Sorghum bicolor and Brachypodiumdistachyon genomes
A DrsquoHont et al Nature 2012 doi101038nature11241
64
Achieving super-DomesticationINTROGRESS-RECREATE-CREATE-DOMESTICATEREDOMESTICATE
bull Mobilizing left out genetic
variation still available in
land races and wild and
weedy species
bull Replaying the evolutionary
tape( resynthesis in
polyploids)
bull Domesticateredomesticate
65
We can admire and emulate how indigenous people still domesticateplants create biodiversity and manage it to sustain their future Thereis no equivalently dynamic or flexible crop breeding in modernagriculture to promote biodiversity we still have much to learn from
traditional knowledge Jan Salick 2012
Domesticated Pearl MilletPennisetum glaucum
better seed recovery and yield but less able to survive in natural conditions compared to wild progenitor
bull more apical dominance (less
branching)
bull compact growth habit
bull flowering at same time (rather
than spread over long period)
bull larger spikes
bull non-shattering spikelets
bull loss of bristles amp glumes around
grains
bull larger seeds
bull non-dormant seeds
bull germinate at same time
15
Ppolystachian
Oats domestication
Wild- A sterilis Modern
sativa
16
Domestication of Sorghum
S helepense
S bicolor17
JR Harlan 1976 Sci American
Divergent
selection for
different
purposes
httpenwikipediaorgwikiImageBrassica_oleracea0jpg
httpwwwcehacukimagesclip_image002_001jpg
Brassica oleracea cabbage
broccoli cauliflower kale
romanesco collards kohrabi
brussels sprouts18
Solanum pimpinellifolium
Solanum lycopersicum19
Domestication of carrot
Queen Annes Lace
Daucus carota
20
Domestication of peanut
A villosulicarpa Hoehne andA Stenosperma (Brazil)
AABB
21
Domestication of Chilli
22
StrawberryBanana
Musa acuminata Musa balbisiana (2n=3x=33) AB genomeFRAGARIA X ANANASSA
Wood berries (F vesca) and Musky strawberries (F moschata)23
Wild SoybeanGlycin soja2n=40
Cultivated soybeanGlycin max 2n=40
24
Increased in domesticate
seed germination
determinate growth
longer pods
bigger (heavier) seeds
earliness
harvest index (seed yieldbiomass)
Decreased in domesticate
seed dispersal
seed dormancy
twining
number of nodes
length of internode
number of pods
photoperiod sensitivity
Common Bean Phaseolus vulgarishttpwwwplantsciencesucdavisedugeptspb143pb143htm
copy Paul Gepts
25
Wild pigeon peaCajanus cajanifolius
Cultivated pigeon pea
26
27
28
WHY OTHERS ARE NOT DOMESTICATED
bull 350000 plants
bull 4629 mammals
bull 9200 birds
bull 10000000 insects
bull 500000 fungi
bull But only 200 plants
bull 15 mammals 5 birds and
bull 2 insects are domesticated
bull Spread of these few species
bull Little change since early
agriculture
bull Repeated domestication of these
species (sometimes)
bull Lack of new species even with
attempts with species known to be
valuable Some groups are good
candidates with no domestication
eg ferns sub-Saharan mammals
29
WHY OTHERS HAVE TO BE DOMESTICATED
New uses and demands ndash biofuels animal feed
medicinalneutraceutical waterclimate food changes
Knowledge why species arenrsquot suitable for domestication or
were not useful
Better understanding of genetics and selection
Sustainability of production
Reliability of production
To meet the food demand in an alternative way
WHY OTHERS HAVE TO BE DOMESTICATED
30
Reallocate biomass for human use
More efficient metabolism and photosynthesis increase
leaf surface decrease root system increase leaf
longevity
Grains more fertile florets larger inflorescence OR
number of ears different ways to get more seed
Larger seeds (automatic vs deliberate selection)
Oil plants increased oil content or more seed
Fiber plants long strong fibers
Domestication syndrome
31
Centers of Domestication
Fuller 2011
32
Centers of Domestication in India
Fuller 2011
33
Indian centers of domestication South India (Deccan centre) Vigna radiate Vigna mungo
Macrotyloma uniflorum (Horse gram) Sataria verticillata S plumila
wheat (macaroni) and two rowed barleybrachiaria ramosa
Orissa (Mahanadi river) Pegion pea Horsegram mung small millets
like Echinichloa Paspalum and Sataria
The Middle Ganges (Harappan civilization) Oryza nivara O rufipogon
and wild sativa Sateria pumia Cannabis sativa even the diffusion of
japonica rice
Saurashtra (Harappan civilization) Eleucine coracana Sateria italica
Paicum spp Brachypodia pearl millet and sorghum Dollicos lablab
The Himalayan foothills of the Punjab region Was a centre of
diversity for Japonica type rice and many temperate fruits and
vegetables
34
DOMESTICATION RELATED TRAITS CONTROLLED BY ONLY FEW GENES
Trait Crops
Plant architecturegrowth habit Ricemaizemilletsbeantomato
Flowering timephotoperiod sensitivity Ricemaizesorghumbeantomato
Fruit size Tomato egg plant
Grain size Ricemaizesorghumbean
Seed dispersal Brassica rice
Inflorescence modification Brassica
Dormancy Bean
35
Genes Crop Moleular and phenotypic function Causative change
Genes identified as controlling domestication traits
tb1 Maize Transcriptional regulator (TCP) plant and
inflorescence structure
regulatory change
tga1 Maize Transcriptional regulator (SBP) seed casing amino acid change
qSH1 Rice Transcriptional regulator (homeodomain)
abscission layer formation shattering
regulatory change
Rc Rice Transcriptional regulator (bHLH) seed color disrupted coding
sequence
sh4 Rice Transcriptional regulator (Myb3) abscission
layer formation shattering
regulatoryamino
acid change
fw22 Tomato Cell signaling fruit weight regulatory change
Q Wheat Transcriptional regulator (AP2) inflorescence
structure
regulatoryamino
acid change
Vrs1 Barley Inflorescence structure Premature stop Amino acid change
Genes of interest in crop domestication and improvement
36
Genes Crop Moleular and phenotypic function Causative change
c1
r1
Maize Transcriptional regulator (MYB) kernel color and
Transcriptional regulator (bHLH) kernel color
regulatory change
sh2 Maize pyrophosphorylase supersweet sweet corn transposon
insertion
su1 Maize isoamylase sweet corn gene amino acid change
brix9-2-5 Tomato Invertase fruit soluble solid content amino acid change
ovate Tomato Unknown fruit shap early stop codon
R Pea Starch branching enzyme seed sugar content transposon
insertion
ehd1 Rice B-type response regulator flowering time amino acid change
hd1 Rice Transcriptional regulator (zinc finger) flowering
time
disrupted coding
sequence
waxy Rice Starch synthase sticky grains intron splicing
defect
rht Wheat Transcriptional regulator (SH2) plant height early stop codon
vrn1 Wheat Transcriptional regulator (MADS) vernalization regulatory change
vrn2 Wheat Transcriptional regulator (ZCCT) vernalization amino acid change
Genes Identified as Controlling Varietal Differences
37
Process of domestication over the yearshellip
Rachel S Meyer 2012
38
Genetic bottleneck
Cultivated crops undergone with narrowing of diversity problem
But this is not the case in weeds
Weeds are the major threats from the beginning of domestication till
today
Problem arose due to
1 Crop mimicry
2 Genetic assimilation
3 Genetic evolution
39
Super- Domestication Vaughn et al (2007) first used the term super-domestication
The processes that lead to a domesticate with dramatically
increased yield that could not be selected in natural environments
from naturally occurring variation without recourse to new
technologies
Super-domesticates can be constructed with knowledge led
approaches based on current needs using the range of new
technologies now available
Plants exploited for continuous selection introduction hybridization
etc which boosted the process of domestication but now the plant
genetic engineering approach is exploiting plants towards synthetic
biology
40
How to use diversitybull Cross two varieties
bull Genome manipulations
Cell fusion hybrids
bull Chromosome manipulation Backcross a new species
bull Generate recombinants Chromosome recombinations
bull Use a new species wild germplasm
Transgenic approach Modern mutagenesis synthetic gene
construction by utilizing green florescent protein genome editing
NGS GWAS sequencing etchelliphelliphelliphelliphellip
41
Reduce genetic bottlenecks through polyplodization
More vigour
SC
Buffering capacity
Heterotic advantage
Enhaced vegetative
charachers
Enhanced oil
Meiotic stability
42
Approach 1 Use one tetraploid and one diploid as parents (4X ndash 2X) followed by the
chromosome doubling of triploid hybrids (Chrom doubling)
a) Cross between B juncea (AjAjBjBj ) and B oleracea (CoCo) to produce hexaploids
(AjAjBjBjCoCo)
(b) Cross between B napus (AnAnCnCn) and B nigra (BniBni) to produce hexaploids
(AnAnBniBniCnCn)
(c) Cross between B carinata (BcaBcaCcaCca) and B rapa (ArAr) to produce hexaploids
(ArArBcaBcaCcaCca)
Approach 2 Use three tetraploids as parents
(a) Cross between B napus (AnAnCnCn) and B carinata (BcaBcaCcaCca) to produce unbalanced
allotetraploids (AnBcaCnCca ndash unreduced gametes gametes with the somatic chromosome
number and cross with B juncea (AjAjBjBj) to obtain allohexaploids (AnAjBcaBjCnCca
(b) Cross between B napus (AnAnCnCn) and B juncea (AjAjBjBj) to produce unbalanced
allotetraploids (AnAjBjCn ndash unreduced gametes) and cross with B carinata (BcaBcaCcaCca) to
obtain allohexaploids (AnAjBcaBjCnCca)
(c) Cross between B carinata (BcaBcaCcaCca) and B juncea (AjAjBjBj) to produce unbalanced
allotetraploids (AjBcaBjCca - ndash unreduced gametes) and cross with B napus (AnAnCnCn) to obtain
allohexaploids (AnAjBcaBjCnCca)
Approach 3 Use three diploids as parents (2X ndash 2X ndash 2X) Cross between B rapa (ArAr) B
nigra (BniBni) and B oleracea (CoCo) sequentially to obtain hexaploid hybrids (ArArBniBniCoCo)
43
Continuehellip
Guijun Yan 2012
44
Applications of Genomic
tools in Super-Domestication
45
Genome Sequencing
Potential methods of sequencing
1 Clone by clone approach
2 Whole genome shotgun approach
3 Combination of the two methods
Till today no of cultivated plants completely sequenced -85
2016- Arachis duranensis
2015- Solanum cumersonii (Wild potato)
(Ref- NCBI)
46
GWAS
It is a study design in which many markers spread across a genome
are genotyped and test a statistical association with a phenotype are
performed locally along the genome
It is also an examination of many common genetic variants in different
individuals to see if any variant is associated with a trait
Used in successfully studying maize sorghum and barley
Method is efficient for large scale low cost genotyping (even with the
minimum number of SNPs)
Cannot be utilized generally because it needs large population size
GWAS identify rare alleles more precisely
If small population we can opt NAM
47
GWAS and whole genome prediction
Xuehui Huang and Bin Han 2014
48
Five high throuput genotyping methods
Xuehui Huang and Bin Han 2014
49
Role of NGS in domestication Capture of novel genes from wild species will be made easier by
understanding the molecular events associated with crop domestication
Re-sequencing of domesticated species can identify low diversity regions
resulting from selection during domestication
To identify gene-specific sequences to aid the cloning of homologues of key
domestication genes from wild relatives
Candidate genes from wild and domesticated plant populations can define
diversity of target genes in wild populations and lead to the discovery of key
genes for important traits by association analysis
NGS of amplicons of large numbers of candidate genes from wild and
domesticated plant populations can define diversity of target genes in
wild populations and lead to the discovery of key genes for important
traits by association analysis
50
NGS
supports the rapid domestication of new plant species and the efficient
identification and capture of novel genetic variation from related species
Allows whole-genome analysis to determine the genetic basis of
phenotypic differences
NGS allows rapid expansion of genomic analysis to investigation of non-
model species
Made rice study easy by related grass
cost-effective method for plant identification
useful strategy to analysis the chloroplast genome sequence from whole-
genome shot-gun sequencing
facilitates managing this diversity and any changes in crop performance
over time due to genetic drift
Patterns of gene expression have been evaluated in hybrids using NGS
51
Two Approaches to Finding Adaptive Genes
Ross-Ibarra et al2007
52
Techniques to re-wild the plants (transgene free)
1 Introgression
breeding
2 Specific
insertion of lost genes
3 Precision
mutagenesis
Palmgren et al2014
53
Process of rewilding
Palmgren et al2014
54
Synthetic biology projects(i) Modifying cereals including wheat to fix atmospheric nitrogen
(ii) Redesigning metabolic pathways to increase the yield of
secondary metabolites or to generate compounds with enhanced
properties
(iii) Transferring the C4 photosynthesis pathway to rice
(iv) Modifying the glycosylation pathway in plants to accommodate
production of therapeutic proteins
(v) Introducing synthetic signal transduction systems that respond to
external cues
55
Synthetic biology projects
Nicholas J Baltes et al 2015
56
Refactoring the N fixation gene cluster from Klebsiella
Karsten Temme et al 2012
57
Continuehellip
Karsten Temme et al 2012
58
Genome editing
Sequence specific nucleases
1 Meganucleases
2 ZFMs ndashZink finger motifs
3 TALENs- Transcription activator like effector nuclease
4 CRISPR CAS-9 ndash Clustered interspaced short palindromic
repeats
qmp4
59Khaoula Belhaj et al 2015
60
Genome modifications achieved in plants using sequence specific nucleases
Type of DNA
modification
Nuclease Delivery
methods
Plants Targets
Trait stacking Megnucleases Bombardment Cotton Intergenic sequence
ZFN Bombardment Maize Transgene
Rewriting host
DNA
Megnucleases Stable integration Maize Intergenic sequence
ZFN Stable integration Soybean Transgene
TALEN Stable integration Barley PAPhy_a
TALEN Agrobacterium
T-DNA (transient)
Oryza sativa SWEET14
TALEN No Protoplasts Arabidopsis
Tobacco
AtTT4 AtADH NbSurB
TALEN Bombardment Wheat MLO
TALEN Protoplasts
Stable integration
Maize PDS IPK1A IPK MRP4
CRISPRCas Protoplasts
Agrobacterium
T-DNA (transient)
Tobacco
Arabidopsis
Sorghum Oryza
OsSWEET14 transgene
61
Type of DNA
modification
Nuclease Delivery
methods
Plants Targets
Rewriting host
DNA large
deletion
Zinc-finger
nuclease
Stable
integration
Tobacco Transgene
CRISPRCas Protoplasts
Stable
integration
Rice Labdane-related
diterpenoid gene
clusters
on Chr 2 4 and 6
Zinc-finger
nuclease
Agrobacterium
T-DNA
(transient)
Tobacco CHN50 transgene
Zinc-finger
nuclease
Whiskers Maize IPK1
CRISPRCas Protoplasts Rice PDS
Controlling gene
expression
TALE repressor
(SRDX)
Stable
integration
Arabidopsis RD29A transgene
Zinc-finger
activator (VP16)
Stable
integration
Brassica
napus
KasII
Continue
62
Application of genome editing1 Introduction of precise and predictable modifications directly in an elite
background
2 multiple traits can be modified simultaneously
3 NHEJ enables gene knockout and targeted modifications
4 Introduction of transgenes at defined loci that promote high-level
transcription and do not interfere with the activity of endogenous genes
5 Site-specific nucleases also allow targeted molecular trait stacking -low
risk of segregation
6 CRISPR Cas is a transgene free approach ndash No regulatory burdens
7 frequency of off-target mutations is well below that caused by chemical
and physical mutagenesis techniques
8 In future can be utilized for metabolic engineering and molecular farming
63
Six-way Venn diagram showing the distribution of shared genefamilies (sequence clusters) among M acuminata P dactyliferaArabidopsis thaliana Oryza sativa Sorghum bicolor and Brachypodiumdistachyon genomes
A DrsquoHont et al Nature 2012 doi101038nature11241
64
Achieving super-DomesticationINTROGRESS-RECREATE-CREATE-DOMESTICATEREDOMESTICATE
bull Mobilizing left out genetic
variation still available in
land races and wild and
weedy species
bull Replaying the evolutionary
tape( resynthesis in
polyploids)
bull Domesticateredomesticate
65
We can admire and emulate how indigenous people still domesticateplants create biodiversity and manage it to sustain their future Thereis no equivalently dynamic or flexible crop breeding in modernagriculture to promote biodiversity we still have much to learn from
traditional knowledge Jan Salick 2012
Oats domestication
Wild- A sterilis Modern
sativa
16
Domestication of Sorghum
S helepense
S bicolor17
JR Harlan 1976 Sci American
Divergent
selection for
different
purposes
httpenwikipediaorgwikiImageBrassica_oleracea0jpg
httpwwwcehacukimagesclip_image002_001jpg
Brassica oleracea cabbage
broccoli cauliflower kale
romanesco collards kohrabi
brussels sprouts18
Solanum pimpinellifolium
Solanum lycopersicum19
Domestication of carrot
Queen Annes Lace
Daucus carota
20
Domestication of peanut
A villosulicarpa Hoehne andA Stenosperma (Brazil)
AABB
21
Domestication of Chilli
22
StrawberryBanana
Musa acuminata Musa balbisiana (2n=3x=33) AB genomeFRAGARIA X ANANASSA
Wood berries (F vesca) and Musky strawberries (F moschata)23
Wild SoybeanGlycin soja2n=40
Cultivated soybeanGlycin max 2n=40
24
Increased in domesticate
seed germination
determinate growth
longer pods
bigger (heavier) seeds
earliness
harvest index (seed yieldbiomass)
Decreased in domesticate
seed dispersal
seed dormancy
twining
number of nodes
length of internode
number of pods
photoperiod sensitivity
Common Bean Phaseolus vulgarishttpwwwplantsciencesucdavisedugeptspb143pb143htm
copy Paul Gepts
25
Wild pigeon peaCajanus cajanifolius
Cultivated pigeon pea
26
27
28
WHY OTHERS ARE NOT DOMESTICATED
bull 350000 plants
bull 4629 mammals
bull 9200 birds
bull 10000000 insects
bull 500000 fungi
bull But only 200 plants
bull 15 mammals 5 birds and
bull 2 insects are domesticated
bull Spread of these few species
bull Little change since early
agriculture
bull Repeated domestication of these
species (sometimes)
bull Lack of new species even with
attempts with species known to be
valuable Some groups are good
candidates with no domestication
eg ferns sub-Saharan mammals
29
WHY OTHERS HAVE TO BE DOMESTICATED
New uses and demands ndash biofuels animal feed
medicinalneutraceutical waterclimate food changes
Knowledge why species arenrsquot suitable for domestication or
were not useful
Better understanding of genetics and selection
Sustainability of production
Reliability of production
To meet the food demand in an alternative way
WHY OTHERS HAVE TO BE DOMESTICATED
30
Reallocate biomass for human use
More efficient metabolism and photosynthesis increase
leaf surface decrease root system increase leaf
longevity
Grains more fertile florets larger inflorescence OR
number of ears different ways to get more seed
Larger seeds (automatic vs deliberate selection)
Oil plants increased oil content or more seed
Fiber plants long strong fibers
Domestication syndrome
31
Centers of Domestication
Fuller 2011
32
Centers of Domestication in India
Fuller 2011
33
Indian centers of domestication South India (Deccan centre) Vigna radiate Vigna mungo
Macrotyloma uniflorum (Horse gram) Sataria verticillata S plumila
wheat (macaroni) and two rowed barleybrachiaria ramosa
Orissa (Mahanadi river) Pegion pea Horsegram mung small millets
like Echinichloa Paspalum and Sataria
The Middle Ganges (Harappan civilization) Oryza nivara O rufipogon
and wild sativa Sateria pumia Cannabis sativa even the diffusion of
japonica rice
Saurashtra (Harappan civilization) Eleucine coracana Sateria italica
Paicum spp Brachypodia pearl millet and sorghum Dollicos lablab
The Himalayan foothills of the Punjab region Was a centre of
diversity for Japonica type rice and many temperate fruits and
vegetables
34
DOMESTICATION RELATED TRAITS CONTROLLED BY ONLY FEW GENES
Trait Crops
Plant architecturegrowth habit Ricemaizemilletsbeantomato
Flowering timephotoperiod sensitivity Ricemaizesorghumbeantomato
Fruit size Tomato egg plant
Grain size Ricemaizesorghumbean
Seed dispersal Brassica rice
Inflorescence modification Brassica
Dormancy Bean
35
Genes Crop Moleular and phenotypic function Causative change
Genes identified as controlling domestication traits
tb1 Maize Transcriptional regulator (TCP) plant and
inflorescence structure
regulatory change
tga1 Maize Transcriptional regulator (SBP) seed casing amino acid change
qSH1 Rice Transcriptional regulator (homeodomain)
abscission layer formation shattering
regulatory change
Rc Rice Transcriptional regulator (bHLH) seed color disrupted coding
sequence
sh4 Rice Transcriptional regulator (Myb3) abscission
layer formation shattering
regulatoryamino
acid change
fw22 Tomato Cell signaling fruit weight regulatory change
Q Wheat Transcriptional regulator (AP2) inflorescence
structure
regulatoryamino
acid change
Vrs1 Barley Inflorescence structure Premature stop Amino acid change
Genes of interest in crop domestication and improvement
36
Genes Crop Moleular and phenotypic function Causative change
c1
r1
Maize Transcriptional regulator (MYB) kernel color and
Transcriptional regulator (bHLH) kernel color
regulatory change
sh2 Maize pyrophosphorylase supersweet sweet corn transposon
insertion
su1 Maize isoamylase sweet corn gene amino acid change
brix9-2-5 Tomato Invertase fruit soluble solid content amino acid change
ovate Tomato Unknown fruit shap early stop codon
R Pea Starch branching enzyme seed sugar content transposon
insertion
ehd1 Rice B-type response regulator flowering time amino acid change
hd1 Rice Transcriptional regulator (zinc finger) flowering
time
disrupted coding
sequence
waxy Rice Starch synthase sticky grains intron splicing
defect
rht Wheat Transcriptional regulator (SH2) plant height early stop codon
vrn1 Wheat Transcriptional regulator (MADS) vernalization regulatory change
vrn2 Wheat Transcriptional regulator (ZCCT) vernalization amino acid change
Genes Identified as Controlling Varietal Differences
37
Process of domestication over the yearshellip
Rachel S Meyer 2012
38
Genetic bottleneck
Cultivated crops undergone with narrowing of diversity problem
But this is not the case in weeds
Weeds are the major threats from the beginning of domestication till
today
Problem arose due to
1 Crop mimicry
2 Genetic assimilation
3 Genetic evolution
39
Super- Domestication Vaughn et al (2007) first used the term super-domestication
The processes that lead to a domesticate with dramatically
increased yield that could not be selected in natural environments
from naturally occurring variation without recourse to new
technologies
Super-domesticates can be constructed with knowledge led
approaches based on current needs using the range of new
technologies now available
Plants exploited for continuous selection introduction hybridization
etc which boosted the process of domestication but now the plant
genetic engineering approach is exploiting plants towards synthetic
biology
40
How to use diversitybull Cross two varieties
bull Genome manipulations
Cell fusion hybrids
bull Chromosome manipulation Backcross a new species
bull Generate recombinants Chromosome recombinations
bull Use a new species wild germplasm
Transgenic approach Modern mutagenesis synthetic gene
construction by utilizing green florescent protein genome editing
NGS GWAS sequencing etchelliphelliphelliphelliphellip
41
Reduce genetic bottlenecks through polyplodization
More vigour
SC
Buffering capacity
Heterotic advantage
Enhaced vegetative
charachers
Enhanced oil
Meiotic stability
42
Approach 1 Use one tetraploid and one diploid as parents (4X ndash 2X) followed by the
chromosome doubling of triploid hybrids (Chrom doubling)
a) Cross between B juncea (AjAjBjBj ) and B oleracea (CoCo) to produce hexaploids
(AjAjBjBjCoCo)
(b) Cross between B napus (AnAnCnCn) and B nigra (BniBni) to produce hexaploids
(AnAnBniBniCnCn)
(c) Cross between B carinata (BcaBcaCcaCca) and B rapa (ArAr) to produce hexaploids
(ArArBcaBcaCcaCca)
Approach 2 Use three tetraploids as parents
(a) Cross between B napus (AnAnCnCn) and B carinata (BcaBcaCcaCca) to produce unbalanced
allotetraploids (AnBcaCnCca ndash unreduced gametes gametes with the somatic chromosome
number and cross with B juncea (AjAjBjBj) to obtain allohexaploids (AnAjBcaBjCnCca
(b) Cross between B napus (AnAnCnCn) and B juncea (AjAjBjBj) to produce unbalanced
allotetraploids (AnAjBjCn ndash unreduced gametes) and cross with B carinata (BcaBcaCcaCca) to
obtain allohexaploids (AnAjBcaBjCnCca)
(c) Cross between B carinata (BcaBcaCcaCca) and B juncea (AjAjBjBj) to produce unbalanced
allotetraploids (AjBcaBjCca - ndash unreduced gametes) and cross with B napus (AnAnCnCn) to obtain
allohexaploids (AnAjBcaBjCnCca)
Approach 3 Use three diploids as parents (2X ndash 2X ndash 2X) Cross between B rapa (ArAr) B
nigra (BniBni) and B oleracea (CoCo) sequentially to obtain hexaploid hybrids (ArArBniBniCoCo)
43
Continuehellip
Guijun Yan 2012
44
Applications of Genomic
tools in Super-Domestication
45
Genome Sequencing
Potential methods of sequencing
1 Clone by clone approach
2 Whole genome shotgun approach
3 Combination of the two methods
Till today no of cultivated plants completely sequenced -85
2016- Arachis duranensis
2015- Solanum cumersonii (Wild potato)
(Ref- NCBI)
46
GWAS
It is a study design in which many markers spread across a genome
are genotyped and test a statistical association with a phenotype are
performed locally along the genome
It is also an examination of many common genetic variants in different
individuals to see if any variant is associated with a trait
Used in successfully studying maize sorghum and barley
Method is efficient for large scale low cost genotyping (even with the
minimum number of SNPs)
Cannot be utilized generally because it needs large population size
GWAS identify rare alleles more precisely
If small population we can opt NAM
47
GWAS and whole genome prediction
Xuehui Huang and Bin Han 2014
48
Five high throuput genotyping methods
Xuehui Huang and Bin Han 2014
49
Role of NGS in domestication Capture of novel genes from wild species will be made easier by
understanding the molecular events associated with crop domestication
Re-sequencing of domesticated species can identify low diversity regions
resulting from selection during domestication
To identify gene-specific sequences to aid the cloning of homologues of key
domestication genes from wild relatives
Candidate genes from wild and domesticated plant populations can define
diversity of target genes in wild populations and lead to the discovery of key
genes for important traits by association analysis
NGS of amplicons of large numbers of candidate genes from wild and
domesticated plant populations can define diversity of target genes in
wild populations and lead to the discovery of key genes for important
traits by association analysis
50
NGS
supports the rapid domestication of new plant species and the efficient
identification and capture of novel genetic variation from related species
Allows whole-genome analysis to determine the genetic basis of
phenotypic differences
NGS allows rapid expansion of genomic analysis to investigation of non-
model species
Made rice study easy by related grass
cost-effective method for plant identification
useful strategy to analysis the chloroplast genome sequence from whole-
genome shot-gun sequencing
facilitates managing this diversity and any changes in crop performance
over time due to genetic drift
Patterns of gene expression have been evaluated in hybrids using NGS
51
Two Approaches to Finding Adaptive Genes
Ross-Ibarra et al2007
52
Techniques to re-wild the plants (transgene free)
1 Introgression
breeding
2 Specific
insertion of lost genes
3 Precision
mutagenesis
Palmgren et al2014
53
Process of rewilding
Palmgren et al2014
54
Synthetic biology projects(i) Modifying cereals including wheat to fix atmospheric nitrogen
(ii) Redesigning metabolic pathways to increase the yield of
secondary metabolites or to generate compounds with enhanced
properties
(iii) Transferring the C4 photosynthesis pathway to rice
(iv) Modifying the glycosylation pathway in plants to accommodate
production of therapeutic proteins
(v) Introducing synthetic signal transduction systems that respond to
external cues
55
Synthetic biology projects
Nicholas J Baltes et al 2015
56
Refactoring the N fixation gene cluster from Klebsiella
Karsten Temme et al 2012
57
Continuehellip
Karsten Temme et al 2012
58
Genome editing
Sequence specific nucleases
1 Meganucleases
2 ZFMs ndashZink finger motifs
3 TALENs- Transcription activator like effector nuclease
4 CRISPR CAS-9 ndash Clustered interspaced short palindromic
repeats
qmp4
59Khaoula Belhaj et al 2015
60
Genome modifications achieved in plants using sequence specific nucleases
Type of DNA
modification
Nuclease Delivery
methods
Plants Targets
Trait stacking Megnucleases Bombardment Cotton Intergenic sequence
ZFN Bombardment Maize Transgene
Rewriting host
DNA
Megnucleases Stable integration Maize Intergenic sequence
ZFN Stable integration Soybean Transgene
TALEN Stable integration Barley PAPhy_a
TALEN Agrobacterium
T-DNA (transient)
Oryza sativa SWEET14
TALEN No Protoplasts Arabidopsis
Tobacco
AtTT4 AtADH NbSurB
TALEN Bombardment Wheat MLO
TALEN Protoplasts
Stable integration
Maize PDS IPK1A IPK MRP4
CRISPRCas Protoplasts
Agrobacterium
T-DNA (transient)
Tobacco
Arabidopsis
Sorghum Oryza
OsSWEET14 transgene
61
Type of DNA
modification
Nuclease Delivery
methods
Plants Targets
Rewriting host
DNA large
deletion
Zinc-finger
nuclease
Stable
integration
Tobacco Transgene
CRISPRCas Protoplasts
Stable
integration
Rice Labdane-related
diterpenoid gene
clusters
on Chr 2 4 and 6
Zinc-finger
nuclease
Agrobacterium
T-DNA
(transient)
Tobacco CHN50 transgene
Zinc-finger
nuclease
Whiskers Maize IPK1
CRISPRCas Protoplasts Rice PDS
Controlling gene
expression
TALE repressor
(SRDX)
Stable
integration
Arabidopsis RD29A transgene
Zinc-finger
activator (VP16)
Stable
integration
Brassica
napus
KasII
Continue
62
Application of genome editing1 Introduction of precise and predictable modifications directly in an elite
background
2 multiple traits can be modified simultaneously
3 NHEJ enables gene knockout and targeted modifications
4 Introduction of transgenes at defined loci that promote high-level
transcription and do not interfere with the activity of endogenous genes
5 Site-specific nucleases also allow targeted molecular trait stacking -low
risk of segregation
6 CRISPR Cas is a transgene free approach ndash No regulatory burdens
7 frequency of off-target mutations is well below that caused by chemical
and physical mutagenesis techniques
8 In future can be utilized for metabolic engineering and molecular farming
63
Six-way Venn diagram showing the distribution of shared genefamilies (sequence clusters) among M acuminata P dactyliferaArabidopsis thaliana Oryza sativa Sorghum bicolor and Brachypodiumdistachyon genomes
A DrsquoHont et al Nature 2012 doi101038nature11241
64
Achieving super-DomesticationINTROGRESS-RECREATE-CREATE-DOMESTICATEREDOMESTICATE
bull Mobilizing left out genetic
variation still available in
land races and wild and
weedy species
bull Replaying the evolutionary
tape( resynthesis in
polyploids)
bull Domesticateredomesticate
65
We can admire and emulate how indigenous people still domesticateplants create biodiversity and manage it to sustain their future Thereis no equivalently dynamic or flexible crop breeding in modernagriculture to promote biodiversity we still have much to learn from
traditional knowledge Jan Salick 2012
Domestication of Sorghum
S helepense
S bicolor17
JR Harlan 1976 Sci American
Divergent
selection for
different
purposes
httpenwikipediaorgwikiImageBrassica_oleracea0jpg
httpwwwcehacukimagesclip_image002_001jpg
Brassica oleracea cabbage
broccoli cauliflower kale
romanesco collards kohrabi
brussels sprouts18
Solanum pimpinellifolium
Solanum lycopersicum19
Domestication of carrot
Queen Annes Lace
Daucus carota
20
Domestication of peanut
A villosulicarpa Hoehne andA Stenosperma (Brazil)
AABB
21
Domestication of Chilli
22
StrawberryBanana
Musa acuminata Musa balbisiana (2n=3x=33) AB genomeFRAGARIA X ANANASSA
Wood berries (F vesca) and Musky strawberries (F moschata)23
Wild SoybeanGlycin soja2n=40
Cultivated soybeanGlycin max 2n=40
24
Increased in domesticate
seed germination
determinate growth
longer pods
bigger (heavier) seeds
earliness
harvest index (seed yieldbiomass)
Decreased in domesticate
seed dispersal
seed dormancy
twining
number of nodes
length of internode
number of pods
photoperiod sensitivity
Common Bean Phaseolus vulgarishttpwwwplantsciencesucdavisedugeptspb143pb143htm
copy Paul Gepts
25
Wild pigeon peaCajanus cajanifolius
Cultivated pigeon pea
26
27
28
WHY OTHERS ARE NOT DOMESTICATED
bull 350000 plants
bull 4629 mammals
bull 9200 birds
bull 10000000 insects
bull 500000 fungi
bull But only 200 plants
bull 15 mammals 5 birds and
bull 2 insects are domesticated
bull Spread of these few species
bull Little change since early
agriculture
bull Repeated domestication of these
species (sometimes)
bull Lack of new species even with
attempts with species known to be
valuable Some groups are good
candidates with no domestication
eg ferns sub-Saharan mammals
29
WHY OTHERS HAVE TO BE DOMESTICATED
New uses and demands ndash biofuels animal feed
medicinalneutraceutical waterclimate food changes
Knowledge why species arenrsquot suitable for domestication or
were not useful
Better understanding of genetics and selection
Sustainability of production
Reliability of production
To meet the food demand in an alternative way
WHY OTHERS HAVE TO BE DOMESTICATED
30
Reallocate biomass for human use
More efficient metabolism and photosynthesis increase
leaf surface decrease root system increase leaf
longevity
Grains more fertile florets larger inflorescence OR
number of ears different ways to get more seed
Larger seeds (automatic vs deliberate selection)
Oil plants increased oil content or more seed
Fiber plants long strong fibers
Domestication syndrome
31
Centers of Domestication
Fuller 2011
32
Centers of Domestication in India
Fuller 2011
33
Indian centers of domestication South India (Deccan centre) Vigna radiate Vigna mungo
Macrotyloma uniflorum (Horse gram) Sataria verticillata S plumila
wheat (macaroni) and two rowed barleybrachiaria ramosa
Orissa (Mahanadi river) Pegion pea Horsegram mung small millets
like Echinichloa Paspalum and Sataria
The Middle Ganges (Harappan civilization) Oryza nivara O rufipogon
and wild sativa Sateria pumia Cannabis sativa even the diffusion of
japonica rice
Saurashtra (Harappan civilization) Eleucine coracana Sateria italica
Paicum spp Brachypodia pearl millet and sorghum Dollicos lablab
The Himalayan foothills of the Punjab region Was a centre of
diversity for Japonica type rice and many temperate fruits and
vegetables
34
DOMESTICATION RELATED TRAITS CONTROLLED BY ONLY FEW GENES
Trait Crops
Plant architecturegrowth habit Ricemaizemilletsbeantomato
Flowering timephotoperiod sensitivity Ricemaizesorghumbeantomato
Fruit size Tomato egg plant
Grain size Ricemaizesorghumbean
Seed dispersal Brassica rice
Inflorescence modification Brassica
Dormancy Bean
35
Genes Crop Moleular and phenotypic function Causative change
Genes identified as controlling domestication traits
tb1 Maize Transcriptional regulator (TCP) plant and
inflorescence structure
regulatory change
tga1 Maize Transcriptional regulator (SBP) seed casing amino acid change
qSH1 Rice Transcriptional regulator (homeodomain)
abscission layer formation shattering
regulatory change
Rc Rice Transcriptional regulator (bHLH) seed color disrupted coding
sequence
sh4 Rice Transcriptional regulator (Myb3) abscission
layer formation shattering
regulatoryamino
acid change
fw22 Tomato Cell signaling fruit weight regulatory change
Q Wheat Transcriptional regulator (AP2) inflorescence
structure
regulatoryamino
acid change
Vrs1 Barley Inflorescence structure Premature stop Amino acid change
Genes of interest in crop domestication and improvement
36
Genes Crop Moleular and phenotypic function Causative change
c1
r1
Maize Transcriptional regulator (MYB) kernel color and
Transcriptional regulator (bHLH) kernel color
regulatory change
sh2 Maize pyrophosphorylase supersweet sweet corn transposon
insertion
su1 Maize isoamylase sweet corn gene amino acid change
brix9-2-5 Tomato Invertase fruit soluble solid content amino acid change
ovate Tomato Unknown fruit shap early stop codon
R Pea Starch branching enzyme seed sugar content transposon
insertion
ehd1 Rice B-type response regulator flowering time amino acid change
hd1 Rice Transcriptional regulator (zinc finger) flowering
time
disrupted coding
sequence
waxy Rice Starch synthase sticky grains intron splicing
defect
rht Wheat Transcriptional regulator (SH2) plant height early stop codon
vrn1 Wheat Transcriptional regulator (MADS) vernalization regulatory change
vrn2 Wheat Transcriptional regulator (ZCCT) vernalization amino acid change
Genes Identified as Controlling Varietal Differences
37
Process of domestication over the yearshellip
Rachel S Meyer 2012
38
Genetic bottleneck
Cultivated crops undergone with narrowing of diversity problem
But this is not the case in weeds
Weeds are the major threats from the beginning of domestication till
today
Problem arose due to
1 Crop mimicry
2 Genetic assimilation
3 Genetic evolution
39
Super- Domestication Vaughn et al (2007) first used the term super-domestication
The processes that lead to a domesticate with dramatically
increased yield that could not be selected in natural environments
from naturally occurring variation without recourse to new
technologies
Super-domesticates can be constructed with knowledge led
approaches based on current needs using the range of new
technologies now available
Plants exploited for continuous selection introduction hybridization
etc which boosted the process of domestication but now the plant
genetic engineering approach is exploiting plants towards synthetic
biology
40
How to use diversitybull Cross two varieties
bull Genome manipulations
Cell fusion hybrids
bull Chromosome manipulation Backcross a new species
bull Generate recombinants Chromosome recombinations
bull Use a new species wild germplasm
Transgenic approach Modern mutagenesis synthetic gene
construction by utilizing green florescent protein genome editing
NGS GWAS sequencing etchelliphelliphelliphelliphellip
41
Reduce genetic bottlenecks through polyplodization
More vigour
SC
Buffering capacity
Heterotic advantage
Enhaced vegetative
charachers
Enhanced oil
Meiotic stability
42
Approach 1 Use one tetraploid and one diploid as parents (4X ndash 2X) followed by the
chromosome doubling of triploid hybrids (Chrom doubling)
a) Cross between B juncea (AjAjBjBj ) and B oleracea (CoCo) to produce hexaploids
(AjAjBjBjCoCo)
(b) Cross between B napus (AnAnCnCn) and B nigra (BniBni) to produce hexaploids
(AnAnBniBniCnCn)
(c) Cross between B carinata (BcaBcaCcaCca) and B rapa (ArAr) to produce hexaploids
(ArArBcaBcaCcaCca)
Approach 2 Use three tetraploids as parents
(a) Cross between B napus (AnAnCnCn) and B carinata (BcaBcaCcaCca) to produce unbalanced
allotetraploids (AnBcaCnCca ndash unreduced gametes gametes with the somatic chromosome
number and cross with B juncea (AjAjBjBj) to obtain allohexaploids (AnAjBcaBjCnCca
(b) Cross between B napus (AnAnCnCn) and B juncea (AjAjBjBj) to produce unbalanced
allotetraploids (AnAjBjCn ndash unreduced gametes) and cross with B carinata (BcaBcaCcaCca) to
obtain allohexaploids (AnAjBcaBjCnCca)
(c) Cross between B carinata (BcaBcaCcaCca) and B juncea (AjAjBjBj) to produce unbalanced
allotetraploids (AjBcaBjCca - ndash unreduced gametes) and cross with B napus (AnAnCnCn) to obtain
allohexaploids (AnAjBcaBjCnCca)
Approach 3 Use three diploids as parents (2X ndash 2X ndash 2X) Cross between B rapa (ArAr) B
nigra (BniBni) and B oleracea (CoCo) sequentially to obtain hexaploid hybrids (ArArBniBniCoCo)
43
Continuehellip
Guijun Yan 2012
44
Applications of Genomic
tools in Super-Domestication
45
Genome Sequencing
Potential methods of sequencing
1 Clone by clone approach
2 Whole genome shotgun approach
3 Combination of the two methods
Till today no of cultivated plants completely sequenced -85
2016- Arachis duranensis
2015- Solanum cumersonii (Wild potato)
(Ref- NCBI)
46
GWAS
It is a study design in which many markers spread across a genome
are genotyped and test a statistical association with a phenotype are
performed locally along the genome
It is also an examination of many common genetic variants in different
individuals to see if any variant is associated with a trait
Used in successfully studying maize sorghum and barley
Method is efficient for large scale low cost genotyping (even with the
minimum number of SNPs)
Cannot be utilized generally because it needs large population size
GWAS identify rare alleles more precisely
If small population we can opt NAM
47
GWAS and whole genome prediction
Xuehui Huang and Bin Han 2014
48
Five high throuput genotyping methods
Xuehui Huang and Bin Han 2014
49
Role of NGS in domestication Capture of novel genes from wild species will be made easier by
understanding the molecular events associated with crop domestication
Re-sequencing of domesticated species can identify low diversity regions
resulting from selection during domestication
To identify gene-specific sequences to aid the cloning of homologues of key
domestication genes from wild relatives
Candidate genes from wild and domesticated plant populations can define
diversity of target genes in wild populations and lead to the discovery of key
genes for important traits by association analysis
NGS of amplicons of large numbers of candidate genes from wild and
domesticated plant populations can define diversity of target genes in
wild populations and lead to the discovery of key genes for important
traits by association analysis
50
NGS
supports the rapid domestication of new plant species and the efficient
identification and capture of novel genetic variation from related species
Allows whole-genome analysis to determine the genetic basis of
phenotypic differences
NGS allows rapid expansion of genomic analysis to investigation of non-
model species
Made rice study easy by related grass
cost-effective method for plant identification
useful strategy to analysis the chloroplast genome sequence from whole-
genome shot-gun sequencing
facilitates managing this diversity and any changes in crop performance
over time due to genetic drift
Patterns of gene expression have been evaluated in hybrids using NGS
51
Two Approaches to Finding Adaptive Genes
Ross-Ibarra et al2007
52
Techniques to re-wild the plants (transgene free)
1 Introgression
breeding
2 Specific
insertion of lost genes
3 Precision
mutagenesis
Palmgren et al2014
53
Process of rewilding
Palmgren et al2014
54
Synthetic biology projects(i) Modifying cereals including wheat to fix atmospheric nitrogen
(ii) Redesigning metabolic pathways to increase the yield of
secondary metabolites or to generate compounds with enhanced
properties
(iii) Transferring the C4 photosynthesis pathway to rice
(iv) Modifying the glycosylation pathway in plants to accommodate
production of therapeutic proteins
(v) Introducing synthetic signal transduction systems that respond to
external cues
55
Synthetic biology projects
Nicholas J Baltes et al 2015
56
Refactoring the N fixation gene cluster from Klebsiella
Karsten Temme et al 2012
57
Continuehellip
Karsten Temme et al 2012
58
Genome editing
Sequence specific nucleases
1 Meganucleases
2 ZFMs ndashZink finger motifs
3 TALENs- Transcription activator like effector nuclease
4 CRISPR CAS-9 ndash Clustered interspaced short palindromic
repeats
qmp4
59Khaoula Belhaj et al 2015
60
Genome modifications achieved in plants using sequence specific nucleases
Type of DNA
modification
Nuclease Delivery
methods
Plants Targets
Trait stacking Megnucleases Bombardment Cotton Intergenic sequence
ZFN Bombardment Maize Transgene
Rewriting host
DNA
Megnucleases Stable integration Maize Intergenic sequence
ZFN Stable integration Soybean Transgene
TALEN Stable integration Barley PAPhy_a
TALEN Agrobacterium
T-DNA (transient)
Oryza sativa SWEET14
TALEN No Protoplasts Arabidopsis
Tobacco
AtTT4 AtADH NbSurB
TALEN Bombardment Wheat MLO
TALEN Protoplasts
Stable integration
Maize PDS IPK1A IPK MRP4
CRISPRCas Protoplasts
Agrobacterium
T-DNA (transient)
Tobacco
Arabidopsis
Sorghum Oryza
OsSWEET14 transgene
61
Type of DNA
modification
Nuclease Delivery
methods
Plants Targets
Rewriting host
DNA large
deletion
Zinc-finger
nuclease
Stable
integration
Tobacco Transgene
CRISPRCas Protoplasts
Stable
integration
Rice Labdane-related
diterpenoid gene
clusters
on Chr 2 4 and 6
Zinc-finger
nuclease
Agrobacterium
T-DNA
(transient)
Tobacco CHN50 transgene
Zinc-finger
nuclease
Whiskers Maize IPK1
CRISPRCas Protoplasts Rice PDS
Controlling gene
expression
TALE repressor
(SRDX)
Stable
integration
Arabidopsis RD29A transgene
Zinc-finger
activator (VP16)
Stable
integration
Brassica
napus
KasII
Continue
62
Application of genome editing1 Introduction of precise and predictable modifications directly in an elite
background
2 multiple traits can be modified simultaneously
3 NHEJ enables gene knockout and targeted modifications
4 Introduction of transgenes at defined loci that promote high-level
transcription and do not interfere with the activity of endogenous genes
5 Site-specific nucleases also allow targeted molecular trait stacking -low
risk of segregation
6 CRISPR Cas is a transgene free approach ndash No regulatory burdens
7 frequency of off-target mutations is well below that caused by chemical
and physical mutagenesis techniques
8 In future can be utilized for metabolic engineering and molecular farming
63
Six-way Venn diagram showing the distribution of shared genefamilies (sequence clusters) among M acuminata P dactyliferaArabidopsis thaliana Oryza sativa Sorghum bicolor and Brachypodiumdistachyon genomes
A DrsquoHont et al Nature 2012 doi101038nature11241
64
Achieving super-DomesticationINTROGRESS-RECREATE-CREATE-DOMESTICATEREDOMESTICATE
bull Mobilizing left out genetic
variation still available in
land races and wild and
weedy species
bull Replaying the evolutionary
tape( resynthesis in
polyploids)
bull Domesticateredomesticate
65
We can admire and emulate how indigenous people still domesticateplants create biodiversity and manage it to sustain their future Thereis no equivalently dynamic or flexible crop breeding in modernagriculture to promote biodiversity we still have much to learn from
traditional knowledge Jan Salick 2012
JR Harlan 1976 Sci American
Divergent
selection for
different
purposes
httpenwikipediaorgwikiImageBrassica_oleracea0jpg
httpwwwcehacukimagesclip_image002_001jpg
Brassica oleracea cabbage
broccoli cauliflower kale
romanesco collards kohrabi
brussels sprouts18
Solanum pimpinellifolium
Solanum lycopersicum19
Domestication of carrot
Queen Annes Lace
Daucus carota
20
Domestication of peanut
A villosulicarpa Hoehne andA Stenosperma (Brazil)
AABB
21
Domestication of Chilli
22
StrawberryBanana
Musa acuminata Musa balbisiana (2n=3x=33) AB genomeFRAGARIA X ANANASSA
Wood berries (F vesca) and Musky strawberries (F moschata)23
Wild SoybeanGlycin soja2n=40
Cultivated soybeanGlycin max 2n=40
24
Increased in domesticate
seed germination
determinate growth
longer pods
bigger (heavier) seeds
earliness
harvest index (seed yieldbiomass)
Decreased in domesticate
seed dispersal
seed dormancy
twining
number of nodes
length of internode
number of pods
photoperiod sensitivity
Common Bean Phaseolus vulgarishttpwwwplantsciencesucdavisedugeptspb143pb143htm
copy Paul Gepts
25
Wild pigeon peaCajanus cajanifolius
Cultivated pigeon pea
26
27
28
WHY OTHERS ARE NOT DOMESTICATED
bull 350000 plants
bull 4629 mammals
bull 9200 birds
bull 10000000 insects
bull 500000 fungi
bull But only 200 plants
bull 15 mammals 5 birds and
bull 2 insects are domesticated
bull Spread of these few species
bull Little change since early
agriculture
bull Repeated domestication of these
species (sometimes)
bull Lack of new species even with
attempts with species known to be
valuable Some groups are good
candidates with no domestication
eg ferns sub-Saharan mammals
29
WHY OTHERS HAVE TO BE DOMESTICATED
New uses and demands ndash biofuels animal feed
medicinalneutraceutical waterclimate food changes
Knowledge why species arenrsquot suitable for domestication or
were not useful
Better understanding of genetics and selection
Sustainability of production
Reliability of production
To meet the food demand in an alternative way
WHY OTHERS HAVE TO BE DOMESTICATED
30
Reallocate biomass for human use
More efficient metabolism and photosynthesis increase
leaf surface decrease root system increase leaf
longevity
Grains more fertile florets larger inflorescence OR
number of ears different ways to get more seed
Larger seeds (automatic vs deliberate selection)
Oil plants increased oil content or more seed
Fiber plants long strong fibers
Domestication syndrome
31
Centers of Domestication
Fuller 2011
32
Centers of Domestication in India
Fuller 2011
33
Indian centers of domestication South India (Deccan centre) Vigna radiate Vigna mungo
Macrotyloma uniflorum (Horse gram) Sataria verticillata S plumila
wheat (macaroni) and two rowed barleybrachiaria ramosa
Orissa (Mahanadi river) Pegion pea Horsegram mung small millets
like Echinichloa Paspalum and Sataria
The Middle Ganges (Harappan civilization) Oryza nivara O rufipogon
and wild sativa Sateria pumia Cannabis sativa even the diffusion of
japonica rice
Saurashtra (Harappan civilization) Eleucine coracana Sateria italica
Paicum spp Brachypodia pearl millet and sorghum Dollicos lablab
The Himalayan foothills of the Punjab region Was a centre of
diversity for Japonica type rice and many temperate fruits and
vegetables
34
DOMESTICATION RELATED TRAITS CONTROLLED BY ONLY FEW GENES
Trait Crops
Plant architecturegrowth habit Ricemaizemilletsbeantomato
Flowering timephotoperiod sensitivity Ricemaizesorghumbeantomato
Fruit size Tomato egg plant
Grain size Ricemaizesorghumbean
Seed dispersal Brassica rice
Inflorescence modification Brassica
Dormancy Bean
35
Genes Crop Moleular and phenotypic function Causative change
Genes identified as controlling domestication traits
tb1 Maize Transcriptional regulator (TCP) plant and
inflorescence structure
regulatory change
tga1 Maize Transcriptional regulator (SBP) seed casing amino acid change
qSH1 Rice Transcriptional regulator (homeodomain)
abscission layer formation shattering
regulatory change
Rc Rice Transcriptional regulator (bHLH) seed color disrupted coding
sequence
sh4 Rice Transcriptional regulator (Myb3) abscission
layer formation shattering
regulatoryamino
acid change
fw22 Tomato Cell signaling fruit weight regulatory change
Q Wheat Transcriptional regulator (AP2) inflorescence
structure
regulatoryamino
acid change
Vrs1 Barley Inflorescence structure Premature stop Amino acid change
Genes of interest in crop domestication and improvement
36
Genes Crop Moleular and phenotypic function Causative change
c1
r1
Maize Transcriptional regulator (MYB) kernel color and
Transcriptional regulator (bHLH) kernel color
regulatory change
sh2 Maize pyrophosphorylase supersweet sweet corn transposon
insertion
su1 Maize isoamylase sweet corn gene amino acid change
brix9-2-5 Tomato Invertase fruit soluble solid content amino acid change
ovate Tomato Unknown fruit shap early stop codon
R Pea Starch branching enzyme seed sugar content transposon
insertion
ehd1 Rice B-type response regulator flowering time amino acid change
hd1 Rice Transcriptional regulator (zinc finger) flowering
time
disrupted coding
sequence
waxy Rice Starch synthase sticky grains intron splicing
defect
rht Wheat Transcriptional regulator (SH2) plant height early stop codon
vrn1 Wheat Transcriptional regulator (MADS) vernalization regulatory change
vrn2 Wheat Transcriptional regulator (ZCCT) vernalization amino acid change
Genes Identified as Controlling Varietal Differences
37
Process of domestication over the yearshellip
Rachel S Meyer 2012
38
Genetic bottleneck
Cultivated crops undergone with narrowing of diversity problem
But this is not the case in weeds
Weeds are the major threats from the beginning of domestication till
today
Problem arose due to
1 Crop mimicry
2 Genetic assimilation
3 Genetic evolution
39
Super- Domestication Vaughn et al (2007) first used the term super-domestication
The processes that lead to a domesticate with dramatically
increased yield that could not be selected in natural environments
from naturally occurring variation without recourse to new
technologies
Super-domesticates can be constructed with knowledge led
approaches based on current needs using the range of new
technologies now available
Plants exploited for continuous selection introduction hybridization
etc which boosted the process of domestication but now the plant
genetic engineering approach is exploiting plants towards synthetic
biology
40
How to use diversitybull Cross two varieties
bull Genome manipulations
Cell fusion hybrids
bull Chromosome manipulation Backcross a new species
bull Generate recombinants Chromosome recombinations
bull Use a new species wild germplasm
Transgenic approach Modern mutagenesis synthetic gene
construction by utilizing green florescent protein genome editing
NGS GWAS sequencing etchelliphelliphelliphelliphellip
41
Reduce genetic bottlenecks through polyplodization
More vigour
SC
Buffering capacity
Heterotic advantage
Enhaced vegetative
charachers
Enhanced oil
Meiotic stability
42
Approach 1 Use one tetraploid and one diploid as parents (4X ndash 2X) followed by the
chromosome doubling of triploid hybrids (Chrom doubling)
a) Cross between B juncea (AjAjBjBj ) and B oleracea (CoCo) to produce hexaploids
(AjAjBjBjCoCo)
(b) Cross between B napus (AnAnCnCn) and B nigra (BniBni) to produce hexaploids
(AnAnBniBniCnCn)
(c) Cross between B carinata (BcaBcaCcaCca) and B rapa (ArAr) to produce hexaploids
(ArArBcaBcaCcaCca)
Approach 2 Use three tetraploids as parents
(a) Cross between B napus (AnAnCnCn) and B carinata (BcaBcaCcaCca) to produce unbalanced
allotetraploids (AnBcaCnCca ndash unreduced gametes gametes with the somatic chromosome
number and cross with B juncea (AjAjBjBj) to obtain allohexaploids (AnAjBcaBjCnCca
(b) Cross between B napus (AnAnCnCn) and B juncea (AjAjBjBj) to produce unbalanced
allotetraploids (AnAjBjCn ndash unreduced gametes) and cross with B carinata (BcaBcaCcaCca) to
obtain allohexaploids (AnAjBcaBjCnCca)
(c) Cross between B carinata (BcaBcaCcaCca) and B juncea (AjAjBjBj) to produce unbalanced
allotetraploids (AjBcaBjCca - ndash unreduced gametes) and cross with B napus (AnAnCnCn) to obtain
allohexaploids (AnAjBcaBjCnCca)
Approach 3 Use three diploids as parents (2X ndash 2X ndash 2X) Cross between B rapa (ArAr) B
nigra (BniBni) and B oleracea (CoCo) sequentially to obtain hexaploid hybrids (ArArBniBniCoCo)
43
Continuehellip
Guijun Yan 2012
44
Applications of Genomic
tools in Super-Domestication
45
Genome Sequencing
Potential methods of sequencing
1 Clone by clone approach
2 Whole genome shotgun approach
3 Combination of the two methods
Till today no of cultivated plants completely sequenced -85
2016- Arachis duranensis
2015- Solanum cumersonii (Wild potato)
(Ref- NCBI)
46
GWAS
It is a study design in which many markers spread across a genome
are genotyped and test a statistical association with a phenotype are
performed locally along the genome
It is also an examination of many common genetic variants in different
individuals to see if any variant is associated with a trait
Used in successfully studying maize sorghum and barley
Method is efficient for large scale low cost genotyping (even with the
minimum number of SNPs)
Cannot be utilized generally because it needs large population size
GWAS identify rare alleles more precisely
If small population we can opt NAM
47
GWAS and whole genome prediction
Xuehui Huang and Bin Han 2014
48
Five high throuput genotyping methods
Xuehui Huang and Bin Han 2014
49
Role of NGS in domestication Capture of novel genes from wild species will be made easier by
understanding the molecular events associated with crop domestication
Re-sequencing of domesticated species can identify low diversity regions
resulting from selection during domestication
To identify gene-specific sequences to aid the cloning of homologues of key
domestication genes from wild relatives
Candidate genes from wild and domesticated plant populations can define
diversity of target genes in wild populations and lead to the discovery of key
genes for important traits by association analysis
NGS of amplicons of large numbers of candidate genes from wild and
domesticated plant populations can define diversity of target genes in
wild populations and lead to the discovery of key genes for important
traits by association analysis
50
NGS
supports the rapid domestication of new plant species and the efficient
identification and capture of novel genetic variation from related species
Allows whole-genome analysis to determine the genetic basis of
phenotypic differences
NGS allows rapid expansion of genomic analysis to investigation of non-
model species
Made rice study easy by related grass
cost-effective method for plant identification
useful strategy to analysis the chloroplast genome sequence from whole-
genome shot-gun sequencing
facilitates managing this diversity and any changes in crop performance
over time due to genetic drift
Patterns of gene expression have been evaluated in hybrids using NGS
51
Two Approaches to Finding Adaptive Genes
Ross-Ibarra et al2007
52
Techniques to re-wild the plants (transgene free)
1 Introgression
breeding
2 Specific
insertion of lost genes
3 Precision
mutagenesis
Palmgren et al2014
53
Process of rewilding
Palmgren et al2014
54
Synthetic biology projects(i) Modifying cereals including wheat to fix atmospheric nitrogen
(ii) Redesigning metabolic pathways to increase the yield of
secondary metabolites or to generate compounds with enhanced
properties
(iii) Transferring the C4 photosynthesis pathway to rice
(iv) Modifying the glycosylation pathway in plants to accommodate
production of therapeutic proteins
(v) Introducing synthetic signal transduction systems that respond to
external cues
55
Synthetic biology projects
Nicholas J Baltes et al 2015
56
Refactoring the N fixation gene cluster from Klebsiella
Karsten Temme et al 2012
57
Continuehellip
Karsten Temme et al 2012
58
Genome editing
Sequence specific nucleases
1 Meganucleases
2 ZFMs ndashZink finger motifs
3 TALENs- Transcription activator like effector nuclease
4 CRISPR CAS-9 ndash Clustered interspaced short palindromic
repeats
qmp4
59Khaoula Belhaj et al 2015
60
Genome modifications achieved in plants using sequence specific nucleases
Type of DNA
modification
Nuclease Delivery
methods
Plants Targets
Trait stacking Megnucleases Bombardment Cotton Intergenic sequence
ZFN Bombardment Maize Transgene
Rewriting host
DNA
Megnucleases Stable integration Maize Intergenic sequence
ZFN Stable integration Soybean Transgene
TALEN Stable integration Barley PAPhy_a
TALEN Agrobacterium
T-DNA (transient)
Oryza sativa SWEET14
TALEN No Protoplasts Arabidopsis
Tobacco
AtTT4 AtADH NbSurB
TALEN Bombardment Wheat MLO
TALEN Protoplasts
Stable integration
Maize PDS IPK1A IPK MRP4
CRISPRCas Protoplasts
Agrobacterium
T-DNA (transient)
Tobacco
Arabidopsis
Sorghum Oryza
OsSWEET14 transgene
61
Type of DNA
modification
Nuclease Delivery
methods
Plants Targets
Rewriting host
DNA large
deletion
Zinc-finger
nuclease
Stable
integration
Tobacco Transgene
CRISPRCas Protoplasts
Stable
integration
Rice Labdane-related
diterpenoid gene
clusters
on Chr 2 4 and 6
Zinc-finger
nuclease
Agrobacterium
T-DNA
(transient)
Tobacco CHN50 transgene
Zinc-finger
nuclease
Whiskers Maize IPK1
CRISPRCas Protoplasts Rice PDS
Controlling gene
expression
TALE repressor
(SRDX)
Stable
integration
Arabidopsis RD29A transgene
Zinc-finger
activator (VP16)
Stable
integration
Brassica
napus
KasII
Continue
62
Application of genome editing1 Introduction of precise and predictable modifications directly in an elite
background
2 multiple traits can be modified simultaneously
3 NHEJ enables gene knockout and targeted modifications
4 Introduction of transgenes at defined loci that promote high-level
transcription and do not interfere with the activity of endogenous genes
5 Site-specific nucleases also allow targeted molecular trait stacking -low
risk of segregation
6 CRISPR Cas is a transgene free approach ndash No regulatory burdens
7 frequency of off-target mutations is well below that caused by chemical
and physical mutagenesis techniques
8 In future can be utilized for metabolic engineering and molecular farming
63
Six-way Venn diagram showing the distribution of shared genefamilies (sequence clusters) among M acuminata P dactyliferaArabidopsis thaliana Oryza sativa Sorghum bicolor and Brachypodiumdistachyon genomes
A DrsquoHont et al Nature 2012 doi101038nature11241
64
Achieving super-DomesticationINTROGRESS-RECREATE-CREATE-DOMESTICATEREDOMESTICATE
bull Mobilizing left out genetic
variation still available in
land races and wild and
weedy species
bull Replaying the evolutionary
tape( resynthesis in
polyploids)
bull Domesticateredomesticate
65
We can admire and emulate how indigenous people still domesticateplants create biodiversity and manage it to sustain their future Thereis no equivalently dynamic or flexible crop breeding in modernagriculture to promote biodiversity we still have much to learn from
traditional knowledge Jan Salick 2012
Solanum pimpinellifolium
Solanum lycopersicum19
Domestication of carrot
Queen Annes Lace
Daucus carota
20
Domestication of peanut
A villosulicarpa Hoehne andA Stenosperma (Brazil)
AABB
21
Domestication of Chilli
22
StrawberryBanana
Musa acuminata Musa balbisiana (2n=3x=33) AB genomeFRAGARIA X ANANASSA
Wood berries (F vesca) and Musky strawberries (F moschata)23
Wild SoybeanGlycin soja2n=40
Cultivated soybeanGlycin max 2n=40
24
Increased in domesticate
seed germination
determinate growth
longer pods
bigger (heavier) seeds
earliness
harvest index (seed yieldbiomass)
Decreased in domesticate
seed dispersal
seed dormancy
twining
number of nodes
length of internode
number of pods
photoperiod sensitivity
Common Bean Phaseolus vulgarishttpwwwplantsciencesucdavisedugeptspb143pb143htm
copy Paul Gepts
25
Wild pigeon peaCajanus cajanifolius
Cultivated pigeon pea
26
27
28
WHY OTHERS ARE NOT DOMESTICATED
bull 350000 plants
bull 4629 mammals
bull 9200 birds
bull 10000000 insects
bull 500000 fungi
bull But only 200 plants
bull 15 mammals 5 birds and
bull 2 insects are domesticated
bull Spread of these few species
bull Little change since early
agriculture
bull Repeated domestication of these
species (sometimes)
bull Lack of new species even with
attempts with species known to be
valuable Some groups are good
candidates with no domestication
eg ferns sub-Saharan mammals
29
WHY OTHERS HAVE TO BE DOMESTICATED
New uses and demands ndash biofuels animal feed
medicinalneutraceutical waterclimate food changes
Knowledge why species arenrsquot suitable for domestication or
were not useful
Better understanding of genetics and selection
Sustainability of production
Reliability of production
To meet the food demand in an alternative way
WHY OTHERS HAVE TO BE DOMESTICATED
30
Reallocate biomass for human use
More efficient metabolism and photosynthesis increase
leaf surface decrease root system increase leaf
longevity
Grains more fertile florets larger inflorescence OR
number of ears different ways to get more seed
Larger seeds (automatic vs deliberate selection)
Oil plants increased oil content or more seed
Fiber plants long strong fibers
Domestication syndrome
31
Centers of Domestication
Fuller 2011
32
Centers of Domestication in India
Fuller 2011
33
Indian centers of domestication South India (Deccan centre) Vigna radiate Vigna mungo
Macrotyloma uniflorum (Horse gram) Sataria verticillata S plumila
wheat (macaroni) and two rowed barleybrachiaria ramosa
Orissa (Mahanadi river) Pegion pea Horsegram mung small millets
like Echinichloa Paspalum and Sataria
The Middle Ganges (Harappan civilization) Oryza nivara O rufipogon
and wild sativa Sateria pumia Cannabis sativa even the diffusion of
japonica rice
Saurashtra (Harappan civilization) Eleucine coracana Sateria italica
Paicum spp Brachypodia pearl millet and sorghum Dollicos lablab
The Himalayan foothills of the Punjab region Was a centre of
diversity for Japonica type rice and many temperate fruits and
vegetables
34
DOMESTICATION RELATED TRAITS CONTROLLED BY ONLY FEW GENES
Trait Crops
Plant architecturegrowth habit Ricemaizemilletsbeantomato
Flowering timephotoperiod sensitivity Ricemaizesorghumbeantomato
Fruit size Tomato egg plant
Grain size Ricemaizesorghumbean
Seed dispersal Brassica rice
Inflorescence modification Brassica
Dormancy Bean
35
Genes Crop Moleular and phenotypic function Causative change
Genes identified as controlling domestication traits
tb1 Maize Transcriptional regulator (TCP) plant and
inflorescence structure
regulatory change
tga1 Maize Transcriptional regulator (SBP) seed casing amino acid change
qSH1 Rice Transcriptional regulator (homeodomain)
abscission layer formation shattering
regulatory change
Rc Rice Transcriptional regulator (bHLH) seed color disrupted coding
sequence
sh4 Rice Transcriptional regulator (Myb3) abscission
layer formation shattering
regulatoryamino
acid change
fw22 Tomato Cell signaling fruit weight regulatory change
Q Wheat Transcriptional regulator (AP2) inflorescence
structure
regulatoryamino
acid change
Vrs1 Barley Inflorescence structure Premature stop Amino acid change
Genes of interest in crop domestication and improvement
36
Genes Crop Moleular and phenotypic function Causative change
c1
r1
Maize Transcriptional regulator (MYB) kernel color and
Transcriptional regulator (bHLH) kernel color
regulatory change
sh2 Maize pyrophosphorylase supersweet sweet corn transposon
insertion
su1 Maize isoamylase sweet corn gene amino acid change
brix9-2-5 Tomato Invertase fruit soluble solid content amino acid change
ovate Tomato Unknown fruit shap early stop codon
R Pea Starch branching enzyme seed sugar content transposon
insertion
ehd1 Rice B-type response regulator flowering time amino acid change
hd1 Rice Transcriptional regulator (zinc finger) flowering
time
disrupted coding
sequence
waxy Rice Starch synthase sticky grains intron splicing
defect
rht Wheat Transcriptional regulator (SH2) plant height early stop codon
vrn1 Wheat Transcriptional regulator (MADS) vernalization regulatory change
vrn2 Wheat Transcriptional regulator (ZCCT) vernalization amino acid change
Genes Identified as Controlling Varietal Differences
37
Process of domestication over the yearshellip
Rachel S Meyer 2012
38
Genetic bottleneck
Cultivated crops undergone with narrowing of diversity problem
But this is not the case in weeds
Weeds are the major threats from the beginning of domestication till
today
Problem arose due to
1 Crop mimicry
2 Genetic assimilation
3 Genetic evolution
39
Super- Domestication Vaughn et al (2007) first used the term super-domestication
The processes that lead to a domesticate with dramatically
increased yield that could not be selected in natural environments
from naturally occurring variation without recourse to new
technologies
Super-domesticates can be constructed with knowledge led
approaches based on current needs using the range of new
technologies now available
Plants exploited for continuous selection introduction hybridization
etc which boosted the process of domestication but now the plant
genetic engineering approach is exploiting plants towards synthetic
biology
40
How to use diversitybull Cross two varieties
bull Genome manipulations
Cell fusion hybrids
bull Chromosome manipulation Backcross a new species
bull Generate recombinants Chromosome recombinations
bull Use a new species wild germplasm
Transgenic approach Modern mutagenesis synthetic gene
construction by utilizing green florescent protein genome editing
NGS GWAS sequencing etchelliphelliphelliphelliphellip
41
Reduce genetic bottlenecks through polyplodization
More vigour
SC
Buffering capacity
Heterotic advantage
Enhaced vegetative
charachers
Enhanced oil
Meiotic stability
42
Approach 1 Use one tetraploid and one diploid as parents (4X ndash 2X) followed by the
chromosome doubling of triploid hybrids (Chrom doubling)
a) Cross between B juncea (AjAjBjBj ) and B oleracea (CoCo) to produce hexaploids
(AjAjBjBjCoCo)
(b) Cross between B napus (AnAnCnCn) and B nigra (BniBni) to produce hexaploids
(AnAnBniBniCnCn)
(c) Cross between B carinata (BcaBcaCcaCca) and B rapa (ArAr) to produce hexaploids
(ArArBcaBcaCcaCca)
Approach 2 Use three tetraploids as parents
(a) Cross between B napus (AnAnCnCn) and B carinata (BcaBcaCcaCca) to produce unbalanced
allotetraploids (AnBcaCnCca ndash unreduced gametes gametes with the somatic chromosome
number and cross with B juncea (AjAjBjBj) to obtain allohexaploids (AnAjBcaBjCnCca
(b) Cross between B napus (AnAnCnCn) and B juncea (AjAjBjBj) to produce unbalanced
allotetraploids (AnAjBjCn ndash unreduced gametes) and cross with B carinata (BcaBcaCcaCca) to
obtain allohexaploids (AnAjBcaBjCnCca)
(c) Cross between B carinata (BcaBcaCcaCca) and B juncea (AjAjBjBj) to produce unbalanced
allotetraploids (AjBcaBjCca - ndash unreduced gametes) and cross with B napus (AnAnCnCn) to obtain
allohexaploids (AnAjBcaBjCnCca)
Approach 3 Use three diploids as parents (2X ndash 2X ndash 2X) Cross between B rapa (ArAr) B
nigra (BniBni) and B oleracea (CoCo) sequentially to obtain hexaploid hybrids (ArArBniBniCoCo)
43
Continuehellip
Guijun Yan 2012
44
Applications of Genomic
tools in Super-Domestication
45
Genome Sequencing
Potential methods of sequencing
1 Clone by clone approach
2 Whole genome shotgun approach
3 Combination of the two methods
Till today no of cultivated plants completely sequenced -85
2016- Arachis duranensis
2015- Solanum cumersonii (Wild potato)
(Ref- NCBI)
46
GWAS
It is a study design in which many markers spread across a genome
are genotyped and test a statistical association with a phenotype are
performed locally along the genome
It is also an examination of many common genetic variants in different
individuals to see if any variant is associated with a trait
Used in successfully studying maize sorghum and barley
Method is efficient for large scale low cost genotyping (even with the
minimum number of SNPs)
Cannot be utilized generally because it needs large population size
GWAS identify rare alleles more precisely
If small population we can opt NAM
47
GWAS and whole genome prediction
Xuehui Huang and Bin Han 2014
48
Five high throuput genotyping methods
Xuehui Huang and Bin Han 2014
49
Role of NGS in domestication Capture of novel genes from wild species will be made easier by
understanding the molecular events associated with crop domestication
Re-sequencing of domesticated species can identify low diversity regions
resulting from selection during domestication
To identify gene-specific sequences to aid the cloning of homologues of key
domestication genes from wild relatives
Candidate genes from wild and domesticated plant populations can define
diversity of target genes in wild populations and lead to the discovery of key
genes for important traits by association analysis
NGS of amplicons of large numbers of candidate genes from wild and
domesticated plant populations can define diversity of target genes in
wild populations and lead to the discovery of key genes for important
traits by association analysis
50
NGS
supports the rapid domestication of new plant species and the efficient
identification and capture of novel genetic variation from related species
Allows whole-genome analysis to determine the genetic basis of
phenotypic differences
NGS allows rapid expansion of genomic analysis to investigation of non-
model species
Made rice study easy by related grass
cost-effective method for plant identification
useful strategy to analysis the chloroplast genome sequence from whole-
genome shot-gun sequencing
facilitates managing this diversity and any changes in crop performance
over time due to genetic drift
Patterns of gene expression have been evaluated in hybrids using NGS
51
Two Approaches to Finding Adaptive Genes
Ross-Ibarra et al2007
52
Techniques to re-wild the plants (transgene free)
1 Introgression
breeding
2 Specific
insertion of lost genes
3 Precision
mutagenesis
Palmgren et al2014
53
Process of rewilding
Palmgren et al2014
54
Synthetic biology projects(i) Modifying cereals including wheat to fix atmospheric nitrogen
(ii) Redesigning metabolic pathways to increase the yield of
secondary metabolites or to generate compounds with enhanced
properties
(iii) Transferring the C4 photosynthesis pathway to rice
(iv) Modifying the glycosylation pathway in plants to accommodate
production of therapeutic proteins
(v) Introducing synthetic signal transduction systems that respond to
external cues
55
Synthetic biology projects
Nicholas J Baltes et al 2015
56
Refactoring the N fixation gene cluster from Klebsiella
Karsten Temme et al 2012
57
Continuehellip
Karsten Temme et al 2012
58
Genome editing
Sequence specific nucleases
1 Meganucleases
2 ZFMs ndashZink finger motifs
3 TALENs- Transcription activator like effector nuclease
4 CRISPR CAS-9 ndash Clustered interspaced short palindromic
repeats
qmp4
59Khaoula Belhaj et al 2015
60
Genome modifications achieved in plants using sequence specific nucleases
Type of DNA
modification
Nuclease Delivery
methods
Plants Targets
Trait stacking Megnucleases Bombardment Cotton Intergenic sequence
ZFN Bombardment Maize Transgene
Rewriting host
DNA
Megnucleases Stable integration Maize Intergenic sequence
ZFN Stable integration Soybean Transgene
TALEN Stable integration Barley PAPhy_a
TALEN Agrobacterium
T-DNA (transient)
Oryza sativa SWEET14
TALEN No Protoplasts Arabidopsis
Tobacco
AtTT4 AtADH NbSurB
TALEN Bombardment Wheat MLO
TALEN Protoplasts
Stable integration
Maize PDS IPK1A IPK MRP4
CRISPRCas Protoplasts
Agrobacterium
T-DNA (transient)
Tobacco
Arabidopsis
Sorghum Oryza
OsSWEET14 transgene
61
Type of DNA
modification
Nuclease Delivery
methods
Plants Targets
Rewriting host
DNA large
deletion
Zinc-finger
nuclease
Stable
integration
Tobacco Transgene
CRISPRCas Protoplasts
Stable
integration
Rice Labdane-related
diterpenoid gene
clusters
on Chr 2 4 and 6
Zinc-finger
nuclease
Agrobacterium
T-DNA
(transient)
Tobacco CHN50 transgene
Zinc-finger
nuclease
Whiskers Maize IPK1
CRISPRCas Protoplasts Rice PDS
Controlling gene
expression
TALE repressor
(SRDX)
Stable
integration
Arabidopsis RD29A transgene
Zinc-finger
activator (VP16)
Stable
integration
Brassica
napus
KasII
Continue
62
Application of genome editing1 Introduction of precise and predictable modifications directly in an elite
background
2 multiple traits can be modified simultaneously
3 NHEJ enables gene knockout and targeted modifications
4 Introduction of transgenes at defined loci that promote high-level
transcription and do not interfere with the activity of endogenous genes
5 Site-specific nucleases also allow targeted molecular trait stacking -low
risk of segregation
6 CRISPR Cas is a transgene free approach ndash No regulatory burdens
7 frequency of off-target mutations is well below that caused by chemical
and physical mutagenesis techniques
8 In future can be utilized for metabolic engineering and molecular farming
63
Six-way Venn diagram showing the distribution of shared genefamilies (sequence clusters) among M acuminata P dactyliferaArabidopsis thaliana Oryza sativa Sorghum bicolor and Brachypodiumdistachyon genomes
A DrsquoHont et al Nature 2012 doi101038nature11241
64
Achieving super-DomesticationINTROGRESS-RECREATE-CREATE-DOMESTICATEREDOMESTICATE
bull Mobilizing left out genetic
variation still available in
land races and wild and
weedy species
bull Replaying the evolutionary
tape( resynthesis in
polyploids)
bull Domesticateredomesticate
65
We can admire and emulate how indigenous people still domesticateplants create biodiversity and manage it to sustain their future Thereis no equivalently dynamic or flexible crop breeding in modernagriculture to promote biodiversity we still have much to learn from
traditional knowledge Jan Salick 2012
Domestication of carrot
Queen Annes Lace
Daucus carota
20
Domestication of peanut
A villosulicarpa Hoehne andA Stenosperma (Brazil)
AABB
21
Domestication of Chilli
22
StrawberryBanana
Musa acuminata Musa balbisiana (2n=3x=33) AB genomeFRAGARIA X ANANASSA
Wood berries (F vesca) and Musky strawberries (F moschata)23
Wild SoybeanGlycin soja2n=40
Cultivated soybeanGlycin max 2n=40
24
Increased in domesticate
seed germination
determinate growth
longer pods
bigger (heavier) seeds
earliness
harvest index (seed yieldbiomass)
Decreased in domesticate
seed dispersal
seed dormancy
twining
number of nodes
length of internode
number of pods
photoperiod sensitivity
Common Bean Phaseolus vulgarishttpwwwplantsciencesucdavisedugeptspb143pb143htm
copy Paul Gepts
25
Wild pigeon peaCajanus cajanifolius
Cultivated pigeon pea
26
27
28
WHY OTHERS ARE NOT DOMESTICATED
bull 350000 plants
bull 4629 mammals
bull 9200 birds
bull 10000000 insects
bull 500000 fungi
bull But only 200 plants
bull 15 mammals 5 birds and
bull 2 insects are domesticated
bull Spread of these few species
bull Little change since early
agriculture
bull Repeated domestication of these
species (sometimes)
bull Lack of new species even with
attempts with species known to be
valuable Some groups are good
candidates with no domestication
eg ferns sub-Saharan mammals
29
WHY OTHERS HAVE TO BE DOMESTICATED
New uses and demands ndash biofuels animal feed
medicinalneutraceutical waterclimate food changes
Knowledge why species arenrsquot suitable for domestication or
were not useful
Better understanding of genetics and selection
Sustainability of production
Reliability of production
To meet the food demand in an alternative way
WHY OTHERS HAVE TO BE DOMESTICATED
30
Reallocate biomass for human use
More efficient metabolism and photosynthesis increase
leaf surface decrease root system increase leaf
longevity
Grains more fertile florets larger inflorescence OR
number of ears different ways to get more seed
Larger seeds (automatic vs deliberate selection)
Oil plants increased oil content or more seed
Fiber plants long strong fibers
Domestication syndrome
31
Centers of Domestication
Fuller 2011
32
Centers of Domestication in India
Fuller 2011
33
Indian centers of domestication South India (Deccan centre) Vigna radiate Vigna mungo
Macrotyloma uniflorum (Horse gram) Sataria verticillata S plumila
wheat (macaroni) and two rowed barleybrachiaria ramosa
Orissa (Mahanadi river) Pegion pea Horsegram mung small millets
like Echinichloa Paspalum and Sataria
The Middle Ganges (Harappan civilization) Oryza nivara O rufipogon
and wild sativa Sateria pumia Cannabis sativa even the diffusion of
japonica rice
Saurashtra (Harappan civilization) Eleucine coracana Sateria italica
Paicum spp Brachypodia pearl millet and sorghum Dollicos lablab
The Himalayan foothills of the Punjab region Was a centre of
diversity for Japonica type rice and many temperate fruits and
vegetables
34
DOMESTICATION RELATED TRAITS CONTROLLED BY ONLY FEW GENES
Trait Crops
Plant architecturegrowth habit Ricemaizemilletsbeantomato
Flowering timephotoperiod sensitivity Ricemaizesorghumbeantomato
Fruit size Tomato egg plant
Grain size Ricemaizesorghumbean
Seed dispersal Brassica rice
Inflorescence modification Brassica
Dormancy Bean
35
Genes Crop Moleular and phenotypic function Causative change
Genes identified as controlling domestication traits
tb1 Maize Transcriptional regulator (TCP) plant and
inflorescence structure
regulatory change
tga1 Maize Transcriptional regulator (SBP) seed casing amino acid change
qSH1 Rice Transcriptional regulator (homeodomain)
abscission layer formation shattering
regulatory change
Rc Rice Transcriptional regulator (bHLH) seed color disrupted coding
sequence
sh4 Rice Transcriptional regulator (Myb3) abscission
layer formation shattering
regulatoryamino
acid change
fw22 Tomato Cell signaling fruit weight regulatory change
Q Wheat Transcriptional regulator (AP2) inflorescence
structure
regulatoryamino
acid change
Vrs1 Barley Inflorescence structure Premature stop Amino acid change
Genes of interest in crop domestication and improvement
36
Genes Crop Moleular and phenotypic function Causative change
c1
r1
Maize Transcriptional regulator (MYB) kernel color and
Transcriptional regulator (bHLH) kernel color
regulatory change
sh2 Maize pyrophosphorylase supersweet sweet corn transposon
insertion
su1 Maize isoamylase sweet corn gene amino acid change
brix9-2-5 Tomato Invertase fruit soluble solid content amino acid change
ovate Tomato Unknown fruit shap early stop codon
R Pea Starch branching enzyme seed sugar content transposon
insertion
ehd1 Rice B-type response regulator flowering time amino acid change
hd1 Rice Transcriptional regulator (zinc finger) flowering
time
disrupted coding
sequence
waxy Rice Starch synthase sticky grains intron splicing
defect
rht Wheat Transcriptional regulator (SH2) plant height early stop codon
vrn1 Wheat Transcriptional regulator (MADS) vernalization regulatory change
vrn2 Wheat Transcriptional regulator (ZCCT) vernalization amino acid change
Genes Identified as Controlling Varietal Differences
37
Process of domestication over the yearshellip
Rachel S Meyer 2012
38
Genetic bottleneck
Cultivated crops undergone with narrowing of diversity problem
But this is not the case in weeds
Weeds are the major threats from the beginning of domestication till
today
Problem arose due to
1 Crop mimicry
2 Genetic assimilation
3 Genetic evolution
39
Super- Domestication Vaughn et al (2007) first used the term super-domestication
The processes that lead to a domesticate with dramatically
increased yield that could not be selected in natural environments
from naturally occurring variation without recourse to new
technologies
Super-domesticates can be constructed with knowledge led
approaches based on current needs using the range of new
technologies now available
Plants exploited for continuous selection introduction hybridization
etc which boosted the process of domestication but now the plant
genetic engineering approach is exploiting plants towards synthetic
biology
40
How to use diversitybull Cross two varieties
bull Genome manipulations
Cell fusion hybrids
bull Chromosome manipulation Backcross a new species
bull Generate recombinants Chromosome recombinations
bull Use a new species wild germplasm
Transgenic approach Modern mutagenesis synthetic gene
construction by utilizing green florescent protein genome editing
NGS GWAS sequencing etchelliphelliphelliphelliphellip
41
Reduce genetic bottlenecks through polyplodization
More vigour
SC
Buffering capacity
Heterotic advantage
Enhaced vegetative
charachers
Enhanced oil
Meiotic stability
42
Approach 1 Use one tetraploid and one diploid as parents (4X ndash 2X) followed by the
chromosome doubling of triploid hybrids (Chrom doubling)
a) Cross between B juncea (AjAjBjBj ) and B oleracea (CoCo) to produce hexaploids
(AjAjBjBjCoCo)
(b) Cross between B napus (AnAnCnCn) and B nigra (BniBni) to produce hexaploids
(AnAnBniBniCnCn)
(c) Cross between B carinata (BcaBcaCcaCca) and B rapa (ArAr) to produce hexaploids
(ArArBcaBcaCcaCca)
Approach 2 Use three tetraploids as parents
(a) Cross between B napus (AnAnCnCn) and B carinata (BcaBcaCcaCca) to produce unbalanced
allotetraploids (AnBcaCnCca ndash unreduced gametes gametes with the somatic chromosome
number and cross with B juncea (AjAjBjBj) to obtain allohexaploids (AnAjBcaBjCnCca
(b) Cross between B napus (AnAnCnCn) and B juncea (AjAjBjBj) to produce unbalanced
allotetraploids (AnAjBjCn ndash unreduced gametes) and cross with B carinata (BcaBcaCcaCca) to
obtain allohexaploids (AnAjBcaBjCnCca)
(c) Cross between B carinata (BcaBcaCcaCca) and B juncea (AjAjBjBj) to produce unbalanced
allotetraploids (AjBcaBjCca - ndash unreduced gametes) and cross with B napus (AnAnCnCn) to obtain
allohexaploids (AnAjBcaBjCnCca)
Approach 3 Use three diploids as parents (2X ndash 2X ndash 2X) Cross between B rapa (ArAr) B
nigra (BniBni) and B oleracea (CoCo) sequentially to obtain hexaploid hybrids (ArArBniBniCoCo)
43
Continuehellip
Guijun Yan 2012
44
Applications of Genomic
tools in Super-Domestication
45
Genome Sequencing
Potential methods of sequencing
1 Clone by clone approach
2 Whole genome shotgun approach
3 Combination of the two methods
Till today no of cultivated plants completely sequenced -85
2016- Arachis duranensis
2015- Solanum cumersonii (Wild potato)
(Ref- NCBI)
46
GWAS
It is a study design in which many markers spread across a genome
are genotyped and test a statistical association with a phenotype are
performed locally along the genome
It is also an examination of many common genetic variants in different
individuals to see if any variant is associated with a trait
Used in successfully studying maize sorghum and barley
Method is efficient for large scale low cost genotyping (even with the
minimum number of SNPs)
Cannot be utilized generally because it needs large population size
GWAS identify rare alleles more precisely
If small population we can opt NAM
47
GWAS and whole genome prediction
Xuehui Huang and Bin Han 2014
48
Five high throuput genotyping methods
Xuehui Huang and Bin Han 2014
49
Role of NGS in domestication Capture of novel genes from wild species will be made easier by
understanding the molecular events associated with crop domestication
Re-sequencing of domesticated species can identify low diversity regions
resulting from selection during domestication
To identify gene-specific sequences to aid the cloning of homologues of key
domestication genes from wild relatives
Candidate genes from wild and domesticated plant populations can define
diversity of target genes in wild populations and lead to the discovery of key
genes for important traits by association analysis
NGS of amplicons of large numbers of candidate genes from wild and
domesticated plant populations can define diversity of target genes in
wild populations and lead to the discovery of key genes for important
traits by association analysis
50
NGS
supports the rapid domestication of new plant species and the efficient
identification and capture of novel genetic variation from related species
Allows whole-genome analysis to determine the genetic basis of
phenotypic differences
NGS allows rapid expansion of genomic analysis to investigation of non-
model species
Made rice study easy by related grass
cost-effective method for plant identification
useful strategy to analysis the chloroplast genome sequence from whole-
genome shot-gun sequencing
facilitates managing this diversity and any changes in crop performance
over time due to genetic drift
Patterns of gene expression have been evaluated in hybrids using NGS
51
Two Approaches to Finding Adaptive Genes
Ross-Ibarra et al2007
52
Techniques to re-wild the plants (transgene free)
1 Introgression
breeding
2 Specific
insertion of lost genes
3 Precision
mutagenesis
Palmgren et al2014
53
Process of rewilding
Palmgren et al2014
54
Synthetic biology projects(i) Modifying cereals including wheat to fix atmospheric nitrogen
(ii) Redesigning metabolic pathways to increase the yield of
secondary metabolites or to generate compounds with enhanced
properties
(iii) Transferring the C4 photosynthesis pathway to rice
(iv) Modifying the glycosylation pathway in plants to accommodate
production of therapeutic proteins
(v) Introducing synthetic signal transduction systems that respond to
external cues
55
Synthetic biology projects
Nicholas J Baltes et al 2015
56
Refactoring the N fixation gene cluster from Klebsiella
Karsten Temme et al 2012
57
Continuehellip
Karsten Temme et al 2012
58
Genome editing
Sequence specific nucleases
1 Meganucleases
2 ZFMs ndashZink finger motifs
3 TALENs- Transcription activator like effector nuclease
4 CRISPR CAS-9 ndash Clustered interspaced short palindromic
repeats
qmp4
59Khaoula Belhaj et al 2015
60
Genome modifications achieved in plants using sequence specific nucleases
Type of DNA
modification
Nuclease Delivery
methods
Plants Targets
Trait stacking Megnucleases Bombardment Cotton Intergenic sequence
ZFN Bombardment Maize Transgene
Rewriting host
DNA
Megnucleases Stable integration Maize Intergenic sequence
ZFN Stable integration Soybean Transgene
TALEN Stable integration Barley PAPhy_a
TALEN Agrobacterium
T-DNA (transient)
Oryza sativa SWEET14
TALEN No Protoplasts Arabidopsis
Tobacco
AtTT4 AtADH NbSurB
TALEN Bombardment Wheat MLO
TALEN Protoplasts
Stable integration
Maize PDS IPK1A IPK MRP4
CRISPRCas Protoplasts
Agrobacterium
T-DNA (transient)
Tobacco
Arabidopsis
Sorghum Oryza
OsSWEET14 transgene
61
Type of DNA
modification
Nuclease Delivery
methods
Plants Targets
Rewriting host
DNA large
deletion
Zinc-finger
nuclease
Stable
integration
Tobacco Transgene
CRISPRCas Protoplasts
Stable
integration
Rice Labdane-related
diterpenoid gene
clusters
on Chr 2 4 and 6
Zinc-finger
nuclease
Agrobacterium
T-DNA
(transient)
Tobacco CHN50 transgene
Zinc-finger
nuclease
Whiskers Maize IPK1
CRISPRCas Protoplasts Rice PDS
Controlling gene
expression
TALE repressor
(SRDX)
Stable
integration
Arabidopsis RD29A transgene
Zinc-finger
activator (VP16)
Stable
integration
Brassica
napus
KasII
Continue
62
Application of genome editing1 Introduction of precise and predictable modifications directly in an elite
background
2 multiple traits can be modified simultaneously
3 NHEJ enables gene knockout and targeted modifications
4 Introduction of transgenes at defined loci that promote high-level
transcription and do not interfere with the activity of endogenous genes
5 Site-specific nucleases also allow targeted molecular trait stacking -low
risk of segregation
6 CRISPR Cas is a transgene free approach ndash No regulatory burdens
7 frequency of off-target mutations is well below that caused by chemical
and physical mutagenesis techniques
8 In future can be utilized for metabolic engineering and molecular farming
63
Six-way Venn diagram showing the distribution of shared genefamilies (sequence clusters) among M acuminata P dactyliferaArabidopsis thaliana Oryza sativa Sorghum bicolor and Brachypodiumdistachyon genomes
A DrsquoHont et al Nature 2012 doi101038nature11241
64
Achieving super-DomesticationINTROGRESS-RECREATE-CREATE-DOMESTICATEREDOMESTICATE
bull Mobilizing left out genetic
variation still available in
land races and wild and
weedy species
bull Replaying the evolutionary
tape( resynthesis in
polyploids)
bull Domesticateredomesticate
65
We can admire and emulate how indigenous people still domesticateplants create biodiversity and manage it to sustain their future Thereis no equivalently dynamic or flexible crop breeding in modernagriculture to promote biodiversity we still have much to learn from
traditional knowledge Jan Salick 2012
Domestication of peanut
A villosulicarpa Hoehne andA Stenosperma (Brazil)
AABB
21
Domestication of Chilli
22
StrawberryBanana
Musa acuminata Musa balbisiana (2n=3x=33) AB genomeFRAGARIA X ANANASSA
Wood berries (F vesca) and Musky strawberries (F moschata)23
Wild SoybeanGlycin soja2n=40
Cultivated soybeanGlycin max 2n=40
24
Increased in domesticate
seed germination
determinate growth
longer pods
bigger (heavier) seeds
earliness
harvest index (seed yieldbiomass)
Decreased in domesticate
seed dispersal
seed dormancy
twining
number of nodes
length of internode
number of pods
photoperiod sensitivity
Common Bean Phaseolus vulgarishttpwwwplantsciencesucdavisedugeptspb143pb143htm
copy Paul Gepts
25
Wild pigeon peaCajanus cajanifolius
Cultivated pigeon pea
26
27
28
WHY OTHERS ARE NOT DOMESTICATED
bull 350000 plants
bull 4629 mammals
bull 9200 birds
bull 10000000 insects
bull 500000 fungi
bull But only 200 plants
bull 15 mammals 5 birds and
bull 2 insects are domesticated
bull Spread of these few species
bull Little change since early
agriculture
bull Repeated domestication of these
species (sometimes)
bull Lack of new species even with
attempts with species known to be
valuable Some groups are good
candidates with no domestication
eg ferns sub-Saharan mammals
29
WHY OTHERS HAVE TO BE DOMESTICATED
New uses and demands ndash biofuels animal feed
medicinalneutraceutical waterclimate food changes
Knowledge why species arenrsquot suitable for domestication or
were not useful
Better understanding of genetics and selection
Sustainability of production
Reliability of production
To meet the food demand in an alternative way
WHY OTHERS HAVE TO BE DOMESTICATED
30
Reallocate biomass for human use
More efficient metabolism and photosynthesis increase
leaf surface decrease root system increase leaf
longevity
Grains more fertile florets larger inflorescence OR
number of ears different ways to get more seed
Larger seeds (automatic vs deliberate selection)
Oil plants increased oil content or more seed
Fiber plants long strong fibers
Domestication syndrome
31
Centers of Domestication
Fuller 2011
32
Centers of Domestication in India
Fuller 2011
33
Indian centers of domestication South India (Deccan centre) Vigna radiate Vigna mungo
Macrotyloma uniflorum (Horse gram) Sataria verticillata S plumila
wheat (macaroni) and two rowed barleybrachiaria ramosa
Orissa (Mahanadi river) Pegion pea Horsegram mung small millets
like Echinichloa Paspalum and Sataria
The Middle Ganges (Harappan civilization) Oryza nivara O rufipogon
and wild sativa Sateria pumia Cannabis sativa even the diffusion of
japonica rice
Saurashtra (Harappan civilization) Eleucine coracana Sateria italica
Paicum spp Brachypodia pearl millet and sorghum Dollicos lablab
The Himalayan foothills of the Punjab region Was a centre of
diversity for Japonica type rice and many temperate fruits and
vegetables
34
DOMESTICATION RELATED TRAITS CONTROLLED BY ONLY FEW GENES
Trait Crops
Plant architecturegrowth habit Ricemaizemilletsbeantomato
Flowering timephotoperiod sensitivity Ricemaizesorghumbeantomato
Fruit size Tomato egg plant
Grain size Ricemaizesorghumbean
Seed dispersal Brassica rice
Inflorescence modification Brassica
Dormancy Bean
35
Genes Crop Moleular and phenotypic function Causative change
Genes identified as controlling domestication traits
tb1 Maize Transcriptional regulator (TCP) plant and
inflorescence structure
regulatory change
tga1 Maize Transcriptional regulator (SBP) seed casing amino acid change
qSH1 Rice Transcriptional regulator (homeodomain)
abscission layer formation shattering
regulatory change
Rc Rice Transcriptional regulator (bHLH) seed color disrupted coding
sequence
sh4 Rice Transcriptional regulator (Myb3) abscission
layer formation shattering
regulatoryamino
acid change
fw22 Tomato Cell signaling fruit weight regulatory change
Q Wheat Transcriptional regulator (AP2) inflorescence
structure
regulatoryamino
acid change
Vrs1 Barley Inflorescence structure Premature stop Amino acid change
Genes of interest in crop domestication and improvement
36
Genes Crop Moleular and phenotypic function Causative change
c1
r1
Maize Transcriptional regulator (MYB) kernel color and
Transcriptional regulator (bHLH) kernel color
regulatory change
sh2 Maize pyrophosphorylase supersweet sweet corn transposon
insertion
su1 Maize isoamylase sweet corn gene amino acid change
brix9-2-5 Tomato Invertase fruit soluble solid content amino acid change
ovate Tomato Unknown fruit shap early stop codon
R Pea Starch branching enzyme seed sugar content transposon
insertion
ehd1 Rice B-type response regulator flowering time amino acid change
hd1 Rice Transcriptional regulator (zinc finger) flowering
time
disrupted coding
sequence
waxy Rice Starch synthase sticky grains intron splicing
defect
rht Wheat Transcriptional regulator (SH2) plant height early stop codon
vrn1 Wheat Transcriptional regulator (MADS) vernalization regulatory change
vrn2 Wheat Transcriptional regulator (ZCCT) vernalization amino acid change
Genes Identified as Controlling Varietal Differences
37
Process of domestication over the yearshellip
Rachel S Meyer 2012
38
Genetic bottleneck
Cultivated crops undergone with narrowing of diversity problem
But this is not the case in weeds
Weeds are the major threats from the beginning of domestication till
today
Problem arose due to
1 Crop mimicry
2 Genetic assimilation
3 Genetic evolution
39
Super- Domestication Vaughn et al (2007) first used the term super-domestication
The processes that lead to a domesticate with dramatically
increased yield that could not be selected in natural environments
from naturally occurring variation without recourse to new
technologies
Super-domesticates can be constructed with knowledge led
approaches based on current needs using the range of new
technologies now available
Plants exploited for continuous selection introduction hybridization
etc which boosted the process of domestication but now the plant
genetic engineering approach is exploiting plants towards synthetic
biology
40
How to use diversitybull Cross two varieties
bull Genome manipulations
Cell fusion hybrids
bull Chromosome manipulation Backcross a new species
bull Generate recombinants Chromosome recombinations
bull Use a new species wild germplasm
Transgenic approach Modern mutagenesis synthetic gene
construction by utilizing green florescent protein genome editing
NGS GWAS sequencing etchelliphelliphelliphelliphellip
41
Reduce genetic bottlenecks through polyplodization
More vigour
SC
Buffering capacity
Heterotic advantage
Enhaced vegetative
charachers
Enhanced oil
Meiotic stability
42
Approach 1 Use one tetraploid and one diploid as parents (4X ndash 2X) followed by the
chromosome doubling of triploid hybrids (Chrom doubling)
a) Cross between B juncea (AjAjBjBj ) and B oleracea (CoCo) to produce hexaploids
(AjAjBjBjCoCo)
(b) Cross between B napus (AnAnCnCn) and B nigra (BniBni) to produce hexaploids
(AnAnBniBniCnCn)
(c) Cross between B carinata (BcaBcaCcaCca) and B rapa (ArAr) to produce hexaploids
(ArArBcaBcaCcaCca)
Approach 2 Use three tetraploids as parents
(a) Cross between B napus (AnAnCnCn) and B carinata (BcaBcaCcaCca) to produce unbalanced
allotetraploids (AnBcaCnCca ndash unreduced gametes gametes with the somatic chromosome
number and cross with B juncea (AjAjBjBj) to obtain allohexaploids (AnAjBcaBjCnCca
(b) Cross between B napus (AnAnCnCn) and B juncea (AjAjBjBj) to produce unbalanced
allotetraploids (AnAjBjCn ndash unreduced gametes) and cross with B carinata (BcaBcaCcaCca) to
obtain allohexaploids (AnAjBcaBjCnCca)
(c) Cross between B carinata (BcaBcaCcaCca) and B juncea (AjAjBjBj) to produce unbalanced
allotetraploids (AjBcaBjCca - ndash unreduced gametes) and cross with B napus (AnAnCnCn) to obtain
allohexaploids (AnAjBcaBjCnCca)
Approach 3 Use three diploids as parents (2X ndash 2X ndash 2X) Cross between B rapa (ArAr) B
nigra (BniBni) and B oleracea (CoCo) sequentially to obtain hexaploid hybrids (ArArBniBniCoCo)
43
Continuehellip
Guijun Yan 2012
44
Applications of Genomic
tools in Super-Domestication
45
Genome Sequencing
Potential methods of sequencing
1 Clone by clone approach
2 Whole genome shotgun approach
3 Combination of the two methods
Till today no of cultivated plants completely sequenced -85
2016- Arachis duranensis
2015- Solanum cumersonii (Wild potato)
(Ref- NCBI)
46
GWAS
It is a study design in which many markers spread across a genome
are genotyped and test a statistical association with a phenotype are
performed locally along the genome
It is also an examination of many common genetic variants in different
individuals to see if any variant is associated with a trait
Used in successfully studying maize sorghum and barley
Method is efficient for large scale low cost genotyping (even with the
minimum number of SNPs)
Cannot be utilized generally because it needs large population size
GWAS identify rare alleles more precisely
If small population we can opt NAM
47
GWAS and whole genome prediction
Xuehui Huang and Bin Han 2014
48
Five high throuput genotyping methods
Xuehui Huang and Bin Han 2014
49
Role of NGS in domestication Capture of novel genes from wild species will be made easier by
understanding the molecular events associated with crop domestication
Re-sequencing of domesticated species can identify low diversity regions
resulting from selection during domestication
To identify gene-specific sequences to aid the cloning of homologues of key
domestication genes from wild relatives
Candidate genes from wild and domesticated plant populations can define
diversity of target genes in wild populations and lead to the discovery of key
genes for important traits by association analysis
NGS of amplicons of large numbers of candidate genes from wild and
domesticated plant populations can define diversity of target genes in
wild populations and lead to the discovery of key genes for important
traits by association analysis
50
NGS
supports the rapid domestication of new plant species and the efficient
identification and capture of novel genetic variation from related species
Allows whole-genome analysis to determine the genetic basis of
phenotypic differences
NGS allows rapid expansion of genomic analysis to investigation of non-
model species
Made rice study easy by related grass
cost-effective method for plant identification
useful strategy to analysis the chloroplast genome sequence from whole-
genome shot-gun sequencing
facilitates managing this diversity and any changes in crop performance
over time due to genetic drift
Patterns of gene expression have been evaluated in hybrids using NGS
51
Two Approaches to Finding Adaptive Genes
Ross-Ibarra et al2007
52
Techniques to re-wild the plants (transgene free)
1 Introgression
breeding
2 Specific
insertion of lost genes
3 Precision
mutagenesis
Palmgren et al2014
53
Process of rewilding
Palmgren et al2014
54
Synthetic biology projects(i) Modifying cereals including wheat to fix atmospheric nitrogen
(ii) Redesigning metabolic pathways to increase the yield of
secondary metabolites or to generate compounds with enhanced
properties
(iii) Transferring the C4 photosynthesis pathway to rice
(iv) Modifying the glycosylation pathway in plants to accommodate
production of therapeutic proteins
(v) Introducing synthetic signal transduction systems that respond to
external cues
55
Synthetic biology projects
Nicholas J Baltes et al 2015
56
Refactoring the N fixation gene cluster from Klebsiella
Karsten Temme et al 2012
57
Continuehellip
Karsten Temme et al 2012
58
Genome editing
Sequence specific nucleases
1 Meganucleases
2 ZFMs ndashZink finger motifs
3 TALENs- Transcription activator like effector nuclease
4 CRISPR CAS-9 ndash Clustered interspaced short palindromic
repeats
qmp4
59Khaoula Belhaj et al 2015
60
Genome modifications achieved in plants using sequence specific nucleases
Type of DNA
modification
Nuclease Delivery
methods
Plants Targets
Trait stacking Megnucleases Bombardment Cotton Intergenic sequence
ZFN Bombardment Maize Transgene
Rewriting host
DNA
Megnucleases Stable integration Maize Intergenic sequence
ZFN Stable integration Soybean Transgene
TALEN Stable integration Barley PAPhy_a
TALEN Agrobacterium
T-DNA (transient)
Oryza sativa SWEET14
TALEN No Protoplasts Arabidopsis
Tobacco
AtTT4 AtADH NbSurB
TALEN Bombardment Wheat MLO
TALEN Protoplasts
Stable integration
Maize PDS IPK1A IPK MRP4
CRISPRCas Protoplasts
Agrobacterium
T-DNA (transient)
Tobacco
Arabidopsis
Sorghum Oryza
OsSWEET14 transgene
61
Type of DNA
modification
Nuclease Delivery
methods
Plants Targets
Rewriting host
DNA large
deletion
Zinc-finger
nuclease
Stable
integration
Tobacco Transgene
CRISPRCas Protoplasts
Stable
integration
Rice Labdane-related
diterpenoid gene
clusters
on Chr 2 4 and 6
Zinc-finger
nuclease
Agrobacterium
T-DNA
(transient)
Tobacco CHN50 transgene
Zinc-finger
nuclease
Whiskers Maize IPK1
CRISPRCas Protoplasts Rice PDS
Controlling gene
expression
TALE repressor
(SRDX)
Stable
integration
Arabidopsis RD29A transgene
Zinc-finger
activator (VP16)
Stable
integration
Brassica
napus
KasII
Continue
62
Application of genome editing1 Introduction of precise and predictable modifications directly in an elite
background
2 multiple traits can be modified simultaneously
3 NHEJ enables gene knockout and targeted modifications
4 Introduction of transgenes at defined loci that promote high-level
transcription and do not interfere with the activity of endogenous genes
5 Site-specific nucleases also allow targeted molecular trait stacking -low
risk of segregation
6 CRISPR Cas is a transgene free approach ndash No regulatory burdens
7 frequency of off-target mutations is well below that caused by chemical
and physical mutagenesis techniques
8 In future can be utilized for metabolic engineering and molecular farming
63
Six-way Venn diagram showing the distribution of shared genefamilies (sequence clusters) among M acuminata P dactyliferaArabidopsis thaliana Oryza sativa Sorghum bicolor and Brachypodiumdistachyon genomes
A DrsquoHont et al Nature 2012 doi101038nature11241
64
Achieving super-DomesticationINTROGRESS-RECREATE-CREATE-DOMESTICATEREDOMESTICATE
bull Mobilizing left out genetic
variation still available in
land races and wild and
weedy species
bull Replaying the evolutionary
tape( resynthesis in
polyploids)
bull Domesticateredomesticate
65
We can admire and emulate how indigenous people still domesticateplants create biodiversity and manage it to sustain their future Thereis no equivalently dynamic or flexible crop breeding in modernagriculture to promote biodiversity we still have much to learn from
traditional knowledge Jan Salick 2012
Domestication of Chilli
22
StrawberryBanana
Musa acuminata Musa balbisiana (2n=3x=33) AB genomeFRAGARIA X ANANASSA
Wood berries (F vesca) and Musky strawberries (F moschata)23
Wild SoybeanGlycin soja2n=40
Cultivated soybeanGlycin max 2n=40
24
Increased in domesticate
seed germination
determinate growth
longer pods
bigger (heavier) seeds
earliness
harvest index (seed yieldbiomass)
Decreased in domesticate
seed dispersal
seed dormancy
twining
number of nodes
length of internode
number of pods
photoperiod sensitivity
Common Bean Phaseolus vulgarishttpwwwplantsciencesucdavisedugeptspb143pb143htm
copy Paul Gepts
25
Wild pigeon peaCajanus cajanifolius
Cultivated pigeon pea
26
27
28
WHY OTHERS ARE NOT DOMESTICATED
bull 350000 plants
bull 4629 mammals
bull 9200 birds
bull 10000000 insects
bull 500000 fungi
bull But only 200 plants
bull 15 mammals 5 birds and
bull 2 insects are domesticated
bull Spread of these few species
bull Little change since early
agriculture
bull Repeated domestication of these
species (sometimes)
bull Lack of new species even with
attempts with species known to be
valuable Some groups are good
candidates with no domestication
eg ferns sub-Saharan mammals
29
WHY OTHERS HAVE TO BE DOMESTICATED
New uses and demands ndash biofuels animal feed
medicinalneutraceutical waterclimate food changes
Knowledge why species arenrsquot suitable for domestication or
were not useful
Better understanding of genetics and selection
Sustainability of production
Reliability of production
To meet the food demand in an alternative way
WHY OTHERS HAVE TO BE DOMESTICATED
30
Reallocate biomass for human use
More efficient metabolism and photosynthesis increase
leaf surface decrease root system increase leaf
longevity
Grains more fertile florets larger inflorescence OR
number of ears different ways to get more seed
Larger seeds (automatic vs deliberate selection)
Oil plants increased oil content or more seed
Fiber plants long strong fibers
Domestication syndrome
31
Centers of Domestication
Fuller 2011
32
Centers of Domestication in India
Fuller 2011
33
Indian centers of domestication South India (Deccan centre) Vigna radiate Vigna mungo
Macrotyloma uniflorum (Horse gram) Sataria verticillata S plumila
wheat (macaroni) and two rowed barleybrachiaria ramosa
Orissa (Mahanadi river) Pegion pea Horsegram mung small millets
like Echinichloa Paspalum and Sataria
The Middle Ganges (Harappan civilization) Oryza nivara O rufipogon
and wild sativa Sateria pumia Cannabis sativa even the diffusion of
japonica rice
Saurashtra (Harappan civilization) Eleucine coracana Sateria italica
Paicum spp Brachypodia pearl millet and sorghum Dollicos lablab
The Himalayan foothills of the Punjab region Was a centre of
diversity for Japonica type rice and many temperate fruits and
vegetables
34
DOMESTICATION RELATED TRAITS CONTROLLED BY ONLY FEW GENES
Trait Crops
Plant architecturegrowth habit Ricemaizemilletsbeantomato
Flowering timephotoperiod sensitivity Ricemaizesorghumbeantomato
Fruit size Tomato egg plant
Grain size Ricemaizesorghumbean
Seed dispersal Brassica rice
Inflorescence modification Brassica
Dormancy Bean
35
Genes Crop Moleular and phenotypic function Causative change
Genes identified as controlling domestication traits
tb1 Maize Transcriptional regulator (TCP) plant and
inflorescence structure
regulatory change
tga1 Maize Transcriptional regulator (SBP) seed casing amino acid change
qSH1 Rice Transcriptional regulator (homeodomain)
abscission layer formation shattering
regulatory change
Rc Rice Transcriptional regulator (bHLH) seed color disrupted coding
sequence
sh4 Rice Transcriptional regulator (Myb3) abscission
layer formation shattering
regulatoryamino
acid change
fw22 Tomato Cell signaling fruit weight regulatory change
Q Wheat Transcriptional regulator (AP2) inflorescence
structure
regulatoryamino
acid change
Vrs1 Barley Inflorescence structure Premature stop Amino acid change
Genes of interest in crop domestication and improvement
36
Genes Crop Moleular and phenotypic function Causative change
c1
r1
Maize Transcriptional regulator (MYB) kernel color and
Transcriptional regulator (bHLH) kernel color
regulatory change
sh2 Maize pyrophosphorylase supersweet sweet corn transposon
insertion
su1 Maize isoamylase sweet corn gene amino acid change
brix9-2-5 Tomato Invertase fruit soluble solid content amino acid change
ovate Tomato Unknown fruit shap early stop codon
R Pea Starch branching enzyme seed sugar content transposon
insertion
ehd1 Rice B-type response regulator flowering time amino acid change
hd1 Rice Transcriptional regulator (zinc finger) flowering
time
disrupted coding
sequence
waxy Rice Starch synthase sticky grains intron splicing
defect
rht Wheat Transcriptional regulator (SH2) plant height early stop codon
vrn1 Wheat Transcriptional regulator (MADS) vernalization regulatory change
vrn2 Wheat Transcriptional regulator (ZCCT) vernalization amino acid change
Genes Identified as Controlling Varietal Differences
37
Process of domestication over the yearshellip
Rachel S Meyer 2012
38
Genetic bottleneck
Cultivated crops undergone with narrowing of diversity problem
But this is not the case in weeds
Weeds are the major threats from the beginning of domestication till
today
Problem arose due to
1 Crop mimicry
2 Genetic assimilation
3 Genetic evolution
39
Super- Domestication Vaughn et al (2007) first used the term super-domestication
The processes that lead to a domesticate with dramatically
increased yield that could not be selected in natural environments
from naturally occurring variation without recourse to new
technologies
Super-domesticates can be constructed with knowledge led
approaches based on current needs using the range of new
technologies now available
Plants exploited for continuous selection introduction hybridization
etc which boosted the process of domestication but now the plant
genetic engineering approach is exploiting plants towards synthetic
biology
40
How to use diversitybull Cross two varieties
bull Genome manipulations
Cell fusion hybrids
bull Chromosome manipulation Backcross a new species
bull Generate recombinants Chromosome recombinations
bull Use a new species wild germplasm
Transgenic approach Modern mutagenesis synthetic gene
construction by utilizing green florescent protein genome editing
NGS GWAS sequencing etchelliphelliphelliphelliphellip
41
Reduce genetic bottlenecks through polyplodization
More vigour
SC
Buffering capacity
Heterotic advantage
Enhaced vegetative
charachers
Enhanced oil
Meiotic stability
42
Approach 1 Use one tetraploid and one diploid as parents (4X ndash 2X) followed by the
chromosome doubling of triploid hybrids (Chrom doubling)
a) Cross between B juncea (AjAjBjBj ) and B oleracea (CoCo) to produce hexaploids
(AjAjBjBjCoCo)
(b) Cross between B napus (AnAnCnCn) and B nigra (BniBni) to produce hexaploids
(AnAnBniBniCnCn)
(c) Cross between B carinata (BcaBcaCcaCca) and B rapa (ArAr) to produce hexaploids
(ArArBcaBcaCcaCca)
Approach 2 Use three tetraploids as parents
(a) Cross between B napus (AnAnCnCn) and B carinata (BcaBcaCcaCca) to produce unbalanced
allotetraploids (AnBcaCnCca ndash unreduced gametes gametes with the somatic chromosome
number and cross with B juncea (AjAjBjBj) to obtain allohexaploids (AnAjBcaBjCnCca
(b) Cross between B napus (AnAnCnCn) and B juncea (AjAjBjBj) to produce unbalanced
allotetraploids (AnAjBjCn ndash unreduced gametes) and cross with B carinata (BcaBcaCcaCca) to
obtain allohexaploids (AnAjBcaBjCnCca)
(c) Cross between B carinata (BcaBcaCcaCca) and B juncea (AjAjBjBj) to produce unbalanced
allotetraploids (AjBcaBjCca - ndash unreduced gametes) and cross with B napus (AnAnCnCn) to obtain
allohexaploids (AnAjBcaBjCnCca)
Approach 3 Use three diploids as parents (2X ndash 2X ndash 2X) Cross between B rapa (ArAr) B
nigra (BniBni) and B oleracea (CoCo) sequentially to obtain hexaploid hybrids (ArArBniBniCoCo)
43
Continuehellip
Guijun Yan 2012
44
Applications of Genomic
tools in Super-Domestication
45
Genome Sequencing
Potential methods of sequencing
1 Clone by clone approach
2 Whole genome shotgun approach
3 Combination of the two methods
Till today no of cultivated plants completely sequenced -85
2016- Arachis duranensis
2015- Solanum cumersonii (Wild potato)
(Ref- NCBI)
46
GWAS
It is a study design in which many markers spread across a genome
are genotyped and test a statistical association with a phenotype are
performed locally along the genome
It is also an examination of many common genetic variants in different
individuals to see if any variant is associated with a trait
Used in successfully studying maize sorghum and barley
Method is efficient for large scale low cost genotyping (even with the
minimum number of SNPs)
Cannot be utilized generally because it needs large population size
GWAS identify rare alleles more precisely
If small population we can opt NAM
47
GWAS and whole genome prediction
Xuehui Huang and Bin Han 2014
48
Five high throuput genotyping methods
Xuehui Huang and Bin Han 2014
49
Role of NGS in domestication Capture of novel genes from wild species will be made easier by
understanding the molecular events associated with crop domestication
Re-sequencing of domesticated species can identify low diversity regions
resulting from selection during domestication
To identify gene-specific sequences to aid the cloning of homologues of key
domestication genes from wild relatives
Candidate genes from wild and domesticated plant populations can define
diversity of target genes in wild populations and lead to the discovery of key
genes for important traits by association analysis
NGS of amplicons of large numbers of candidate genes from wild and
domesticated plant populations can define diversity of target genes in
wild populations and lead to the discovery of key genes for important
traits by association analysis
50
NGS
supports the rapid domestication of new plant species and the efficient
identification and capture of novel genetic variation from related species
Allows whole-genome analysis to determine the genetic basis of
phenotypic differences
NGS allows rapid expansion of genomic analysis to investigation of non-
model species
Made rice study easy by related grass
cost-effective method for plant identification
useful strategy to analysis the chloroplast genome sequence from whole-
genome shot-gun sequencing
facilitates managing this diversity and any changes in crop performance
over time due to genetic drift
Patterns of gene expression have been evaluated in hybrids using NGS
51
Two Approaches to Finding Adaptive Genes
Ross-Ibarra et al2007
52
Techniques to re-wild the plants (transgene free)
1 Introgression
breeding
2 Specific
insertion of lost genes
3 Precision
mutagenesis
Palmgren et al2014
53
Process of rewilding
Palmgren et al2014
54
Synthetic biology projects(i) Modifying cereals including wheat to fix atmospheric nitrogen
(ii) Redesigning metabolic pathways to increase the yield of
secondary metabolites or to generate compounds with enhanced
properties
(iii) Transferring the C4 photosynthesis pathway to rice
(iv) Modifying the glycosylation pathway in plants to accommodate
production of therapeutic proteins
(v) Introducing synthetic signal transduction systems that respond to
external cues
55
Synthetic biology projects
Nicholas J Baltes et al 2015
56
Refactoring the N fixation gene cluster from Klebsiella
Karsten Temme et al 2012
57
Continuehellip
Karsten Temme et al 2012
58
Genome editing
Sequence specific nucleases
1 Meganucleases
2 ZFMs ndashZink finger motifs
3 TALENs- Transcription activator like effector nuclease
4 CRISPR CAS-9 ndash Clustered interspaced short palindromic
repeats
qmp4
59Khaoula Belhaj et al 2015
60
Genome modifications achieved in plants using sequence specific nucleases
Type of DNA
modification
Nuclease Delivery
methods
Plants Targets
Trait stacking Megnucleases Bombardment Cotton Intergenic sequence
ZFN Bombardment Maize Transgene
Rewriting host
DNA
Megnucleases Stable integration Maize Intergenic sequence
ZFN Stable integration Soybean Transgene
TALEN Stable integration Barley PAPhy_a
TALEN Agrobacterium
T-DNA (transient)
Oryza sativa SWEET14
TALEN No Protoplasts Arabidopsis
Tobacco
AtTT4 AtADH NbSurB
TALEN Bombardment Wheat MLO
TALEN Protoplasts
Stable integration
Maize PDS IPK1A IPK MRP4
CRISPRCas Protoplasts
Agrobacterium
T-DNA (transient)
Tobacco
Arabidopsis
Sorghum Oryza
OsSWEET14 transgene
61
Type of DNA
modification
Nuclease Delivery
methods
Plants Targets
Rewriting host
DNA large
deletion
Zinc-finger
nuclease
Stable
integration
Tobacco Transgene
CRISPRCas Protoplasts
Stable
integration
Rice Labdane-related
diterpenoid gene
clusters
on Chr 2 4 and 6
Zinc-finger
nuclease
Agrobacterium
T-DNA
(transient)
Tobacco CHN50 transgene
Zinc-finger
nuclease
Whiskers Maize IPK1
CRISPRCas Protoplasts Rice PDS
Controlling gene
expression
TALE repressor
(SRDX)
Stable
integration
Arabidopsis RD29A transgene
Zinc-finger
activator (VP16)
Stable
integration
Brassica
napus
KasII
Continue
62
Application of genome editing1 Introduction of precise and predictable modifications directly in an elite
background
2 multiple traits can be modified simultaneously
3 NHEJ enables gene knockout and targeted modifications
4 Introduction of transgenes at defined loci that promote high-level
transcription and do not interfere with the activity of endogenous genes
5 Site-specific nucleases also allow targeted molecular trait stacking -low
risk of segregation
6 CRISPR Cas is a transgene free approach ndash No regulatory burdens
7 frequency of off-target mutations is well below that caused by chemical
and physical mutagenesis techniques
8 In future can be utilized for metabolic engineering and molecular farming
63
Six-way Venn diagram showing the distribution of shared genefamilies (sequence clusters) among M acuminata P dactyliferaArabidopsis thaliana Oryza sativa Sorghum bicolor and Brachypodiumdistachyon genomes
A DrsquoHont et al Nature 2012 doi101038nature11241
64
Achieving super-DomesticationINTROGRESS-RECREATE-CREATE-DOMESTICATEREDOMESTICATE
bull Mobilizing left out genetic
variation still available in
land races and wild and
weedy species
bull Replaying the evolutionary
tape( resynthesis in
polyploids)
bull Domesticateredomesticate
65
We can admire and emulate how indigenous people still domesticateplants create biodiversity and manage it to sustain their future Thereis no equivalently dynamic or flexible crop breeding in modernagriculture to promote biodiversity we still have much to learn from
traditional knowledge Jan Salick 2012
StrawberryBanana
Musa acuminata Musa balbisiana (2n=3x=33) AB genomeFRAGARIA X ANANASSA
Wood berries (F vesca) and Musky strawberries (F moschata)23
Wild SoybeanGlycin soja2n=40
Cultivated soybeanGlycin max 2n=40
24
Increased in domesticate
seed germination
determinate growth
longer pods
bigger (heavier) seeds
earliness
harvest index (seed yieldbiomass)
Decreased in domesticate
seed dispersal
seed dormancy
twining
number of nodes
length of internode
number of pods
photoperiod sensitivity
Common Bean Phaseolus vulgarishttpwwwplantsciencesucdavisedugeptspb143pb143htm
copy Paul Gepts
25
Wild pigeon peaCajanus cajanifolius
Cultivated pigeon pea
26
27
28
WHY OTHERS ARE NOT DOMESTICATED
bull 350000 plants
bull 4629 mammals
bull 9200 birds
bull 10000000 insects
bull 500000 fungi
bull But only 200 plants
bull 15 mammals 5 birds and
bull 2 insects are domesticated
bull Spread of these few species
bull Little change since early
agriculture
bull Repeated domestication of these
species (sometimes)
bull Lack of new species even with
attempts with species known to be
valuable Some groups are good
candidates with no domestication
eg ferns sub-Saharan mammals
29
WHY OTHERS HAVE TO BE DOMESTICATED
New uses and demands ndash biofuels animal feed
medicinalneutraceutical waterclimate food changes
Knowledge why species arenrsquot suitable for domestication or
were not useful
Better understanding of genetics and selection
Sustainability of production
Reliability of production
To meet the food demand in an alternative way
WHY OTHERS HAVE TO BE DOMESTICATED
30
Reallocate biomass for human use
More efficient metabolism and photosynthesis increase
leaf surface decrease root system increase leaf
longevity
Grains more fertile florets larger inflorescence OR
number of ears different ways to get more seed
Larger seeds (automatic vs deliberate selection)
Oil plants increased oil content or more seed
Fiber plants long strong fibers
Domestication syndrome
31
Centers of Domestication
Fuller 2011
32
Centers of Domestication in India
Fuller 2011
33
Indian centers of domestication South India (Deccan centre) Vigna radiate Vigna mungo
Macrotyloma uniflorum (Horse gram) Sataria verticillata S plumila
wheat (macaroni) and two rowed barleybrachiaria ramosa
Orissa (Mahanadi river) Pegion pea Horsegram mung small millets
like Echinichloa Paspalum and Sataria
The Middle Ganges (Harappan civilization) Oryza nivara O rufipogon
and wild sativa Sateria pumia Cannabis sativa even the diffusion of
japonica rice
Saurashtra (Harappan civilization) Eleucine coracana Sateria italica
Paicum spp Brachypodia pearl millet and sorghum Dollicos lablab
The Himalayan foothills of the Punjab region Was a centre of
diversity for Japonica type rice and many temperate fruits and
vegetables
34
DOMESTICATION RELATED TRAITS CONTROLLED BY ONLY FEW GENES
Trait Crops
Plant architecturegrowth habit Ricemaizemilletsbeantomato
Flowering timephotoperiod sensitivity Ricemaizesorghumbeantomato
Fruit size Tomato egg plant
Grain size Ricemaizesorghumbean
Seed dispersal Brassica rice
Inflorescence modification Brassica
Dormancy Bean
35
Genes Crop Moleular and phenotypic function Causative change
Genes identified as controlling domestication traits
tb1 Maize Transcriptional regulator (TCP) plant and
inflorescence structure
regulatory change
tga1 Maize Transcriptional regulator (SBP) seed casing amino acid change
qSH1 Rice Transcriptional regulator (homeodomain)
abscission layer formation shattering
regulatory change
Rc Rice Transcriptional regulator (bHLH) seed color disrupted coding
sequence
sh4 Rice Transcriptional regulator (Myb3) abscission
layer formation shattering
regulatoryamino
acid change
fw22 Tomato Cell signaling fruit weight regulatory change
Q Wheat Transcriptional regulator (AP2) inflorescence
structure
regulatoryamino
acid change
Vrs1 Barley Inflorescence structure Premature stop Amino acid change
Genes of interest in crop domestication and improvement
36
Genes Crop Moleular and phenotypic function Causative change
c1
r1
Maize Transcriptional regulator (MYB) kernel color and
Transcriptional regulator (bHLH) kernel color
regulatory change
sh2 Maize pyrophosphorylase supersweet sweet corn transposon
insertion
su1 Maize isoamylase sweet corn gene amino acid change
brix9-2-5 Tomato Invertase fruit soluble solid content amino acid change
ovate Tomato Unknown fruit shap early stop codon
R Pea Starch branching enzyme seed sugar content transposon
insertion
ehd1 Rice B-type response regulator flowering time amino acid change
hd1 Rice Transcriptional regulator (zinc finger) flowering
time
disrupted coding
sequence
waxy Rice Starch synthase sticky grains intron splicing
defect
rht Wheat Transcriptional regulator (SH2) plant height early stop codon
vrn1 Wheat Transcriptional regulator (MADS) vernalization regulatory change
vrn2 Wheat Transcriptional regulator (ZCCT) vernalization amino acid change
Genes Identified as Controlling Varietal Differences
37
Process of domestication over the yearshellip
Rachel S Meyer 2012
38
Genetic bottleneck
Cultivated crops undergone with narrowing of diversity problem
But this is not the case in weeds
Weeds are the major threats from the beginning of domestication till
today
Problem arose due to
1 Crop mimicry
2 Genetic assimilation
3 Genetic evolution
39
Super- Domestication Vaughn et al (2007) first used the term super-domestication
The processes that lead to a domesticate with dramatically
increased yield that could not be selected in natural environments
from naturally occurring variation without recourse to new
technologies
Super-domesticates can be constructed with knowledge led
approaches based on current needs using the range of new
technologies now available
Plants exploited for continuous selection introduction hybridization
etc which boosted the process of domestication but now the plant
genetic engineering approach is exploiting plants towards synthetic
biology
40
How to use diversitybull Cross two varieties
bull Genome manipulations
Cell fusion hybrids
bull Chromosome manipulation Backcross a new species
bull Generate recombinants Chromosome recombinations
bull Use a new species wild germplasm
Transgenic approach Modern mutagenesis synthetic gene
construction by utilizing green florescent protein genome editing
NGS GWAS sequencing etchelliphelliphelliphelliphellip
41
Reduce genetic bottlenecks through polyplodization
More vigour
SC
Buffering capacity
Heterotic advantage
Enhaced vegetative
charachers
Enhanced oil
Meiotic stability
42
Approach 1 Use one tetraploid and one diploid as parents (4X ndash 2X) followed by the
chromosome doubling of triploid hybrids (Chrom doubling)
a) Cross between B juncea (AjAjBjBj ) and B oleracea (CoCo) to produce hexaploids
(AjAjBjBjCoCo)
(b) Cross between B napus (AnAnCnCn) and B nigra (BniBni) to produce hexaploids
(AnAnBniBniCnCn)
(c) Cross between B carinata (BcaBcaCcaCca) and B rapa (ArAr) to produce hexaploids
(ArArBcaBcaCcaCca)
Approach 2 Use three tetraploids as parents
(a) Cross between B napus (AnAnCnCn) and B carinata (BcaBcaCcaCca) to produce unbalanced
allotetraploids (AnBcaCnCca ndash unreduced gametes gametes with the somatic chromosome
number and cross with B juncea (AjAjBjBj) to obtain allohexaploids (AnAjBcaBjCnCca
(b) Cross between B napus (AnAnCnCn) and B juncea (AjAjBjBj) to produce unbalanced
allotetraploids (AnAjBjCn ndash unreduced gametes) and cross with B carinata (BcaBcaCcaCca) to
obtain allohexaploids (AnAjBcaBjCnCca)
(c) Cross between B carinata (BcaBcaCcaCca) and B juncea (AjAjBjBj) to produce unbalanced
allotetraploids (AjBcaBjCca - ndash unreduced gametes) and cross with B napus (AnAnCnCn) to obtain
allohexaploids (AnAjBcaBjCnCca)
Approach 3 Use three diploids as parents (2X ndash 2X ndash 2X) Cross between B rapa (ArAr) B
nigra (BniBni) and B oleracea (CoCo) sequentially to obtain hexaploid hybrids (ArArBniBniCoCo)
43
Continuehellip
Guijun Yan 2012
44
Applications of Genomic
tools in Super-Domestication
45
Genome Sequencing
Potential methods of sequencing
1 Clone by clone approach
2 Whole genome shotgun approach
3 Combination of the two methods
Till today no of cultivated plants completely sequenced -85
2016- Arachis duranensis
2015- Solanum cumersonii (Wild potato)
(Ref- NCBI)
46
GWAS
It is a study design in which many markers spread across a genome
are genotyped and test a statistical association with a phenotype are
performed locally along the genome
It is also an examination of many common genetic variants in different
individuals to see if any variant is associated with a trait
Used in successfully studying maize sorghum and barley
Method is efficient for large scale low cost genotyping (even with the
minimum number of SNPs)
Cannot be utilized generally because it needs large population size
GWAS identify rare alleles more precisely
If small population we can opt NAM
47
GWAS and whole genome prediction
Xuehui Huang and Bin Han 2014
48
Five high throuput genotyping methods
Xuehui Huang and Bin Han 2014
49
Role of NGS in domestication Capture of novel genes from wild species will be made easier by
understanding the molecular events associated with crop domestication
Re-sequencing of domesticated species can identify low diversity regions
resulting from selection during domestication
To identify gene-specific sequences to aid the cloning of homologues of key
domestication genes from wild relatives
Candidate genes from wild and domesticated plant populations can define
diversity of target genes in wild populations and lead to the discovery of key
genes for important traits by association analysis
NGS of amplicons of large numbers of candidate genes from wild and
domesticated plant populations can define diversity of target genes in
wild populations and lead to the discovery of key genes for important
traits by association analysis
50
NGS
supports the rapid domestication of new plant species and the efficient
identification and capture of novel genetic variation from related species
Allows whole-genome analysis to determine the genetic basis of
phenotypic differences
NGS allows rapid expansion of genomic analysis to investigation of non-
model species
Made rice study easy by related grass
cost-effective method for plant identification
useful strategy to analysis the chloroplast genome sequence from whole-
genome shot-gun sequencing
facilitates managing this diversity and any changes in crop performance
over time due to genetic drift
Patterns of gene expression have been evaluated in hybrids using NGS
51
Two Approaches to Finding Adaptive Genes
Ross-Ibarra et al2007
52
Techniques to re-wild the plants (transgene free)
1 Introgression
breeding
2 Specific
insertion of lost genes
3 Precision
mutagenesis
Palmgren et al2014
53
Process of rewilding
Palmgren et al2014
54
Synthetic biology projects(i) Modifying cereals including wheat to fix atmospheric nitrogen
(ii) Redesigning metabolic pathways to increase the yield of
secondary metabolites or to generate compounds with enhanced
properties
(iii) Transferring the C4 photosynthesis pathway to rice
(iv) Modifying the glycosylation pathway in plants to accommodate
production of therapeutic proteins
(v) Introducing synthetic signal transduction systems that respond to
external cues
55
Synthetic biology projects
Nicholas J Baltes et al 2015
56
Refactoring the N fixation gene cluster from Klebsiella
Karsten Temme et al 2012
57
Continuehellip
Karsten Temme et al 2012
58
Genome editing
Sequence specific nucleases
1 Meganucleases
2 ZFMs ndashZink finger motifs
3 TALENs- Transcription activator like effector nuclease
4 CRISPR CAS-9 ndash Clustered interspaced short palindromic
repeats
qmp4
59Khaoula Belhaj et al 2015
60
Genome modifications achieved in plants using sequence specific nucleases
Type of DNA
modification
Nuclease Delivery
methods
Plants Targets
Trait stacking Megnucleases Bombardment Cotton Intergenic sequence
ZFN Bombardment Maize Transgene
Rewriting host
DNA
Megnucleases Stable integration Maize Intergenic sequence
ZFN Stable integration Soybean Transgene
TALEN Stable integration Barley PAPhy_a
TALEN Agrobacterium
T-DNA (transient)
Oryza sativa SWEET14
TALEN No Protoplasts Arabidopsis
Tobacco
AtTT4 AtADH NbSurB
TALEN Bombardment Wheat MLO
TALEN Protoplasts
Stable integration
Maize PDS IPK1A IPK MRP4
CRISPRCas Protoplasts
Agrobacterium
T-DNA (transient)
Tobacco
Arabidopsis
Sorghum Oryza
OsSWEET14 transgene
61
Type of DNA
modification
Nuclease Delivery
methods
Plants Targets
Rewriting host
DNA large
deletion
Zinc-finger
nuclease
Stable
integration
Tobacco Transgene
CRISPRCas Protoplasts
Stable
integration
Rice Labdane-related
diterpenoid gene
clusters
on Chr 2 4 and 6
Zinc-finger
nuclease
Agrobacterium
T-DNA
(transient)
Tobacco CHN50 transgene
Zinc-finger
nuclease
Whiskers Maize IPK1
CRISPRCas Protoplasts Rice PDS
Controlling gene
expression
TALE repressor
(SRDX)
Stable
integration
Arabidopsis RD29A transgene
Zinc-finger
activator (VP16)
Stable
integration
Brassica
napus
KasII
Continue
62
Application of genome editing1 Introduction of precise and predictable modifications directly in an elite
background
2 multiple traits can be modified simultaneously
3 NHEJ enables gene knockout and targeted modifications
4 Introduction of transgenes at defined loci that promote high-level
transcription and do not interfere with the activity of endogenous genes
5 Site-specific nucleases also allow targeted molecular trait stacking -low
risk of segregation
6 CRISPR Cas is a transgene free approach ndash No regulatory burdens
7 frequency of off-target mutations is well below that caused by chemical
and physical mutagenesis techniques
8 In future can be utilized for metabolic engineering and molecular farming
63
Six-way Venn diagram showing the distribution of shared genefamilies (sequence clusters) among M acuminata P dactyliferaArabidopsis thaliana Oryza sativa Sorghum bicolor and Brachypodiumdistachyon genomes
A DrsquoHont et al Nature 2012 doi101038nature11241
64
Achieving super-DomesticationINTROGRESS-RECREATE-CREATE-DOMESTICATEREDOMESTICATE
bull Mobilizing left out genetic
variation still available in
land races and wild and
weedy species
bull Replaying the evolutionary
tape( resynthesis in
polyploids)
bull Domesticateredomesticate
65
We can admire and emulate how indigenous people still domesticateplants create biodiversity and manage it to sustain their future Thereis no equivalently dynamic or flexible crop breeding in modernagriculture to promote biodiversity we still have much to learn from
traditional knowledge Jan Salick 2012
Wild SoybeanGlycin soja2n=40
Cultivated soybeanGlycin max 2n=40
24
Increased in domesticate
seed germination
determinate growth
longer pods
bigger (heavier) seeds
earliness
harvest index (seed yieldbiomass)
Decreased in domesticate
seed dispersal
seed dormancy
twining
number of nodes
length of internode
number of pods
photoperiod sensitivity
Common Bean Phaseolus vulgarishttpwwwplantsciencesucdavisedugeptspb143pb143htm
copy Paul Gepts
25
Wild pigeon peaCajanus cajanifolius
Cultivated pigeon pea
26
27
28
WHY OTHERS ARE NOT DOMESTICATED
bull 350000 plants
bull 4629 mammals
bull 9200 birds
bull 10000000 insects
bull 500000 fungi
bull But only 200 plants
bull 15 mammals 5 birds and
bull 2 insects are domesticated
bull Spread of these few species
bull Little change since early
agriculture
bull Repeated domestication of these
species (sometimes)
bull Lack of new species even with
attempts with species known to be
valuable Some groups are good
candidates with no domestication
eg ferns sub-Saharan mammals
29
WHY OTHERS HAVE TO BE DOMESTICATED
New uses and demands ndash biofuels animal feed
medicinalneutraceutical waterclimate food changes
Knowledge why species arenrsquot suitable for domestication or
were not useful
Better understanding of genetics and selection
Sustainability of production
Reliability of production
To meet the food demand in an alternative way
WHY OTHERS HAVE TO BE DOMESTICATED
30
Reallocate biomass for human use
More efficient metabolism and photosynthesis increase
leaf surface decrease root system increase leaf
longevity
Grains more fertile florets larger inflorescence OR
number of ears different ways to get more seed
Larger seeds (automatic vs deliberate selection)
Oil plants increased oil content or more seed
Fiber plants long strong fibers
Domestication syndrome
31
Centers of Domestication
Fuller 2011
32
Centers of Domestication in India
Fuller 2011
33
Indian centers of domestication South India (Deccan centre) Vigna radiate Vigna mungo
Macrotyloma uniflorum (Horse gram) Sataria verticillata S plumila
wheat (macaroni) and two rowed barleybrachiaria ramosa
Orissa (Mahanadi river) Pegion pea Horsegram mung small millets
like Echinichloa Paspalum and Sataria
The Middle Ganges (Harappan civilization) Oryza nivara O rufipogon
and wild sativa Sateria pumia Cannabis sativa even the diffusion of
japonica rice
Saurashtra (Harappan civilization) Eleucine coracana Sateria italica
Paicum spp Brachypodia pearl millet and sorghum Dollicos lablab
The Himalayan foothills of the Punjab region Was a centre of
diversity for Japonica type rice and many temperate fruits and
vegetables
34
DOMESTICATION RELATED TRAITS CONTROLLED BY ONLY FEW GENES
Trait Crops
Plant architecturegrowth habit Ricemaizemilletsbeantomato
Flowering timephotoperiod sensitivity Ricemaizesorghumbeantomato
Fruit size Tomato egg plant
Grain size Ricemaizesorghumbean
Seed dispersal Brassica rice
Inflorescence modification Brassica
Dormancy Bean
35
Genes Crop Moleular and phenotypic function Causative change
Genes identified as controlling domestication traits
tb1 Maize Transcriptional regulator (TCP) plant and
inflorescence structure
regulatory change
tga1 Maize Transcriptional regulator (SBP) seed casing amino acid change
qSH1 Rice Transcriptional regulator (homeodomain)
abscission layer formation shattering
regulatory change
Rc Rice Transcriptional regulator (bHLH) seed color disrupted coding
sequence
sh4 Rice Transcriptional regulator (Myb3) abscission
layer formation shattering
regulatoryamino
acid change
fw22 Tomato Cell signaling fruit weight regulatory change
Q Wheat Transcriptional regulator (AP2) inflorescence
structure
regulatoryamino
acid change
Vrs1 Barley Inflorescence structure Premature stop Amino acid change
Genes of interest in crop domestication and improvement
36
Genes Crop Moleular and phenotypic function Causative change
c1
r1
Maize Transcriptional regulator (MYB) kernel color and
Transcriptional regulator (bHLH) kernel color
regulatory change
sh2 Maize pyrophosphorylase supersweet sweet corn transposon
insertion
su1 Maize isoamylase sweet corn gene amino acid change
brix9-2-5 Tomato Invertase fruit soluble solid content amino acid change
ovate Tomato Unknown fruit shap early stop codon
R Pea Starch branching enzyme seed sugar content transposon
insertion
ehd1 Rice B-type response regulator flowering time amino acid change
hd1 Rice Transcriptional regulator (zinc finger) flowering
time
disrupted coding
sequence
waxy Rice Starch synthase sticky grains intron splicing
defect
rht Wheat Transcriptional regulator (SH2) plant height early stop codon
vrn1 Wheat Transcriptional regulator (MADS) vernalization regulatory change
vrn2 Wheat Transcriptional regulator (ZCCT) vernalization amino acid change
Genes Identified as Controlling Varietal Differences
37
Process of domestication over the yearshellip
Rachel S Meyer 2012
38
Genetic bottleneck
Cultivated crops undergone with narrowing of diversity problem
But this is not the case in weeds
Weeds are the major threats from the beginning of domestication till
today
Problem arose due to
1 Crop mimicry
2 Genetic assimilation
3 Genetic evolution
39
Super- Domestication Vaughn et al (2007) first used the term super-domestication
The processes that lead to a domesticate with dramatically
increased yield that could not be selected in natural environments
from naturally occurring variation without recourse to new
technologies
Super-domesticates can be constructed with knowledge led
approaches based on current needs using the range of new
technologies now available
Plants exploited for continuous selection introduction hybridization
etc which boosted the process of domestication but now the plant
genetic engineering approach is exploiting plants towards synthetic
biology
40
How to use diversitybull Cross two varieties
bull Genome manipulations
Cell fusion hybrids
bull Chromosome manipulation Backcross a new species
bull Generate recombinants Chromosome recombinations
bull Use a new species wild germplasm
Transgenic approach Modern mutagenesis synthetic gene
construction by utilizing green florescent protein genome editing
NGS GWAS sequencing etchelliphelliphelliphelliphellip
41
Reduce genetic bottlenecks through polyplodization
More vigour
SC
Buffering capacity
Heterotic advantage
Enhaced vegetative
charachers
Enhanced oil
Meiotic stability
42
Approach 1 Use one tetraploid and one diploid as parents (4X ndash 2X) followed by the
chromosome doubling of triploid hybrids (Chrom doubling)
a) Cross between B juncea (AjAjBjBj ) and B oleracea (CoCo) to produce hexaploids
(AjAjBjBjCoCo)
(b) Cross between B napus (AnAnCnCn) and B nigra (BniBni) to produce hexaploids
(AnAnBniBniCnCn)
(c) Cross between B carinata (BcaBcaCcaCca) and B rapa (ArAr) to produce hexaploids
(ArArBcaBcaCcaCca)
Approach 2 Use three tetraploids as parents
(a) Cross between B napus (AnAnCnCn) and B carinata (BcaBcaCcaCca) to produce unbalanced
allotetraploids (AnBcaCnCca ndash unreduced gametes gametes with the somatic chromosome
number and cross with B juncea (AjAjBjBj) to obtain allohexaploids (AnAjBcaBjCnCca
(b) Cross between B napus (AnAnCnCn) and B juncea (AjAjBjBj) to produce unbalanced
allotetraploids (AnAjBjCn ndash unreduced gametes) and cross with B carinata (BcaBcaCcaCca) to
obtain allohexaploids (AnAjBcaBjCnCca)
(c) Cross between B carinata (BcaBcaCcaCca) and B juncea (AjAjBjBj) to produce unbalanced
allotetraploids (AjBcaBjCca - ndash unreduced gametes) and cross with B napus (AnAnCnCn) to obtain
allohexaploids (AnAjBcaBjCnCca)
Approach 3 Use three diploids as parents (2X ndash 2X ndash 2X) Cross between B rapa (ArAr) B
nigra (BniBni) and B oleracea (CoCo) sequentially to obtain hexaploid hybrids (ArArBniBniCoCo)
43
Continuehellip
Guijun Yan 2012
44
Applications of Genomic
tools in Super-Domestication
45
Genome Sequencing
Potential methods of sequencing
1 Clone by clone approach
2 Whole genome shotgun approach
3 Combination of the two methods
Till today no of cultivated plants completely sequenced -85
2016- Arachis duranensis
2015- Solanum cumersonii (Wild potato)
(Ref- NCBI)
46
GWAS
It is a study design in which many markers spread across a genome
are genotyped and test a statistical association with a phenotype are
performed locally along the genome
It is also an examination of many common genetic variants in different
individuals to see if any variant is associated with a trait
Used in successfully studying maize sorghum and barley
Method is efficient for large scale low cost genotyping (even with the
minimum number of SNPs)
Cannot be utilized generally because it needs large population size
GWAS identify rare alleles more precisely
If small population we can opt NAM
47
GWAS and whole genome prediction
Xuehui Huang and Bin Han 2014
48
Five high throuput genotyping methods
Xuehui Huang and Bin Han 2014
49
Role of NGS in domestication Capture of novel genes from wild species will be made easier by
understanding the molecular events associated with crop domestication
Re-sequencing of domesticated species can identify low diversity regions
resulting from selection during domestication
To identify gene-specific sequences to aid the cloning of homologues of key
domestication genes from wild relatives
Candidate genes from wild and domesticated plant populations can define
diversity of target genes in wild populations and lead to the discovery of key
genes for important traits by association analysis
NGS of amplicons of large numbers of candidate genes from wild and
domesticated plant populations can define diversity of target genes in
wild populations and lead to the discovery of key genes for important
traits by association analysis
50
NGS
supports the rapid domestication of new plant species and the efficient
identification and capture of novel genetic variation from related species
Allows whole-genome analysis to determine the genetic basis of
phenotypic differences
NGS allows rapid expansion of genomic analysis to investigation of non-
model species
Made rice study easy by related grass
cost-effective method for plant identification
useful strategy to analysis the chloroplast genome sequence from whole-
genome shot-gun sequencing
facilitates managing this diversity and any changes in crop performance
over time due to genetic drift
Patterns of gene expression have been evaluated in hybrids using NGS
51
Two Approaches to Finding Adaptive Genes
Ross-Ibarra et al2007
52
Techniques to re-wild the plants (transgene free)
1 Introgression
breeding
2 Specific
insertion of lost genes
3 Precision
mutagenesis
Palmgren et al2014
53
Process of rewilding
Palmgren et al2014
54
Synthetic biology projects(i) Modifying cereals including wheat to fix atmospheric nitrogen
(ii) Redesigning metabolic pathways to increase the yield of
secondary metabolites or to generate compounds with enhanced
properties
(iii) Transferring the C4 photosynthesis pathway to rice
(iv) Modifying the glycosylation pathway in plants to accommodate
production of therapeutic proteins
(v) Introducing synthetic signal transduction systems that respond to
external cues
55
Synthetic biology projects
Nicholas J Baltes et al 2015
56
Refactoring the N fixation gene cluster from Klebsiella
Karsten Temme et al 2012
57
Continuehellip
Karsten Temme et al 2012
58
Genome editing
Sequence specific nucleases
1 Meganucleases
2 ZFMs ndashZink finger motifs
3 TALENs- Transcription activator like effector nuclease
4 CRISPR CAS-9 ndash Clustered interspaced short palindromic
repeats
qmp4
59Khaoula Belhaj et al 2015
60
Genome modifications achieved in plants using sequence specific nucleases
Type of DNA
modification
Nuclease Delivery
methods
Plants Targets
Trait stacking Megnucleases Bombardment Cotton Intergenic sequence
ZFN Bombardment Maize Transgene
Rewriting host
DNA
Megnucleases Stable integration Maize Intergenic sequence
ZFN Stable integration Soybean Transgene
TALEN Stable integration Barley PAPhy_a
TALEN Agrobacterium
T-DNA (transient)
Oryza sativa SWEET14
TALEN No Protoplasts Arabidopsis
Tobacco
AtTT4 AtADH NbSurB
TALEN Bombardment Wheat MLO
TALEN Protoplasts
Stable integration
Maize PDS IPK1A IPK MRP4
CRISPRCas Protoplasts
Agrobacterium
T-DNA (transient)
Tobacco
Arabidopsis
Sorghum Oryza
OsSWEET14 transgene
61
Type of DNA
modification
Nuclease Delivery
methods
Plants Targets
Rewriting host
DNA large
deletion
Zinc-finger
nuclease
Stable
integration
Tobacco Transgene
CRISPRCas Protoplasts
Stable
integration
Rice Labdane-related
diterpenoid gene
clusters
on Chr 2 4 and 6
Zinc-finger
nuclease
Agrobacterium
T-DNA
(transient)
Tobacco CHN50 transgene
Zinc-finger
nuclease
Whiskers Maize IPK1
CRISPRCas Protoplasts Rice PDS
Controlling gene
expression
TALE repressor
(SRDX)
Stable
integration
Arabidopsis RD29A transgene
Zinc-finger
activator (VP16)
Stable
integration
Brassica
napus
KasII
Continue
62
Application of genome editing1 Introduction of precise and predictable modifications directly in an elite
background
2 multiple traits can be modified simultaneously
3 NHEJ enables gene knockout and targeted modifications
4 Introduction of transgenes at defined loci that promote high-level
transcription and do not interfere with the activity of endogenous genes
5 Site-specific nucleases also allow targeted molecular trait stacking -low
risk of segregation
6 CRISPR Cas is a transgene free approach ndash No regulatory burdens
7 frequency of off-target mutations is well below that caused by chemical
and physical mutagenesis techniques
8 In future can be utilized for metabolic engineering and molecular farming
63
Six-way Venn diagram showing the distribution of shared genefamilies (sequence clusters) among M acuminata P dactyliferaArabidopsis thaliana Oryza sativa Sorghum bicolor and Brachypodiumdistachyon genomes
A DrsquoHont et al Nature 2012 doi101038nature11241
64
Achieving super-DomesticationINTROGRESS-RECREATE-CREATE-DOMESTICATEREDOMESTICATE
bull Mobilizing left out genetic
variation still available in
land races and wild and
weedy species
bull Replaying the evolutionary
tape( resynthesis in
polyploids)
bull Domesticateredomesticate
65
We can admire and emulate how indigenous people still domesticateplants create biodiversity and manage it to sustain their future Thereis no equivalently dynamic or flexible crop breeding in modernagriculture to promote biodiversity we still have much to learn from
traditional knowledge Jan Salick 2012
Increased in domesticate
seed germination
determinate growth
longer pods
bigger (heavier) seeds
earliness
harvest index (seed yieldbiomass)
Decreased in domesticate
seed dispersal
seed dormancy
twining
number of nodes
length of internode
number of pods
photoperiod sensitivity
Common Bean Phaseolus vulgarishttpwwwplantsciencesucdavisedugeptspb143pb143htm
copy Paul Gepts
25
Wild pigeon peaCajanus cajanifolius
Cultivated pigeon pea
26
27
28
WHY OTHERS ARE NOT DOMESTICATED
bull 350000 plants
bull 4629 mammals
bull 9200 birds
bull 10000000 insects
bull 500000 fungi
bull But only 200 plants
bull 15 mammals 5 birds and
bull 2 insects are domesticated
bull Spread of these few species
bull Little change since early
agriculture
bull Repeated domestication of these
species (sometimes)
bull Lack of new species even with
attempts with species known to be
valuable Some groups are good
candidates with no domestication
eg ferns sub-Saharan mammals
29
WHY OTHERS HAVE TO BE DOMESTICATED
New uses and demands ndash biofuels animal feed
medicinalneutraceutical waterclimate food changes
Knowledge why species arenrsquot suitable for domestication or
were not useful
Better understanding of genetics and selection
Sustainability of production
Reliability of production
To meet the food demand in an alternative way
WHY OTHERS HAVE TO BE DOMESTICATED
30
Reallocate biomass for human use
More efficient metabolism and photosynthesis increase
leaf surface decrease root system increase leaf
longevity
Grains more fertile florets larger inflorescence OR
number of ears different ways to get more seed
Larger seeds (automatic vs deliberate selection)
Oil plants increased oil content or more seed
Fiber plants long strong fibers
Domestication syndrome
31
Centers of Domestication
Fuller 2011
32
Centers of Domestication in India
Fuller 2011
33
Indian centers of domestication South India (Deccan centre) Vigna radiate Vigna mungo
Macrotyloma uniflorum (Horse gram) Sataria verticillata S plumila
wheat (macaroni) and two rowed barleybrachiaria ramosa
Orissa (Mahanadi river) Pegion pea Horsegram mung small millets
like Echinichloa Paspalum and Sataria
The Middle Ganges (Harappan civilization) Oryza nivara O rufipogon
and wild sativa Sateria pumia Cannabis sativa even the diffusion of
japonica rice
Saurashtra (Harappan civilization) Eleucine coracana Sateria italica
Paicum spp Brachypodia pearl millet and sorghum Dollicos lablab
The Himalayan foothills of the Punjab region Was a centre of
diversity for Japonica type rice and many temperate fruits and
vegetables
34
DOMESTICATION RELATED TRAITS CONTROLLED BY ONLY FEW GENES
Trait Crops
Plant architecturegrowth habit Ricemaizemilletsbeantomato
Flowering timephotoperiod sensitivity Ricemaizesorghumbeantomato
Fruit size Tomato egg plant
Grain size Ricemaizesorghumbean
Seed dispersal Brassica rice
Inflorescence modification Brassica
Dormancy Bean
35
Genes Crop Moleular and phenotypic function Causative change
Genes identified as controlling domestication traits
tb1 Maize Transcriptional regulator (TCP) plant and
inflorescence structure
regulatory change
tga1 Maize Transcriptional regulator (SBP) seed casing amino acid change
qSH1 Rice Transcriptional regulator (homeodomain)
abscission layer formation shattering
regulatory change
Rc Rice Transcriptional regulator (bHLH) seed color disrupted coding
sequence
sh4 Rice Transcriptional regulator (Myb3) abscission
layer formation shattering
regulatoryamino
acid change
fw22 Tomato Cell signaling fruit weight regulatory change
Q Wheat Transcriptional regulator (AP2) inflorescence
structure
regulatoryamino
acid change
Vrs1 Barley Inflorescence structure Premature stop Amino acid change
Genes of interest in crop domestication and improvement
36
Genes Crop Moleular and phenotypic function Causative change
c1
r1
Maize Transcriptional regulator (MYB) kernel color and
Transcriptional regulator (bHLH) kernel color
regulatory change
sh2 Maize pyrophosphorylase supersweet sweet corn transposon
insertion
su1 Maize isoamylase sweet corn gene amino acid change
brix9-2-5 Tomato Invertase fruit soluble solid content amino acid change
ovate Tomato Unknown fruit shap early stop codon
R Pea Starch branching enzyme seed sugar content transposon
insertion
ehd1 Rice B-type response regulator flowering time amino acid change
hd1 Rice Transcriptional regulator (zinc finger) flowering
time
disrupted coding
sequence
waxy Rice Starch synthase sticky grains intron splicing
defect
rht Wheat Transcriptional regulator (SH2) plant height early stop codon
vrn1 Wheat Transcriptional regulator (MADS) vernalization regulatory change
vrn2 Wheat Transcriptional regulator (ZCCT) vernalization amino acid change
Genes Identified as Controlling Varietal Differences
37
Process of domestication over the yearshellip
Rachel S Meyer 2012
38
Genetic bottleneck
Cultivated crops undergone with narrowing of diversity problem
But this is not the case in weeds
Weeds are the major threats from the beginning of domestication till
today
Problem arose due to
1 Crop mimicry
2 Genetic assimilation
3 Genetic evolution
39
Super- Domestication Vaughn et al (2007) first used the term super-domestication
The processes that lead to a domesticate with dramatically
increased yield that could not be selected in natural environments
from naturally occurring variation without recourse to new
technologies
Super-domesticates can be constructed with knowledge led
approaches based on current needs using the range of new
technologies now available
Plants exploited for continuous selection introduction hybridization
etc which boosted the process of domestication but now the plant
genetic engineering approach is exploiting plants towards synthetic
biology
40
How to use diversitybull Cross two varieties
bull Genome manipulations
Cell fusion hybrids
bull Chromosome manipulation Backcross a new species
bull Generate recombinants Chromosome recombinations
bull Use a new species wild germplasm
Transgenic approach Modern mutagenesis synthetic gene
construction by utilizing green florescent protein genome editing
NGS GWAS sequencing etchelliphelliphelliphelliphellip
41
Reduce genetic bottlenecks through polyplodization
More vigour
SC
Buffering capacity
Heterotic advantage
Enhaced vegetative
charachers
Enhanced oil
Meiotic stability
42
Approach 1 Use one tetraploid and one diploid as parents (4X ndash 2X) followed by the
chromosome doubling of triploid hybrids (Chrom doubling)
a) Cross between B juncea (AjAjBjBj ) and B oleracea (CoCo) to produce hexaploids
(AjAjBjBjCoCo)
(b) Cross between B napus (AnAnCnCn) and B nigra (BniBni) to produce hexaploids
(AnAnBniBniCnCn)
(c) Cross between B carinata (BcaBcaCcaCca) and B rapa (ArAr) to produce hexaploids
(ArArBcaBcaCcaCca)
Approach 2 Use three tetraploids as parents
(a) Cross between B napus (AnAnCnCn) and B carinata (BcaBcaCcaCca) to produce unbalanced
allotetraploids (AnBcaCnCca ndash unreduced gametes gametes with the somatic chromosome
number and cross with B juncea (AjAjBjBj) to obtain allohexaploids (AnAjBcaBjCnCca
(b) Cross between B napus (AnAnCnCn) and B juncea (AjAjBjBj) to produce unbalanced
allotetraploids (AnAjBjCn ndash unreduced gametes) and cross with B carinata (BcaBcaCcaCca) to
obtain allohexaploids (AnAjBcaBjCnCca)
(c) Cross between B carinata (BcaBcaCcaCca) and B juncea (AjAjBjBj) to produce unbalanced
allotetraploids (AjBcaBjCca - ndash unreduced gametes) and cross with B napus (AnAnCnCn) to obtain
allohexaploids (AnAjBcaBjCnCca)
Approach 3 Use three diploids as parents (2X ndash 2X ndash 2X) Cross between B rapa (ArAr) B
nigra (BniBni) and B oleracea (CoCo) sequentially to obtain hexaploid hybrids (ArArBniBniCoCo)
43
Continuehellip
Guijun Yan 2012
44
Applications of Genomic
tools in Super-Domestication
45
Genome Sequencing
Potential methods of sequencing
1 Clone by clone approach
2 Whole genome shotgun approach
3 Combination of the two methods
Till today no of cultivated plants completely sequenced -85
2016- Arachis duranensis
2015- Solanum cumersonii (Wild potato)
(Ref- NCBI)
46
GWAS
It is a study design in which many markers spread across a genome
are genotyped and test a statistical association with a phenotype are
performed locally along the genome
It is also an examination of many common genetic variants in different
individuals to see if any variant is associated with a trait
Used in successfully studying maize sorghum and barley
Method is efficient for large scale low cost genotyping (even with the
minimum number of SNPs)
Cannot be utilized generally because it needs large population size
GWAS identify rare alleles more precisely
If small population we can opt NAM
47
GWAS and whole genome prediction
Xuehui Huang and Bin Han 2014
48
Five high throuput genotyping methods
Xuehui Huang and Bin Han 2014
49
Role of NGS in domestication Capture of novel genes from wild species will be made easier by
understanding the molecular events associated with crop domestication
Re-sequencing of domesticated species can identify low diversity regions
resulting from selection during domestication
To identify gene-specific sequences to aid the cloning of homologues of key
domestication genes from wild relatives
Candidate genes from wild and domesticated plant populations can define
diversity of target genes in wild populations and lead to the discovery of key
genes for important traits by association analysis
NGS of amplicons of large numbers of candidate genes from wild and
domesticated plant populations can define diversity of target genes in
wild populations and lead to the discovery of key genes for important
traits by association analysis
50
NGS
supports the rapid domestication of new plant species and the efficient
identification and capture of novel genetic variation from related species
Allows whole-genome analysis to determine the genetic basis of
phenotypic differences
NGS allows rapid expansion of genomic analysis to investigation of non-
model species
Made rice study easy by related grass
cost-effective method for plant identification
useful strategy to analysis the chloroplast genome sequence from whole-
genome shot-gun sequencing
facilitates managing this diversity and any changes in crop performance
over time due to genetic drift
Patterns of gene expression have been evaluated in hybrids using NGS
51
Two Approaches to Finding Adaptive Genes
Ross-Ibarra et al2007
52
Techniques to re-wild the plants (transgene free)
1 Introgression
breeding
2 Specific
insertion of lost genes
3 Precision
mutagenesis
Palmgren et al2014
53
Process of rewilding
Palmgren et al2014
54
Synthetic biology projects(i) Modifying cereals including wheat to fix atmospheric nitrogen
(ii) Redesigning metabolic pathways to increase the yield of
secondary metabolites or to generate compounds with enhanced
properties
(iii) Transferring the C4 photosynthesis pathway to rice
(iv) Modifying the glycosylation pathway in plants to accommodate
production of therapeutic proteins
(v) Introducing synthetic signal transduction systems that respond to
external cues
55
Synthetic biology projects
Nicholas J Baltes et al 2015
56
Refactoring the N fixation gene cluster from Klebsiella
Karsten Temme et al 2012
57
Continuehellip
Karsten Temme et al 2012
58
Genome editing
Sequence specific nucleases
1 Meganucleases
2 ZFMs ndashZink finger motifs
3 TALENs- Transcription activator like effector nuclease
4 CRISPR CAS-9 ndash Clustered interspaced short palindromic
repeats
qmp4
59Khaoula Belhaj et al 2015
60
Genome modifications achieved in plants using sequence specific nucleases
Type of DNA
modification
Nuclease Delivery
methods
Plants Targets
Trait stacking Megnucleases Bombardment Cotton Intergenic sequence
ZFN Bombardment Maize Transgene
Rewriting host
DNA
Megnucleases Stable integration Maize Intergenic sequence
ZFN Stable integration Soybean Transgene
TALEN Stable integration Barley PAPhy_a
TALEN Agrobacterium
T-DNA (transient)
Oryza sativa SWEET14
TALEN No Protoplasts Arabidopsis
Tobacco
AtTT4 AtADH NbSurB
TALEN Bombardment Wheat MLO
TALEN Protoplasts
Stable integration
Maize PDS IPK1A IPK MRP4
CRISPRCas Protoplasts
Agrobacterium
T-DNA (transient)
Tobacco
Arabidopsis
Sorghum Oryza
OsSWEET14 transgene
61
Type of DNA
modification
Nuclease Delivery
methods
Plants Targets
Rewriting host
DNA large
deletion
Zinc-finger
nuclease
Stable
integration
Tobacco Transgene
CRISPRCas Protoplasts
Stable
integration
Rice Labdane-related
diterpenoid gene
clusters
on Chr 2 4 and 6
Zinc-finger
nuclease
Agrobacterium
T-DNA
(transient)
Tobacco CHN50 transgene
Zinc-finger
nuclease
Whiskers Maize IPK1
CRISPRCas Protoplasts Rice PDS
Controlling gene
expression
TALE repressor
(SRDX)
Stable
integration
Arabidopsis RD29A transgene
Zinc-finger
activator (VP16)
Stable
integration
Brassica
napus
KasII
Continue
62
Application of genome editing1 Introduction of precise and predictable modifications directly in an elite
background
2 multiple traits can be modified simultaneously
3 NHEJ enables gene knockout and targeted modifications
4 Introduction of transgenes at defined loci that promote high-level
transcription and do not interfere with the activity of endogenous genes
5 Site-specific nucleases also allow targeted molecular trait stacking -low
risk of segregation
6 CRISPR Cas is a transgene free approach ndash No regulatory burdens
7 frequency of off-target mutations is well below that caused by chemical
and physical mutagenesis techniques
8 In future can be utilized for metabolic engineering and molecular farming
63
Six-way Venn diagram showing the distribution of shared genefamilies (sequence clusters) among M acuminata P dactyliferaArabidopsis thaliana Oryza sativa Sorghum bicolor and Brachypodiumdistachyon genomes
A DrsquoHont et al Nature 2012 doi101038nature11241
64
Achieving super-DomesticationINTROGRESS-RECREATE-CREATE-DOMESTICATEREDOMESTICATE
bull Mobilizing left out genetic
variation still available in
land races and wild and
weedy species
bull Replaying the evolutionary
tape( resynthesis in
polyploids)
bull Domesticateredomesticate
65
We can admire and emulate how indigenous people still domesticateplants create biodiversity and manage it to sustain their future Thereis no equivalently dynamic or flexible crop breeding in modernagriculture to promote biodiversity we still have much to learn from
traditional knowledge Jan Salick 2012
Wild pigeon peaCajanus cajanifolius
Cultivated pigeon pea
26
27
28
WHY OTHERS ARE NOT DOMESTICATED
bull 350000 plants
bull 4629 mammals
bull 9200 birds
bull 10000000 insects
bull 500000 fungi
bull But only 200 plants
bull 15 mammals 5 birds and
bull 2 insects are domesticated
bull Spread of these few species
bull Little change since early
agriculture
bull Repeated domestication of these
species (sometimes)
bull Lack of new species even with
attempts with species known to be
valuable Some groups are good
candidates with no domestication
eg ferns sub-Saharan mammals
29
WHY OTHERS HAVE TO BE DOMESTICATED
New uses and demands ndash biofuels animal feed
medicinalneutraceutical waterclimate food changes
Knowledge why species arenrsquot suitable for domestication or
were not useful
Better understanding of genetics and selection
Sustainability of production
Reliability of production
To meet the food demand in an alternative way
WHY OTHERS HAVE TO BE DOMESTICATED
30
Reallocate biomass for human use
More efficient metabolism and photosynthesis increase
leaf surface decrease root system increase leaf
longevity
Grains more fertile florets larger inflorescence OR
number of ears different ways to get more seed
Larger seeds (automatic vs deliberate selection)
Oil plants increased oil content or more seed
Fiber plants long strong fibers
Domestication syndrome
31
Centers of Domestication
Fuller 2011
32
Centers of Domestication in India
Fuller 2011
33
Indian centers of domestication South India (Deccan centre) Vigna radiate Vigna mungo
Macrotyloma uniflorum (Horse gram) Sataria verticillata S plumila
wheat (macaroni) and two rowed barleybrachiaria ramosa
Orissa (Mahanadi river) Pegion pea Horsegram mung small millets
like Echinichloa Paspalum and Sataria
The Middle Ganges (Harappan civilization) Oryza nivara O rufipogon
and wild sativa Sateria pumia Cannabis sativa even the diffusion of
japonica rice
Saurashtra (Harappan civilization) Eleucine coracana Sateria italica
Paicum spp Brachypodia pearl millet and sorghum Dollicos lablab
The Himalayan foothills of the Punjab region Was a centre of
diversity for Japonica type rice and many temperate fruits and
vegetables
34
DOMESTICATION RELATED TRAITS CONTROLLED BY ONLY FEW GENES
Trait Crops
Plant architecturegrowth habit Ricemaizemilletsbeantomato
Flowering timephotoperiod sensitivity Ricemaizesorghumbeantomato
Fruit size Tomato egg plant
Grain size Ricemaizesorghumbean
Seed dispersal Brassica rice
Inflorescence modification Brassica
Dormancy Bean
35
Genes Crop Moleular and phenotypic function Causative change
Genes identified as controlling domestication traits
tb1 Maize Transcriptional regulator (TCP) plant and
inflorescence structure
regulatory change
tga1 Maize Transcriptional regulator (SBP) seed casing amino acid change
qSH1 Rice Transcriptional regulator (homeodomain)
abscission layer formation shattering
regulatory change
Rc Rice Transcriptional regulator (bHLH) seed color disrupted coding
sequence
sh4 Rice Transcriptional regulator (Myb3) abscission
layer formation shattering
regulatoryamino
acid change
fw22 Tomato Cell signaling fruit weight regulatory change
Q Wheat Transcriptional regulator (AP2) inflorescence
structure
regulatoryamino
acid change
Vrs1 Barley Inflorescence structure Premature stop Amino acid change
Genes of interest in crop domestication and improvement
36
Genes Crop Moleular and phenotypic function Causative change
c1
r1
Maize Transcriptional regulator (MYB) kernel color and
Transcriptional regulator (bHLH) kernel color
regulatory change
sh2 Maize pyrophosphorylase supersweet sweet corn transposon
insertion
su1 Maize isoamylase sweet corn gene amino acid change
brix9-2-5 Tomato Invertase fruit soluble solid content amino acid change
ovate Tomato Unknown fruit shap early stop codon
R Pea Starch branching enzyme seed sugar content transposon
insertion
ehd1 Rice B-type response regulator flowering time amino acid change
hd1 Rice Transcriptional regulator (zinc finger) flowering
time
disrupted coding
sequence
waxy Rice Starch synthase sticky grains intron splicing
defect
rht Wheat Transcriptional regulator (SH2) plant height early stop codon
vrn1 Wheat Transcriptional regulator (MADS) vernalization regulatory change
vrn2 Wheat Transcriptional regulator (ZCCT) vernalization amino acid change
Genes Identified as Controlling Varietal Differences
37
Process of domestication over the yearshellip
Rachel S Meyer 2012
38
Genetic bottleneck
Cultivated crops undergone with narrowing of diversity problem
But this is not the case in weeds
Weeds are the major threats from the beginning of domestication till
today
Problem arose due to
1 Crop mimicry
2 Genetic assimilation
3 Genetic evolution
39
Super- Domestication Vaughn et al (2007) first used the term super-domestication
The processes that lead to a domesticate with dramatically
increased yield that could not be selected in natural environments
from naturally occurring variation without recourse to new
technologies
Super-domesticates can be constructed with knowledge led
approaches based on current needs using the range of new
technologies now available
Plants exploited for continuous selection introduction hybridization
etc which boosted the process of domestication but now the plant
genetic engineering approach is exploiting plants towards synthetic
biology
40
How to use diversitybull Cross two varieties
bull Genome manipulations
Cell fusion hybrids
bull Chromosome manipulation Backcross a new species
bull Generate recombinants Chromosome recombinations
bull Use a new species wild germplasm
Transgenic approach Modern mutagenesis synthetic gene
construction by utilizing green florescent protein genome editing
NGS GWAS sequencing etchelliphelliphelliphelliphellip
41
Reduce genetic bottlenecks through polyplodization
More vigour
SC
Buffering capacity
Heterotic advantage
Enhaced vegetative
charachers
Enhanced oil
Meiotic stability
42
Approach 1 Use one tetraploid and one diploid as parents (4X ndash 2X) followed by the
chromosome doubling of triploid hybrids (Chrom doubling)
a) Cross between B juncea (AjAjBjBj ) and B oleracea (CoCo) to produce hexaploids
(AjAjBjBjCoCo)
(b) Cross between B napus (AnAnCnCn) and B nigra (BniBni) to produce hexaploids
(AnAnBniBniCnCn)
(c) Cross between B carinata (BcaBcaCcaCca) and B rapa (ArAr) to produce hexaploids
(ArArBcaBcaCcaCca)
Approach 2 Use three tetraploids as parents
(a) Cross between B napus (AnAnCnCn) and B carinata (BcaBcaCcaCca) to produce unbalanced
allotetraploids (AnBcaCnCca ndash unreduced gametes gametes with the somatic chromosome
number and cross with B juncea (AjAjBjBj) to obtain allohexaploids (AnAjBcaBjCnCca
(b) Cross between B napus (AnAnCnCn) and B juncea (AjAjBjBj) to produce unbalanced
allotetraploids (AnAjBjCn ndash unreduced gametes) and cross with B carinata (BcaBcaCcaCca) to
obtain allohexaploids (AnAjBcaBjCnCca)
(c) Cross between B carinata (BcaBcaCcaCca) and B juncea (AjAjBjBj) to produce unbalanced
allotetraploids (AjBcaBjCca - ndash unreduced gametes) and cross with B napus (AnAnCnCn) to obtain
allohexaploids (AnAjBcaBjCnCca)
Approach 3 Use three diploids as parents (2X ndash 2X ndash 2X) Cross between B rapa (ArAr) B
nigra (BniBni) and B oleracea (CoCo) sequentially to obtain hexaploid hybrids (ArArBniBniCoCo)
43
Continuehellip
Guijun Yan 2012
44
Applications of Genomic
tools in Super-Domestication
45
Genome Sequencing
Potential methods of sequencing
1 Clone by clone approach
2 Whole genome shotgun approach
3 Combination of the two methods
Till today no of cultivated plants completely sequenced -85
2016- Arachis duranensis
2015- Solanum cumersonii (Wild potato)
(Ref- NCBI)
46
GWAS
It is a study design in which many markers spread across a genome
are genotyped and test a statistical association with a phenotype are
performed locally along the genome
It is also an examination of many common genetic variants in different
individuals to see if any variant is associated with a trait
Used in successfully studying maize sorghum and barley
Method is efficient for large scale low cost genotyping (even with the
minimum number of SNPs)
Cannot be utilized generally because it needs large population size
GWAS identify rare alleles more precisely
If small population we can opt NAM
47
GWAS and whole genome prediction
Xuehui Huang and Bin Han 2014
48
Five high throuput genotyping methods
Xuehui Huang and Bin Han 2014
49
Role of NGS in domestication Capture of novel genes from wild species will be made easier by
understanding the molecular events associated with crop domestication
Re-sequencing of domesticated species can identify low diversity regions
resulting from selection during domestication
To identify gene-specific sequences to aid the cloning of homologues of key
domestication genes from wild relatives
Candidate genes from wild and domesticated plant populations can define
diversity of target genes in wild populations and lead to the discovery of key
genes for important traits by association analysis
NGS of amplicons of large numbers of candidate genes from wild and
domesticated plant populations can define diversity of target genes in
wild populations and lead to the discovery of key genes for important
traits by association analysis
50
NGS
supports the rapid domestication of new plant species and the efficient
identification and capture of novel genetic variation from related species
Allows whole-genome analysis to determine the genetic basis of
phenotypic differences
NGS allows rapid expansion of genomic analysis to investigation of non-
model species
Made rice study easy by related grass
cost-effective method for plant identification
useful strategy to analysis the chloroplast genome sequence from whole-
genome shot-gun sequencing
facilitates managing this diversity and any changes in crop performance
over time due to genetic drift
Patterns of gene expression have been evaluated in hybrids using NGS
51
Two Approaches to Finding Adaptive Genes
Ross-Ibarra et al2007
52
Techniques to re-wild the plants (transgene free)
1 Introgression
breeding
2 Specific
insertion of lost genes
3 Precision
mutagenesis
Palmgren et al2014
53
Process of rewilding
Palmgren et al2014
54
Synthetic biology projects(i) Modifying cereals including wheat to fix atmospheric nitrogen
(ii) Redesigning metabolic pathways to increase the yield of
secondary metabolites or to generate compounds with enhanced
properties
(iii) Transferring the C4 photosynthesis pathway to rice
(iv) Modifying the glycosylation pathway in plants to accommodate
production of therapeutic proteins
(v) Introducing synthetic signal transduction systems that respond to
external cues
55
Synthetic biology projects
Nicholas J Baltes et al 2015
56
Refactoring the N fixation gene cluster from Klebsiella
Karsten Temme et al 2012
57
Continuehellip
Karsten Temme et al 2012
58
Genome editing
Sequence specific nucleases
1 Meganucleases
2 ZFMs ndashZink finger motifs
3 TALENs- Transcription activator like effector nuclease
4 CRISPR CAS-9 ndash Clustered interspaced short palindromic
repeats
qmp4
59Khaoula Belhaj et al 2015
60
Genome modifications achieved in plants using sequence specific nucleases
Type of DNA
modification
Nuclease Delivery
methods
Plants Targets
Trait stacking Megnucleases Bombardment Cotton Intergenic sequence
ZFN Bombardment Maize Transgene
Rewriting host
DNA
Megnucleases Stable integration Maize Intergenic sequence
ZFN Stable integration Soybean Transgene
TALEN Stable integration Barley PAPhy_a
TALEN Agrobacterium
T-DNA (transient)
Oryza sativa SWEET14
TALEN No Protoplasts Arabidopsis
Tobacco
AtTT4 AtADH NbSurB
TALEN Bombardment Wheat MLO
TALEN Protoplasts
Stable integration
Maize PDS IPK1A IPK MRP4
CRISPRCas Protoplasts
Agrobacterium
T-DNA (transient)
Tobacco
Arabidopsis
Sorghum Oryza
OsSWEET14 transgene
61
Type of DNA
modification
Nuclease Delivery
methods
Plants Targets
Rewriting host
DNA large
deletion
Zinc-finger
nuclease
Stable
integration
Tobacco Transgene
CRISPRCas Protoplasts
Stable
integration
Rice Labdane-related
diterpenoid gene
clusters
on Chr 2 4 and 6
Zinc-finger
nuclease
Agrobacterium
T-DNA
(transient)
Tobacco CHN50 transgene
Zinc-finger
nuclease
Whiskers Maize IPK1
CRISPRCas Protoplasts Rice PDS
Controlling gene
expression
TALE repressor
(SRDX)
Stable
integration
Arabidopsis RD29A transgene
Zinc-finger
activator (VP16)
Stable
integration
Brassica
napus
KasII
Continue
62
Application of genome editing1 Introduction of precise and predictable modifications directly in an elite
background
2 multiple traits can be modified simultaneously
3 NHEJ enables gene knockout and targeted modifications
4 Introduction of transgenes at defined loci that promote high-level
transcription and do not interfere with the activity of endogenous genes
5 Site-specific nucleases also allow targeted molecular trait stacking -low
risk of segregation
6 CRISPR Cas is a transgene free approach ndash No regulatory burdens
7 frequency of off-target mutations is well below that caused by chemical
and physical mutagenesis techniques
8 In future can be utilized for metabolic engineering and molecular farming
63
Six-way Venn diagram showing the distribution of shared genefamilies (sequence clusters) among M acuminata P dactyliferaArabidopsis thaliana Oryza sativa Sorghum bicolor and Brachypodiumdistachyon genomes
A DrsquoHont et al Nature 2012 doi101038nature11241
64
Achieving super-DomesticationINTROGRESS-RECREATE-CREATE-DOMESTICATEREDOMESTICATE
bull Mobilizing left out genetic
variation still available in
land races and wild and
weedy species
bull Replaying the evolutionary
tape( resynthesis in
polyploids)
bull Domesticateredomesticate
65
We can admire and emulate how indigenous people still domesticateplants create biodiversity and manage it to sustain their future Thereis no equivalently dynamic or flexible crop breeding in modernagriculture to promote biodiversity we still have much to learn from
traditional knowledge Jan Salick 2012
27
28
WHY OTHERS ARE NOT DOMESTICATED
bull 350000 plants
bull 4629 mammals
bull 9200 birds
bull 10000000 insects
bull 500000 fungi
bull But only 200 plants
bull 15 mammals 5 birds and
bull 2 insects are domesticated
bull Spread of these few species
bull Little change since early
agriculture
bull Repeated domestication of these
species (sometimes)
bull Lack of new species even with
attempts with species known to be
valuable Some groups are good
candidates with no domestication
eg ferns sub-Saharan mammals
29
WHY OTHERS HAVE TO BE DOMESTICATED
New uses and demands ndash biofuels animal feed
medicinalneutraceutical waterclimate food changes
Knowledge why species arenrsquot suitable for domestication or
were not useful
Better understanding of genetics and selection
Sustainability of production
Reliability of production
To meet the food demand in an alternative way
WHY OTHERS HAVE TO BE DOMESTICATED
30
Reallocate biomass for human use
More efficient metabolism and photosynthesis increase
leaf surface decrease root system increase leaf
longevity
Grains more fertile florets larger inflorescence OR
number of ears different ways to get more seed
Larger seeds (automatic vs deliberate selection)
Oil plants increased oil content or more seed
Fiber plants long strong fibers
Domestication syndrome
31
Centers of Domestication
Fuller 2011
32
Centers of Domestication in India
Fuller 2011
33
Indian centers of domestication South India (Deccan centre) Vigna radiate Vigna mungo
Macrotyloma uniflorum (Horse gram) Sataria verticillata S plumila
wheat (macaroni) and two rowed barleybrachiaria ramosa
Orissa (Mahanadi river) Pegion pea Horsegram mung small millets
like Echinichloa Paspalum and Sataria
The Middle Ganges (Harappan civilization) Oryza nivara O rufipogon
and wild sativa Sateria pumia Cannabis sativa even the diffusion of
japonica rice
Saurashtra (Harappan civilization) Eleucine coracana Sateria italica
Paicum spp Brachypodia pearl millet and sorghum Dollicos lablab
The Himalayan foothills of the Punjab region Was a centre of
diversity for Japonica type rice and many temperate fruits and
vegetables
34
DOMESTICATION RELATED TRAITS CONTROLLED BY ONLY FEW GENES
Trait Crops
Plant architecturegrowth habit Ricemaizemilletsbeantomato
Flowering timephotoperiod sensitivity Ricemaizesorghumbeantomato
Fruit size Tomato egg plant
Grain size Ricemaizesorghumbean
Seed dispersal Brassica rice
Inflorescence modification Brassica
Dormancy Bean
35
Genes Crop Moleular and phenotypic function Causative change
Genes identified as controlling domestication traits
tb1 Maize Transcriptional regulator (TCP) plant and
inflorescence structure
regulatory change
tga1 Maize Transcriptional regulator (SBP) seed casing amino acid change
qSH1 Rice Transcriptional regulator (homeodomain)
abscission layer formation shattering
regulatory change
Rc Rice Transcriptional regulator (bHLH) seed color disrupted coding
sequence
sh4 Rice Transcriptional regulator (Myb3) abscission
layer formation shattering
regulatoryamino
acid change
fw22 Tomato Cell signaling fruit weight regulatory change
Q Wheat Transcriptional regulator (AP2) inflorescence
structure
regulatoryamino
acid change
Vrs1 Barley Inflorescence structure Premature stop Amino acid change
Genes of interest in crop domestication and improvement
36
Genes Crop Moleular and phenotypic function Causative change
c1
r1
Maize Transcriptional regulator (MYB) kernel color and
Transcriptional regulator (bHLH) kernel color
regulatory change
sh2 Maize pyrophosphorylase supersweet sweet corn transposon
insertion
su1 Maize isoamylase sweet corn gene amino acid change
brix9-2-5 Tomato Invertase fruit soluble solid content amino acid change
ovate Tomato Unknown fruit shap early stop codon
R Pea Starch branching enzyme seed sugar content transposon
insertion
ehd1 Rice B-type response regulator flowering time amino acid change
hd1 Rice Transcriptional regulator (zinc finger) flowering
time
disrupted coding
sequence
waxy Rice Starch synthase sticky grains intron splicing
defect
rht Wheat Transcriptional regulator (SH2) plant height early stop codon
vrn1 Wheat Transcriptional regulator (MADS) vernalization regulatory change
vrn2 Wheat Transcriptional regulator (ZCCT) vernalization amino acid change
Genes Identified as Controlling Varietal Differences
37
Process of domestication over the yearshellip
Rachel S Meyer 2012
38
Genetic bottleneck
Cultivated crops undergone with narrowing of diversity problem
But this is not the case in weeds
Weeds are the major threats from the beginning of domestication till
today
Problem arose due to
1 Crop mimicry
2 Genetic assimilation
3 Genetic evolution
39
Super- Domestication Vaughn et al (2007) first used the term super-domestication
The processes that lead to a domesticate with dramatically
increased yield that could not be selected in natural environments
from naturally occurring variation without recourse to new
technologies
Super-domesticates can be constructed with knowledge led
approaches based on current needs using the range of new
technologies now available
Plants exploited for continuous selection introduction hybridization
etc which boosted the process of domestication but now the plant
genetic engineering approach is exploiting plants towards synthetic
biology
40
How to use diversitybull Cross two varieties
bull Genome manipulations
Cell fusion hybrids
bull Chromosome manipulation Backcross a new species
bull Generate recombinants Chromosome recombinations
bull Use a new species wild germplasm
Transgenic approach Modern mutagenesis synthetic gene
construction by utilizing green florescent protein genome editing
NGS GWAS sequencing etchelliphelliphelliphelliphellip
41
Reduce genetic bottlenecks through polyplodization
More vigour
SC
Buffering capacity
Heterotic advantage
Enhaced vegetative
charachers
Enhanced oil
Meiotic stability
42
Approach 1 Use one tetraploid and one diploid as parents (4X ndash 2X) followed by the
chromosome doubling of triploid hybrids (Chrom doubling)
a) Cross between B juncea (AjAjBjBj ) and B oleracea (CoCo) to produce hexaploids
(AjAjBjBjCoCo)
(b) Cross between B napus (AnAnCnCn) and B nigra (BniBni) to produce hexaploids
(AnAnBniBniCnCn)
(c) Cross between B carinata (BcaBcaCcaCca) and B rapa (ArAr) to produce hexaploids
(ArArBcaBcaCcaCca)
Approach 2 Use three tetraploids as parents
(a) Cross between B napus (AnAnCnCn) and B carinata (BcaBcaCcaCca) to produce unbalanced
allotetraploids (AnBcaCnCca ndash unreduced gametes gametes with the somatic chromosome
number and cross with B juncea (AjAjBjBj) to obtain allohexaploids (AnAjBcaBjCnCca
(b) Cross between B napus (AnAnCnCn) and B juncea (AjAjBjBj) to produce unbalanced
allotetraploids (AnAjBjCn ndash unreduced gametes) and cross with B carinata (BcaBcaCcaCca) to
obtain allohexaploids (AnAjBcaBjCnCca)
(c) Cross between B carinata (BcaBcaCcaCca) and B juncea (AjAjBjBj) to produce unbalanced
allotetraploids (AjBcaBjCca - ndash unreduced gametes) and cross with B napus (AnAnCnCn) to obtain
allohexaploids (AnAjBcaBjCnCca)
Approach 3 Use three diploids as parents (2X ndash 2X ndash 2X) Cross between B rapa (ArAr) B
nigra (BniBni) and B oleracea (CoCo) sequentially to obtain hexaploid hybrids (ArArBniBniCoCo)
43
Continuehellip
Guijun Yan 2012
44
Applications of Genomic
tools in Super-Domestication
45
Genome Sequencing
Potential methods of sequencing
1 Clone by clone approach
2 Whole genome shotgun approach
3 Combination of the two methods
Till today no of cultivated plants completely sequenced -85
2016- Arachis duranensis
2015- Solanum cumersonii (Wild potato)
(Ref- NCBI)
46
GWAS
It is a study design in which many markers spread across a genome
are genotyped and test a statistical association with a phenotype are
performed locally along the genome
It is also an examination of many common genetic variants in different
individuals to see if any variant is associated with a trait
Used in successfully studying maize sorghum and barley
Method is efficient for large scale low cost genotyping (even with the
minimum number of SNPs)
Cannot be utilized generally because it needs large population size
GWAS identify rare alleles more precisely
If small population we can opt NAM
47
GWAS and whole genome prediction
Xuehui Huang and Bin Han 2014
48
Five high throuput genotyping methods
Xuehui Huang and Bin Han 2014
49
Role of NGS in domestication Capture of novel genes from wild species will be made easier by
understanding the molecular events associated with crop domestication
Re-sequencing of domesticated species can identify low diversity regions
resulting from selection during domestication
To identify gene-specific sequences to aid the cloning of homologues of key
domestication genes from wild relatives
Candidate genes from wild and domesticated plant populations can define
diversity of target genes in wild populations and lead to the discovery of key
genes for important traits by association analysis
NGS of amplicons of large numbers of candidate genes from wild and
domesticated plant populations can define diversity of target genes in
wild populations and lead to the discovery of key genes for important
traits by association analysis
50
NGS
supports the rapid domestication of new plant species and the efficient
identification and capture of novel genetic variation from related species
Allows whole-genome analysis to determine the genetic basis of
phenotypic differences
NGS allows rapid expansion of genomic analysis to investigation of non-
model species
Made rice study easy by related grass
cost-effective method for plant identification
useful strategy to analysis the chloroplast genome sequence from whole-
genome shot-gun sequencing
facilitates managing this diversity and any changes in crop performance
over time due to genetic drift
Patterns of gene expression have been evaluated in hybrids using NGS
51
Two Approaches to Finding Adaptive Genes
Ross-Ibarra et al2007
52
Techniques to re-wild the plants (transgene free)
1 Introgression
breeding
2 Specific
insertion of lost genes
3 Precision
mutagenesis
Palmgren et al2014
53
Process of rewilding
Palmgren et al2014
54
Synthetic biology projects(i) Modifying cereals including wheat to fix atmospheric nitrogen
(ii) Redesigning metabolic pathways to increase the yield of
secondary metabolites or to generate compounds with enhanced
properties
(iii) Transferring the C4 photosynthesis pathway to rice
(iv) Modifying the glycosylation pathway in plants to accommodate
production of therapeutic proteins
(v) Introducing synthetic signal transduction systems that respond to
external cues
55
Synthetic biology projects
Nicholas J Baltes et al 2015
56
Refactoring the N fixation gene cluster from Klebsiella
Karsten Temme et al 2012
57
Continuehellip
Karsten Temme et al 2012
58
Genome editing
Sequence specific nucleases
1 Meganucleases
2 ZFMs ndashZink finger motifs
3 TALENs- Transcription activator like effector nuclease
4 CRISPR CAS-9 ndash Clustered interspaced short palindromic
repeats
qmp4
59Khaoula Belhaj et al 2015
60
Genome modifications achieved in plants using sequence specific nucleases
Type of DNA
modification
Nuclease Delivery
methods
Plants Targets
Trait stacking Megnucleases Bombardment Cotton Intergenic sequence
ZFN Bombardment Maize Transgene
Rewriting host
DNA
Megnucleases Stable integration Maize Intergenic sequence
ZFN Stable integration Soybean Transgene
TALEN Stable integration Barley PAPhy_a
TALEN Agrobacterium
T-DNA (transient)
Oryza sativa SWEET14
TALEN No Protoplasts Arabidopsis
Tobacco
AtTT4 AtADH NbSurB
TALEN Bombardment Wheat MLO
TALEN Protoplasts
Stable integration
Maize PDS IPK1A IPK MRP4
CRISPRCas Protoplasts
Agrobacterium
T-DNA (transient)
Tobacco
Arabidopsis
Sorghum Oryza
OsSWEET14 transgene
61
Type of DNA
modification
Nuclease Delivery
methods
Plants Targets
Rewriting host
DNA large
deletion
Zinc-finger
nuclease
Stable
integration
Tobacco Transgene
CRISPRCas Protoplasts
Stable
integration
Rice Labdane-related
diterpenoid gene
clusters
on Chr 2 4 and 6
Zinc-finger
nuclease
Agrobacterium
T-DNA
(transient)
Tobacco CHN50 transgene
Zinc-finger
nuclease
Whiskers Maize IPK1
CRISPRCas Protoplasts Rice PDS
Controlling gene
expression
TALE repressor
(SRDX)
Stable
integration
Arabidopsis RD29A transgene
Zinc-finger
activator (VP16)
Stable
integration
Brassica
napus
KasII
Continue
62
Application of genome editing1 Introduction of precise and predictable modifications directly in an elite
background
2 multiple traits can be modified simultaneously
3 NHEJ enables gene knockout and targeted modifications
4 Introduction of transgenes at defined loci that promote high-level
transcription and do not interfere with the activity of endogenous genes
5 Site-specific nucleases also allow targeted molecular trait stacking -low
risk of segregation
6 CRISPR Cas is a transgene free approach ndash No regulatory burdens
7 frequency of off-target mutations is well below that caused by chemical
and physical mutagenesis techniques
8 In future can be utilized for metabolic engineering and molecular farming
63
Six-way Venn diagram showing the distribution of shared genefamilies (sequence clusters) among M acuminata P dactyliferaArabidopsis thaliana Oryza sativa Sorghum bicolor and Brachypodiumdistachyon genomes
A DrsquoHont et al Nature 2012 doi101038nature11241
64
Achieving super-DomesticationINTROGRESS-RECREATE-CREATE-DOMESTICATEREDOMESTICATE
bull Mobilizing left out genetic
variation still available in
land races and wild and
weedy species
bull Replaying the evolutionary
tape( resynthesis in
polyploids)
bull Domesticateredomesticate
65
We can admire and emulate how indigenous people still domesticateplants create biodiversity and manage it to sustain their future Thereis no equivalently dynamic or flexible crop breeding in modernagriculture to promote biodiversity we still have much to learn from
traditional knowledge Jan Salick 2012
28
WHY OTHERS ARE NOT DOMESTICATED
bull 350000 plants
bull 4629 mammals
bull 9200 birds
bull 10000000 insects
bull 500000 fungi
bull But only 200 plants
bull 15 mammals 5 birds and
bull 2 insects are domesticated
bull Spread of these few species
bull Little change since early
agriculture
bull Repeated domestication of these
species (sometimes)
bull Lack of new species even with
attempts with species known to be
valuable Some groups are good
candidates with no domestication
eg ferns sub-Saharan mammals
29
WHY OTHERS HAVE TO BE DOMESTICATED
New uses and demands ndash biofuels animal feed
medicinalneutraceutical waterclimate food changes
Knowledge why species arenrsquot suitable for domestication or
were not useful
Better understanding of genetics and selection
Sustainability of production
Reliability of production
To meet the food demand in an alternative way
WHY OTHERS HAVE TO BE DOMESTICATED
30
Reallocate biomass for human use
More efficient metabolism and photosynthesis increase
leaf surface decrease root system increase leaf
longevity
Grains more fertile florets larger inflorescence OR
number of ears different ways to get more seed
Larger seeds (automatic vs deliberate selection)
Oil plants increased oil content or more seed
Fiber plants long strong fibers
Domestication syndrome
31
Centers of Domestication
Fuller 2011
32
Centers of Domestication in India
Fuller 2011
33
Indian centers of domestication South India (Deccan centre) Vigna radiate Vigna mungo
Macrotyloma uniflorum (Horse gram) Sataria verticillata S plumila
wheat (macaroni) and two rowed barleybrachiaria ramosa
Orissa (Mahanadi river) Pegion pea Horsegram mung small millets
like Echinichloa Paspalum and Sataria
The Middle Ganges (Harappan civilization) Oryza nivara O rufipogon
and wild sativa Sateria pumia Cannabis sativa even the diffusion of
japonica rice
Saurashtra (Harappan civilization) Eleucine coracana Sateria italica
Paicum spp Brachypodia pearl millet and sorghum Dollicos lablab
The Himalayan foothills of the Punjab region Was a centre of
diversity for Japonica type rice and many temperate fruits and
vegetables
34
DOMESTICATION RELATED TRAITS CONTROLLED BY ONLY FEW GENES
Trait Crops
Plant architecturegrowth habit Ricemaizemilletsbeantomato
Flowering timephotoperiod sensitivity Ricemaizesorghumbeantomato
Fruit size Tomato egg plant
Grain size Ricemaizesorghumbean
Seed dispersal Brassica rice
Inflorescence modification Brassica
Dormancy Bean
35
Genes Crop Moleular and phenotypic function Causative change
Genes identified as controlling domestication traits
tb1 Maize Transcriptional regulator (TCP) plant and
inflorescence structure
regulatory change
tga1 Maize Transcriptional regulator (SBP) seed casing amino acid change
qSH1 Rice Transcriptional regulator (homeodomain)
abscission layer formation shattering
regulatory change
Rc Rice Transcriptional regulator (bHLH) seed color disrupted coding
sequence
sh4 Rice Transcriptional regulator (Myb3) abscission
layer formation shattering
regulatoryamino
acid change
fw22 Tomato Cell signaling fruit weight regulatory change
Q Wheat Transcriptional regulator (AP2) inflorescence
structure
regulatoryamino
acid change
Vrs1 Barley Inflorescence structure Premature stop Amino acid change
Genes of interest in crop domestication and improvement
36
Genes Crop Moleular and phenotypic function Causative change
c1
r1
Maize Transcriptional regulator (MYB) kernel color and
Transcriptional regulator (bHLH) kernel color
regulatory change
sh2 Maize pyrophosphorylase supersweet sweet corn transposon
insertion
su1 Maize isoamylase sweet corn gene amino acid change
brix9-2-5 Tomato Invertase fruit soluble solid content amino acid change
ovate Tomato Unknown fruit shap early stop codon
R Pea Starch branching enzyme seed sugar content transposon
insertion
ehd1 Rice B-type response regulator flowering time amino acid change
hd1 Rice Transcriptional regulator (zinc finger) flowering
time
disrupted coding
sequence
waxy Rice Starch synthase sticky grains intron splicing
defect
rht Wheat Transcriptional regulator (SH2) plant height early stop codon
vrn1 Wheat Transcriptional regulator (MADS) vernalization regulatory change
vrn2 Wheat Transcriptional regulator (ZCCT) vernalization amino acid change
Genes Identified as Controlling Varietal Differences
37
Process of domestication over the yearshellip
Rachel S Meyer 2012
38
Genetic bottleneck
Cultivated crops undergone with narrowing of diversity problem
But this is not the case in weeds
Weeds are the major threats from the beginning of domestication till
today
Problem arose due to
1 Crop mimicry
2 Genetic assimilation
3 Genetic evolution
39
Super- Domestication Vaughn et al (2007) first used the term super-domestication
The processes that lead to a domesticate with dramatically
increased yield that could not be selected in natural environments
from naturally occurring variation without recourse to new
technologies
Super-domesticates can be constructed with knowledge led
approaches based on current needs using the range of new
technologies now available
Plants exploited for continuous selection introduction hybridization
etc which boosted the process of domestication but now the plant
genetic engineering approach is exploiting plants towards synthetic
biology
40
How to use diversitybull Cross two varieties
bull Genome manipulations
Cell fusion hybrids
bull Chromosome manipulation Backcross a new species
bull Generate recombinants Chromosome recombinations
bull Use a new species wild germplasm
Transgenic approach Modern mutagenesis synthetic gene
construction by utilizing green florescent protein genome editing
NGS GWAS sequencing etchelliphelliphelliphelliphellip
41
Reduce genetic bottlenecks through polyplodization
More vigour
SC
Buffering capacity
Heterotic advantage
Enhaced vegetative
charachers
Enhanced oil
Meiotic stability
42
Approach 1 Use one tetraploid and one diploid as parents (4X ndash 2X) followed by the
chromosome doubling of triploid hybrids (Chrom doubling)
a) Cross between B juncea (AjAjBjBj ) and B oleracea (CoCo) to produce hexaploids
(AjAjBjBjCoCo)
(b) Cross between B napus (AnAnCnCn) and B nigra (BniBni) to produce hexaploids
(AnAnBniBniCnCn)
(c) Cross between B carinata (BcaBcaCcaCca) and B rapa (ArAr) to produce hexaploids
(ArArBcaBcaCcaCca)
Approach 2 Use three tetraploids as parents
(a) Cross between B napus (AnAnCnCn) and B carinata (BcaBcaCcaCca) to produce unbalanced
allotetraploids (AnBcaCnCca ndash unreduced gametes gametes with the somatic chromosome
number and cross with B juncea (AjAjBjBj) to obtain allohexaploids (AnAjBcaBjCnCca
(b) Cross between B napus (AnAnCnCn) and B juncea (AjAjBjBj) to produce unbalanced
allotetraploids (AnAjBjCn ndash unreduced gametes) and cross with B carinata (BcaBcaCcaCca) to
obtain allohexaploids (AnAjBcaBjCnCca)
(c) Cross between B carinata (BcaBcaCcaCca) and B juncea (AjAjBjBj) to produce unbalanced
allotetraploids (AjBcaBjCca - ndash unreduced gametes) and cross with B napus (AnAnCnCn) to obtain
allohexaploids (AnAjBcaBjCnCca)
Approach 3 Use three diploids as parents (2X ndash 2X ndash 2X) Cross between B rapa (ArAr) B
nigra (BniBni) and B oleracea (CoCo) sequentially to obtain hexaploid hybrids (ArArBniBniCoCo)
43
Continuehellip
Guijun Yan 2012
44
Applications of Genomic
tools in Super-Domestication
45
Genome Sequencing
Potential methods of sequencing
1 Clone by clone approach
2 Whole genome shotgun approach
3 Combination of the two methods
Till today no of cultivated plants completely sequenced -85
2016- Arachis duranensis
2015- Solanum cumersonii (Wild potato)
(Ref- NCBI)
46
GWAS
It is a study design in which many markers spread across a genome
are genotyped and test a statistical association with a phenotype are
performed locally along the genome
It is also an examination of many common genetic variants in different
individuals to see if any variant is associated with a trait
Used in successfully studying maize sorghum and barley
Method is efficient for large scale low cost genotyping (even with the
minimum number of SNPs)
Cannot be utilized generally because it needs large population size
GWAS identify rare alleles more precisely
If small population we can opt NAM
47
GWAS and whole genome prediction
Xuehui Huang and Bin Han 2014
48
Five high throuput genotyping methods
Xuehui Huang and Bin Han 2014
49
Role of NGS in domestication Capture of novel genes from wild species will be made easier by
understanding the molecular events associated with crop domestication
Re-sequencing of domesticated species can identify low diversity regions
resulting from selection during domestication
To identify gene-specific sequences to aid the cloning of homologues of key
domestication genes from wild relatives
Candidate genes from wild and domesticated plant populations can define
diversity of target genes in wild populations and lead to the discovery of key
genes for important traits by association analysis
NGS of amplicons of large numbers of candidate genes from wild and
domesticated plant populations can define diversity of target genes in
wild populations and lead to the discovery of key genes for important
traits by association analysis
50
NGS
supports the rapid domestication of new plant species and the efficient
identification and capture of novel genetic variation from related species
Allows whole-genome analysis to determine the genetic basis of
phenotypic differences
NGS allows rapid expansion of genomic analysis to investigation of non-
model species
Made rice study easy by related grass
cost-effective method for plant identification
useful strategy to analysis the chloroplast genome sequence from whole-
genome shot-gun sequencing
facilitates managing this diversity and any changes in crop performance
over time due to genetic drift
Patterns of gene expression have been evaluated in hybrids using NGS
51
Two Approaches to Finding Adaptive Genes
Ross-Ibarra et al2007
52
Techniques to re-wild the plants (transgene free)
1 Introgression
breeding
2 Specific
insertion of lost genes
3 Precision
mutagenesis
Palmgren et al2014
53
Process of rewilding
Palmgren et al2014
54
Synthetic biology projects(i) Modifying cereals including wheat to fix atmospheric nitrogen
(ii) Redesigning metabolic pathways to increase the yield of
secondary metabolites or to generate compounds with enhanced
properties
(iii) Transferring the C4 photosynthesis pathway to rice
(iv) Modifying the glycosylation pathway in plants to accommodate
production of therapeutic proteins
(v) Introducing synthetic signal transduction systems that respond to
external cues
55
Synthetic biology projects
Nicholas J Baltes et al 2015
56
Refactoring the N fixation gene cluster from Klebsiella
Karsten Temme et al 2012
57
Continuehellip
Karsten Temme et al 2012
58
Genome editing
Sequence specific nucleases
1 Meganucleases
2 ZFMs ndashZink finger motifs
3 TALENs- Transcription activator like effector nuclease
4 CRISPR CAS-9 ndash Clustered interspaced short palindromic
repeats
qmp4
59Khaoula Belhaj et al 2015
60
Genome modifications achieved in plants using sequence specific nucleases
Type of DNA
modification
Nuclease Delivery
methods
Plants Targets
Trait stacking Megnucleases Bombardment Cotton Intergenic sequence
ZFN Bombardment Maize Transgene
Rewriting host
DNA
Megnucleases Stable integration Maize Intergenic sequence
ZFN Stable integration Soybean Transgene
TALEN Stable integration Barley PAPhy_a
TALEN Agrobacterium
T-DNA (transient)
Oryza sativa SWEET14
TALEN No Protoplasts Arabidopsis
Tobacco
AtTT4 AtADH NbSurB
TALEN Bombardment Wheat MLO
TALEN Protoplasts
Stable integration
Maize PDS IPK1A IPK MRP4
CRISPRCas Protoplasts
Agrobacterium
T-DNA (transient)
Tobacco
Arabidopsis
Sorghum Oryza
OsSWEET14 transgene
61
Type of DNA
modification
Nuclease Delivery
methods
Plants Targets
Rewriting host
DNA large
deletion
Zinc-finger
nuclease
Stable
integration
Tobacco Transgene
CRISPRCas Protoplasts
Stable
integration
Rice Labdane-related
diterpenoid gene
clusters
on Chr 2 4 and 6
Zinc-finger
nuclease
Agrobacterium
T-DNA
(transient)
Tobacco CHN50 transgene
Zinc-finger
nuclease
Whiskers Maize IPK1
CRISPRCas Protoplasts Rice PDS
Controlling gene
expression
TALE repressor
(SRDX)
Stable
integration
Arabidopsis RD29A transgene
Zinc-finger
activator (VP16)
Stable
integration
Brassica
napus
KasII
Continue
62
Application of genome editing1 Introduction of precise and predictable modifications directly in an elite
background
2 multiple traits can be modified simultaneously
3 NHEJ enables gene knockout and targeted modifications
4 Introduction of transgenes at defined loci that promote high-level
transcription and do not interfere with the activity of endogenous genes
5 Site-specific nucleases also allow targeted molecular trait stacking -low
risk of segregation
6 CRISPR Cas is a transgene free approach ndash No regulatory burdens
7 frequency of off-target mutations is well below that caused by chemical
and physical mutagenesis techniques
8 In future can be utilized for metabolic engineering and molecular farming
63
Six-way Venn diagram showing the distribution of shared genefamilies (sequence clusters) among M acuminata P dactyliferaArabidopsis thaliana Oryza sativa Sorghum bicolor and Brachypodiumdistachyon genomes
A DrsquoHont et al Nature 2012 doi101038nature11241
64
Achieving super-DomesticationINTROGRESS-RECREATE-CREATE-DOMESTICATEREDOMESTICATE
bull Mobilizing left out genetic
variation still available in
land races and wild and
weedy species
bull Replaying the evolutionary
tape( resynthesis in
polyploids)
bull Domesticateredomesticate
65
We can admire and emulate how indigenous people still domesticateplants create biodiversity and manage it to sustain their future Thereis no equivalently dynamic or flexible crop breeding in modernagriculture to promote biodiversity we still have much to learn from
traditional knowledge Jan Salick 2012
29
WHY OTHERS HAVE TO BE DOMESTICATED
New uses and demands ndash biofuels animal feed
medicinalneutraceutical waterclimate food changes
Knowledge why species arenrsquot suitable for domestication or
were not useful
Better understanding of genetics and selection
Sustainability of production
Reliability of production
To meet the food demand in an alternative way
WHY OTHERS HAVE TO BE DOMESTICATED
30
Reallocate biomass for human use
More efficient metabolism and photosynthesis increase
leaf surface decrease root system increase leaf
longevity
Grains more fertile florets larger inflorescence OR
number of ears different ways to get more seed
Larger seeds (automatic vs deliberate selection)
Oil plants increased oil content or more seed
Fiber plants long strong fibers
Domestication syndrome
31
Centers of Domestication
Fuller 2011
32
Centers of Domestication in India
Fuller 2011
33
Indian centers of domestication South India (Deccan centre) Vigna radiate Vigna mungo
Macrotyloma uniflorum (Horse gram) Sataria verticillata S plumila
wheat (macaroni) and two rowed barleybrachiaria ramosa
Orissa (Mahanadi river) Pegion pea Horsegram mung small millets
like Echinichloa Paspalum and Sataria
The Middle Ganges (Harappan civilization) Oryza nivara O rufipogon
and wild sativa Sateria pumia Cannabis sativa even the diffusion of
japonica rice
Saurashtra (Harappan civilization) Eleucine coracana Sateria italica
Paicum spp Brachypodia pearl millet and sorghum Dollicos lablab
The Himalayan foothills of the Punjab region Was a centre of
diversity for Japonica type rice and many temperate fruits and
vegetables
34
DOMESTICATION RELATED TRAITS CONTROLLED BY ONLY FEW GENES
Trait Crops
Plant architecturegrowth habit Ricemaizemilletsbeantomato
Flowering timephotoperiod sensitivity Ricemaizesorghumbeantomato
Fruit size Tomato egg plant
Grain size Ricemaizesorghumbean
Seed dispersal Brassica rice
Inflorescence modification Brassica
Dormancy Bean
35
Genes Crop Moleular and phenotypic function Causative change
Genes identified as controlling domestication traits
tb1 Maize Transcriptional regulator (TCP) plant and
inflorescence structure
regulatory change
tga1 Maize Transcriptional regulator (SBP) seed casing amino acid change
qSH1 Rice Transcriptional regulator (homeodomain)
abscission layer formation shattering
regulatory change
Rc Rice Transcriptional regulator (bHLH) seed color disrupted coding
sequence
sh4 Rice Transcriptional regulator (Myb3) abscission
layer formation shattering
regulatoryamino
acid change
fw22 Tomato Cell signaling fruit weight regulatory change
Q Wheat Transcriptional regulator (AP2) inflorescence
structure
regulatoryamino
acid change
Vrs1 Barley Inflorescence structure Premature stop Amino acid change
Genes of interest in crop domestication and improvement
36
Genes Crop Moleular and phenotypic function Causative change
c1
r1
Maize Transcriptional regulator (MYB) kernel color and
Transcriptional regulator (bHLH) kernel color
regulatory change
sh2 Maize pyrophosphorylase supersweet sweet corn transposon
insertion
su1 Maize isoamylase sweet corn gene amino acid change
brix9-2-5 Tomato Invertase fruit soluble solid content amino acid change
ovate Tomato Unknown fruit shap early stop codon
R Pea Starch branching enzyme seed sugar content transposon
insertion
ehd1 Rice B-type response regulator flowering time amino acid change
hd1 Rice Transcriptional regulator (zinc finger) flowering
time
disrupted coding
sequence
waxy Rice Starch synthase sticky grains intron splicing
defect
rht Wheat Transcriptional regulator (SH2) plant height early stop codon
vrn1 Wheat Transcriptional regulator (MADS) vernalization regulatory change
vrn2 Wheat Transcriptional regulator (ZCCT) vernalization amino acid change
Genes Identified as Controlling Varietal Differences
37
Process of domestication over the yearshellip
Rachel S Meyer 2012
38
Genetic bottleneck
Cultivated crops undergone with narrowing of diversity problem
But this is not the case in weeds
Weeds are the major threats from the beginning of domestication till
today
Problem arose due to
1 Crop mimicry
2 Genetic assimilation
3 Genetic evolution
39
Super- Domestication Vaughn et al (2007) first used the term super-domestication
The processes that lead to a domesticate with dramatically
increased yield that could not be selected in natural environments
from naturally occurring variation without recourse to new
technologies
Super-domesticates can be constructed with knowledge led
approaches based on current needs using the range of new
technologies now available
Plants exploited for continuous selection introduction hybridization
etc which boosted the process of domestication but now the plant
genetic engineering approach is exploiting plants towards synthetic
biology
40
How to use diversitybull Cross two varieties
bull Genome manipulations
Cell fusion hybrids
bull Chromosome manipulation Backcross a new species
bull Generate recombinants Chromosome recombinations
bull Use a new species wild germplasm
Transgenic approach Modern mutagenesis synthetic gene
construction by utilizing green florescent protein genome editing
NGS GWAS sequencing etchelliphelliphelliphelliphellip
41
Reduce genetic bottlenecks through polyplodization
More vigour
SC
Buffering capacity
Heterotic advantage
Enhaced vegetative
charachers
Enhanced oil
Meiotic stability
42
Approach 1 Use one tetraploid and one diploid as parents (4X ndash 2X) followed by the
chromosome doubling of triploid hybrids (Chrom doubling)
a) Cross between B juncea (AjAjBjBj ) and B oleracea (CoCo) to produce hexaploids
(AjAjBjBjCoCo)
(b) Cross between B napus (AnAnCnCn) and B nigra (BniBni) to produce hexaploids
(AnAnBniBniCnCn)
(c) Cross between B carinata (BcaBcaCcaCca) and B rapa (ArAr) to produce hexaploids
(ArArBcaBcaCcaCca)
Approach 2 Use three tetraploids as parents
(a) Cross between B napus (AnAnCnCn) and B carinata (BcaBcaCcaCca) to produce unbalanced
allotetraploids (AnBcaCnCca ndash unreduced gametes gametes with the somatic chromosome
number and cross with B juncea (AjAjBjBj) to obtain allohexaploids (AnAjBcaBjCnCca
(b) Cross between B napus (AnAnCnCn) and B juncea (AjAjBjBj) to produce unbalanced
allotetraploids (AnAjBjCn ndash unreduced gametes) and cross with B carinata (BcaBcaCcaCca) to
obtain allohexaploids (AnAjBcaBjCnCca)
(c) Cross between B carinata (BcaBcaCcaCca) and B juncea (AjAjBjBj) to produce unbalanced
allotetraploids (AjBcaBjCca - ndash unreduced gametes) and cross with B napus (AnAnCnCn) to obtain
allohexaploids (AnAjBcaBjCnCca)
Approach 3 Use three diploids as parents (2X ndash 2X ndash 2X) Cross between B rapa (ArAr) B
nigra (BniBni) and B oleracea (CoCo) sequentially to obtain hexaploid hybrids (ArArBniBniCoCo)
43
Continuehellip
Guijun Yan 2012
44
Applications of Genomic
tools in Super-Domestication
45
Genome Sequencing
Potential methods of sequencing
1 Clone by clone approach
2 Whole genome shotgun approach
3 Combination of the two methods
Till today no of cultivated plants completely sequenced -85
2016- Arachis duranensis
2015- Solanum cumersonii (Wild potato)
(Ref- NCBI)
46
GWAS
It is a study design in which many markers spread across a genome
are genotyped and test a statistical association with a phenotype are
performed locally along the genome
It is also an examination of many common genetic variants in different
individuals to see if any variant is associated with a trait
Used in successfully studying maize sorghum and barley
Method is efficient for large scale low cost genotyping (even with the
minimum number of SNPs)
Cannot be utilized generally because it needs large population size
GWAS identify rare alleles more precisely
If small population we can opt NAM
47
GWAS and whole genome prediction
Xuehui Huang and Bin Han 2014
48
Five high throuput genotyping methods
Xuehui Huang and Bin Han 2014
49
Role of NGS in domestication Capture of novel genes from wild species will be made easier by
understanding the molecular events associated with crop domestication
Re-sequencing of domesticated species can identify low diversity regions
resulting from selection during domestication
To identify gene-specific sequences to aid the cloning of homologues of key
domestication genes from wild relatives
Candidate genes from wild and domesticated plant populations can define
diversity of target genes in wild populations and lead to the discovery of key
genes for important traits by association analysis
NGS of amplicons of large numbers of candidate genes from wild and
domesticated plant populations can define diversity of target genes in
wild populations and lead to the discovery of key genes for important
traits by association analysis
50
NGS
supports the rapid domestication of new plant species and the efficient
identification and capture of novel genetic variation from related species
Allows whole-genome analysis to determine the genetic basis of
phenotypic differences
NGS allows rapid expansion of genomic analysis to investigation of non-
model species
Made rice study easy by related grass
cost-effective method for plant identification
useful strategy to analysis the chloroplast genome sequence from whole-
genome shot-gun sequencing
facilitates managing this diversity and any changes in crop performance
over time due to genetic drift
Patterns of gene expression have been evaluated in hybrids using NGS
51
Two Approaches to Finding Adaptive Genes
Ross-Ibarra et al2007
52
Techniques to re-wild the plants (transgene free)
1 Introgression
breeding
2 Specific
insertion of lost genes
3 Precision
mutagenesis
Palmgren et al2014
53
Process of rewilding
Palmgren et al2014
54
Synthetic biology projects(i) Modifying cereals including wheat to fix atmospheric nitrogen
(ii) Redesigning metabolic pathways to increase the yield of
secondary metabolites or to generate compounds with enhanced
properties
(iii) Transferring the C4 photosynthesis pathway to rice
(iv) Modifying the glycosylation pathway in plants to accommodate
production of therapeutic proteins
(v) Introducing synthetic signal transduction systems that respond to
external cues
55
Synthetic biology projects
Nicholas J Baltes et al 2015
56
Refactoring the N fixation gene cluster from Klebsiella
Karsten Temme et al 2012
57
Continuehellip
Karsten Temme et al 2012
58
Genome editing
Sequence specific nucleases
1 Meganucleases
2 ZFMs ndashZink finger motifs
3 TALENs- Transcription activator like effector nuclease
4 CRISPR CAS-9 ndash Clustered interspaced short palindromic
repeats
qmp4
59Khaoula Belhaj et al 2015
60
Genome modifications achieved in plants using sequence specific nucleases
Type of DNA
modification
Nuclease Delivery
methods
Plants Targets
Trait stacking Megnucleases Bombardment Cotton Intergenic sequence
ZFN Bombardment Maize Transgene
Rewriting host
DNA
Megnucleases Stable integration Maize Intergenic sequence
ZFN Stable integration Soybean Transgene
TALEN Stable integration Barley PAPhy_a
TALEN Agrobacterium
T-DNA (transient)
Oryza sativa SWEET14
TALEN No Protoplasts Arabidopsis
Tobacco
AtTT4 AtADH NbSurB
TALEN Bombardment Wheat MLO
TALEN Protoplasts
Stable integration
Maize PDS IPK1A IPK MRP4
CRISPRCas Protoplasts
Agrobacterium
T-DNA (transient)
Tobacco
Arabidopsis
Sorghum Oryza
OsSWEET14 transgene
61
Type of DNA
modification
Nuclease Delivery
methods
Plants Targets
Rewriting host
DNA large
deletion
Zinc-finger
nuclease
Stable
integration
Tobacco Transgene
CRISPRCas Protoplasts
Stable
integration
Rice Labdane-related
diterpenoid gene
clusters
on Chr 2 4 and 6
Zinc-finger
nuclease
Agrobacterium
T-DNA
(transient)
Tobacco CHN50 transgene
Zinc-finger
nuclease
Whiskers Maize IPK1
CRISPRCas Protoplasts Rice PDS
Controlling gene
expression
TALE repressor
(SRDX)
Stable
integration
Arabidopsis RD29A transgene
Zinc-finger
activator (VP16)
Stable
integration
Brassica
napus
KasII
Continue
62
Application of genome editing1 Introduction of precise and predictable modifications directly in an elite
background
2 multiple traits can be modified simultaneously
3 NHEJ enables gene knockout and targeted modifications
4 Introduction of transgenes at defined loci that promote high-level
transcription and do not interfere with the activity of endogenous genes
5 Site-specific nucleases also allow targeted molecular trait stacking -low
risk of segregation
6 CRISPR Cas is a transgene free approach ndash No regulatory burdens
7 frequency of off-target mutations is well below that caused by chemical
and physical mutagenesis techniques
8 In future can be utilized for metabolic engineering and molecular farming
63
Six-way Venn diagram showing the distribution of shared genefamilies (sequence clusters) among M acuminata P dactyliferaArabidopsis thaliana Oryza sativa Sorghum bicolor and Brachypodiumdistachyon genomes
A DrsquoHont et al Nature 2012 doi101038nature11241
64
Achieving super-DomesticationINTROGRESS-RECREATE-CREATE-DOMESTICATEREDOMESTICATE
bull Mobilizing left out genetic
variation still available in
land races and wild and
weedy species
bull Replaying the evolutionary
tape( resynthesis in
polyploids)
bull Domesticateredomesticate
65
We can admire and emulate how indigenous people still domesticateplants create biodiversity and manage it to sustain their future Thereis no equivalently dynamic or flexible crop breeding in modernagriculture to promote biodiversity we still have much to learn from
traditional knowledge Jan Salick 2012
30
Reallocate biomass for human use
More efficient metabolism and photosynthesis increase
leaf surface decrease root system increase leaf
longevity
Grains more fertile florets larger inflorescence OR
number of ears different ways to get more seed
Larger seeds (automatic vs deliberate selection)
Oil plants increased oil content or more seed
Fiber plants long strong fibers
Domestication syndrome
31
Centers of Domestication
Fuller 2011
32
Centers of Domestication in India
Fuller 2011
33
Indian centers of domestication South India (Deccan centre) Vigna radiate Vigna mungo
Macrotyloma uniflorum (Horse gram) Sataria verticillata S plumila
wheat (macaroni) and two rowed barleybrachiaria ramosa
Orissa (Mahanadi river) Pegion pea Horsegram mung small millets
like Echinichloa Paspalum and Sataria
The Middle Ganges (Harappan civilization) Oryza nivara O rufipogon
and wild sativa Sateria pumia Cannabis sativa even the diffusion of
japonica rice
Saurashtra (Harappan civilization) Eleucine coracana Sateria italica
Paicum spp Brachypodia pearl millet and sorghum Dollicos lablab
The Himalayan foothills of the Punjab region Was a centre of
diversity for Japonica type rice and many temperate fruits and
vegetables
34
DOMESTICATION RELATED TRAITS CONTROLLED BY ONLY FEW GENES
Trait Crops
Plant architecturegrowth habit Ricemaizemilletsbeantomato
Flowering timephotoperiod sensitivity Ricemaizesorghumbeantomato
Fruit size Tomato egg plant
Grain size Ricemaizesorghumbean
Seed dispersal Brassica rice
Inflorescence modification Brassica
Dormancy Bean
35
Genes Crop Moleular and phenotypic function Causative change
Genes identified as controlling domestication traits
tb1 Maize Transcriptional regulator (TCP) plant and
inflorescence structure
regulatory change
tga1 Maize Transcriptional regulator (SBP) seed casing amino acid change
qSH1 Rice Transcriptional regulator (homeodomain)
abscission layer formation shattering
regulatory change
Rc Rice Transcriptional regulator (bHLH) seed color disrupted coding
sequence
sh4 Rice Transcriptional regulator (Myb3) abscission
layer formation shattering
regulatoryamino
acid change
fw22 Tomato Cell signaling fruit weight regulatory change
Q Wheat Transcriptional regulator (AP2) inflorescence
structure
regulatoryamino
acid change
Vrs1 Barley Inflorescence structure Premature stop Amino acid change
Genes of interest in crop domestication and improvement
36
Genes Crop Moleular and phenotypic function Causative change
c1
r1
Maize Transcriptional regulator (MYB) kernel color and
Transcriptional regulator (bHLH) kernel color
regulatory change
sh2 Maize pyrophosphorylase supersweet sweet corn transposon
insertion
su1 Maize isoamylase sweet corn gene amino acid change
brix9-2-5 Tomato Invertase fruit soluble solid content amino acid change
ovate Tomato Unknown fruit shap early stop codon
R Pea Starch branching enzyme seed sugar content transposon
insertion
ehd1 Rice B-type response regulator flowering time amino acid change
hd1 Rice Transcriptional regulator (zinc finger) flowering
time
disrupted coding
sequence
waxy Rice Starch synthase sticky grains intron splicing
defect
rht Wheat Transcriptional regulator (SH2) plant height early stop codon
vrn1 Wheat Transcriptional regulator (MADS) vernalization regulatory change
vrn2 Wheat Transcriptional regulator (ZCCT) vernalization amino acid change
Genes Identified as Controlling Varietal Differences
37
Process of domestication over the yearshellip
Rachel S Meyer 2012
38
Genetic bottleneck
Cultivated crops undergone with narrowing of diversity problem
But this is not the case in weeds
Weeds are the major threats from the beginning of domestication till
today
Problem arose due to
1 Crop mimicry
2 Genetic assimilation
3 Genetic evolution
39
Super- Domestication Vaughn et al (2007) first used the term super-domestication
The processes that lead to a domesticate with dramatically
increased yield that could not be selected in natural environments
from naturally occurring variation without recourse to new
technologies
Super-domesticates can be constructed with knowledge led
approaches based on current needs using the range of new
technologies now available
Plants exploited for continuous selection introduction hybridization
etc which boosted the process of domestication but now the plant
genetic engineering approach is exploiting plants towards synthetic
biology
40
How to use diversitybull Cross two varieties
bull Genome manipulations
Cell fusion hybrids
bull Chromosome manipulation Backcross a new species
bull Generate recombinants Chromosome recombinations
bull Use a new species wild germplasm
Transgenic approach Modern mutagenesis synthetic gene
construction by utilizing green florescent protein genome editing
NGS GWAS sequencing etchelliphelliphelliphelliphellip
41
Reduce genetic bottlenecks through polyplodization
More vigour
SC
Buffering capacity
Heterotic advantage
Enhaced vegetative
charachers
Enhanced oil
Meiotic stability
42
Approach 1 Use one tetraploid and one diploid as parents (4X ndash 2X) followed by the
chromosome doubling of triploid hybrids (Chrom doubling)
a) Cross between B juncea (AjAjBjBj ) and B oleracea (CoCo) to produce hexaploids
(AjAjBjBjCoCo)
(b) Cross between B napus (AnAnCnCn) and B nigra (BniBni) to produce hexaploids
(AnAnBniBniCnCn)
(c) Cross between B carinata (BcaBcaCcaCca) and B rapa (ArAr) to produce hexaploids
(ArArBcaBcaCcaCca)
Approach 2 Use three tetraploids as parents
(a) Cross between B napus (AnAnCnCn) and B carinata (BcaBcaCcaCca) to produce unbalanced
allotetraploids (AnBcaCnCca ndash unreduced gametes gametes with the somatic chromosome
number and cross with B juncea (AjAjBjBj) to obtain allohexaploids (AnAjBcaBjCnCca
(b) Cross between B napus (AnAnCnCn) and B juncea (AjAjBjBj) to produce unbalanced
allotetraploids (AnAjBjCn ndash unreduced gametes) and cross with B carinata (BcaBcaCcaCca) to
obtain allohexaploids (AnAjBcaBjCnCca)
(c) Cross between B carinata (BcaBcaCcaCca) and B juncea (AjAjBjBj) to produce unbalanced
allotetraploids (AjBcaBjCca - ndash unreduced gametes) and cross with B napus (AnAnCnCn) to obtain
allohexaploids (AnAjBcaBjCnCca)
Approach 3 Use three diploids as parents (2X ndash 2X ndash 2X) Cross between B rapa (ArAr) B
nigra (BniBni) and B oleracea (CoCo) sequentially to obtain hexaploid hybrids (ArArBniBniCoCo)
43
Continuehellip
Guijun Yan 2012
44
Applications of Genomic
tools in Super-Domestication
45
Genome Sequencing
Potential methods of sequencing
1 Clone by clone approach
2 Whole genome shotgun approach
3 Combination of the two methods
Till today no of cultivated plants completely sequenced -85
2016- Arachis duranensis
2015- Solanum cumersonii (Wild potato)
(Ref- NCBI)
46
GWAS
It is a study design in which many markers spread across a genome
are genotyped and test a statistical association with a phenotype are
performed locally along the genome
It is also an examination of many common genetic variants in different
individuals to see if any variant is associated with a trait
Used in successfully studying maize sorghum and barley
Method is efficient for large scale low cost genotyping (even with the
minimum number of SNPs)
Cannot be utilized generally because it needs large population size
GWAS identify rare alleles more precisely
If small population we can opt NAM
47
GWAS and whole genome prediction
Xuehui Huang and Bin Han 2014
48
Five high throuput genotyping methods
Xuehui Huang and Bin Han 2014
49
Role of NGS in domestication Capture of novel genes from wild species will be made easier by
understanding the molecular events associated with crop domestication
Re-sequencing of domesticated species can identify low diversity regions
resulting from selection during domestication
To identify gene-specific sequences to aid the cloning of homologues of key
domestication genes from wild relatives
Candidate genes from wild and domesticated plant populations can define
diversity of target genes in wild populations and lead to the discovery of key
genes for important traits by association analysis
NGS of amplicons of large numbers of candidate genes from wild and
domesticated plant populations can define diversity of target genes in
wild populations and lead to the discovery of key genes for important
traits by association analysis
50
NGS
supports the rapid domestication of new plant species and the efficient
identification and capture of novel genetic variation from related species
Allows whole-genome analysis to determine the genetic basis of
phenotypic differences
NGS allows rapid expansion of genomic analysis to investigation of non-
model species
Made rice study easy by related grass
cost-effective method for plant identification
useful strategy to analysis the chloroplast genome sequence from whole-
genome shot-gun sequencing
facilitates managing this diversity and any changes in crop performance
over time due to genetic drift
Patterns of gene expression have been evaluated in hybrids using NGS
51
Two Approaches to Finding Adaptive Genes
Ross-Ibarra et al2007
52
Techniques to re-wild the plants (transgene free)
1 Introgression
breeding
2 Specific
insertion of lost genes
3 Precision
mutagenesis
Palmgren et al2014
53
Process of rewilding
Palmgren et al2014
54
Synthetic biology projects(i) Modifying cereals including wheat to fix atmospheric nitrogen
(ii) Redesigning metabolic pathways to increase the yield of
secondary metabolites or to generate compounds with enhanced
properties
(iii) Transferring the C4 photosynthesis pathway to rice
(iv) Modifying the glycosylation pathway in plants to accommodate
production of therapeutic proteins
(v) Introducing synthetic signal transduction systems that respond to
external cues
55
Synthetic biology projects
Nicholas J Baltes et al 2015
56
Refactoring the N fixation gene cluster from Klebsiella
Karsten Temme et al 2012
57
Continuehellip
Karsten Temme et al 2012
58
Genome editing
Sequence specific nucleases
1 Meganucleases
2 ZFMs ndashZink finger motifs
3 TALENs- Transcription activator like effector nuclease
4 CRISPR CAS-9 ndash Clustered interspaced short palindromic
repeats
qmp4
59Khaoula Belhaj et al 2015
60
Genome modifications achieved in plants using sequence specific nucleases
Type of DNA
modification
Nuclease Delivery
methods
Plants Targets
Trait stacking Megnucleases Bombardment Cotton Intergenic sequence
ZFN Bombardment Maize Transgene
Rewriting host
DNA
Megnucleases Stable integration Maize Intergenic sequence
ZFN Stable integration Soybean Transgene
TALEN Stable integration Barley PAPhy_a
TALEN Agrobacterium
T-DNA (transient)
Oryza sativa SWEET14
TALEN No Protoplasts Arabidopsis
Tobacco
AtTT4 AtADH NbSurB
TALEN Bombardment Wheat MLO
TALEN Protoplasts
Stable integration
Maize PDS IPK1A IPK MRP4
CRISPRCas Protoplasts
Agrobacterium
T-DNA (transient)
Tobacco
Arabidopsis
Sorghum Oryza
OsSWEET14 transgene
61
Type of DNA
modification
Nuclease Delivery
methods
Plants Targets
Rewriting host
DNA large
deletion
Zinc-finger
nuclease
Stable
integration
Tobacco Transgene
CRISPRCas Protoplasts
Stable
integration
Rice Labdane-related
diterpenoid gene
clusters
on Chr 2 4 and 6
Zinc-finger
nuclease
Agrobacterium
T-DNA
(transient)
Tobacco CHN50 transgene
Zinc-finger
nuclease
Whiskers Maize IPK1
CRISPRCas Protoplasts Rice PDS
Controlling gene
expression
TALE repressor
(SRDX)
Stable
integration
Arabidopsis RD29A transgene
Zinc-finger
activator (VP16)
Stable
integration
Brassica
napus
KasII
Continue
62
Application of genome editing1 Introduction of precise and predictable modifications directly in an elite
background
2 multiple traits can be modified simultaneously
3 NHEJ enables gene knockout and targeted modifications
4 Introduction of transgenes at defined loci that promote high-level
transcription and do not interfere with the activity of endogenous genes
5 Site-specific nucleases also allow targeted molecular trait stacking -low
risk of segregation
6 CRISPR Cas is a transgene free approach ndash No regulatory burdens
7 frequency of off-target mutations is well below that caused by chemical
and physical mutagenesis techniques
8 In future can be utilized for metabolic engineering and molecular farming
63
Six-way Venn diagram showing the distribution of shared genefamilies (sequence clusters) among M acuminata P dactyliferaArabidopsis thaliana Oryza sativa Sorghum bicolor and Brachypodiumdistachyon genomes
A DrsquoHont et al Nature 2012 doi101038nature11241
64
Achieving super-DomesticationINTROGRESS-RECREATE-CREATE-DOMESTICATEREDOMESTICATE
bull Mobilizing left out genetic
variation still available in
land races and wild and
weedy species
bull Replaying the evolutionary
tape( resynthesis in
polyploids)
bull Domesticateredomesticate
65
We can admire and emulate how indigenous people still domesticateplants create biodiversity and manage it to sustain their future Thereis no equivalently dynamic or flexible crop breeding in modernagriculture to promote biodiversity we still have much to learn from
traditional knowledge Jan Salick 2012
31
Centers of Domestication
Fuller 2011
32
Centers of Domestication in India
Fuller 2011
33
Indian centers of domestication South India (Deccan centre) Vigna radiate Vigna mungo
Macrotyloma uniflorum (Horse gram) Sataria verticillata S plumila
wheat (macaroni) and two rowed barleybrachiaria ramosa
Orissa (Mahanadi river) Pegion pea Horsegram mung small millets
like Echinichloa Paspalum and Sataria
The Middle Ganges (Harappan civilization) Oryza nivara O rufipogon
and wild sativa Sateria pumia Cannabis sativa even the diffusion of
japonica rice
Saurashtra (Harappan civilization) Eleucine coracana Sateria italica
Paicum spp Brachypodia pearl millet and sorghum Dollicos lablab
The Himalayan foothills of the Punjab region Was a centre of
diversity for Japonica type rice and many temperate fruits and
vegetables
34
DOMESTICATION RELATED TRAITS CONTROLLED BY ONLY FEW GENES
Trait Crops
Plant architecturegrowth habit Ricemaizemilletsbeantomato
Flowering timephotoperiod sensitivity Ricemaizesorghumbeantomato
Fruit size Tomato egg plant
Grain size Ricemaizesorghumbean
Seed dispersal Brassica rice
Inflorescence modification Brassica
Dormancy Bean
35
Genes Crop Moleular and phenotypic function Causative change
Genes identified as controlling domestication traits
tb1 Maize Transcriptional regulator (TCP) plant and
inflorescence structure
regulatory change
tga1 Maize Transcriptional regulator (SBP) seed casing amino acid change
qSH1 Rice Transcriptional regulator (homeodomain)
abscission layer formation shattering
regulatory change
Rc Rice Transcriptional regulator (bHLH) seed color disrupted coding
sequence
sh4 Rice Transcriptional regulator (Myb3) abscission
layer formation shattering
regulatoryamino
acid change
fw22 Tomato Cell signaling fruit weight regulatory change
Q Wheat Transcriptional regulator (AP2) inflorescence
structure
regulatoryamino
acid change
Vrs1 Barley Inflorescence structure Premature stop Amino acid change
Genes of interest in crop domestication and improvement
36
Genes Crop Moleular and phenotypic function Causative change
c1
r1
Maize Transcriptional regulator (MYB) kernel color and
Transcriptional regulator (bHLH) kernel color
regulatory change
sh2 Maize pyrophosphorylase supersweet sweet corn transposon
insertion
su1 Maize isoamylase sweet corn gene amino acid change
brix9-2-5 Tomato Invertase fruit soluble solid content amino acid change
ovate Tomato Unknown fruit shap early stop codon
R Pea Starch branching enzyme seed sugar content transposon
insertion
ehd1 Rice B-type response regulator flowering time amino acid change
hd1 Rice Transcriptional regulator (zinc finger) flowering
time
disrupted coding
sequence
waxy Rice Starch synthase sticky grains intron splicing
defect
rht Wheat Transcriptional regulator (SH2) plant height early stop codon
vrn1 Wheat Transcriptional regulator (MADS) vernalization regulatory change
vrn2 Wheat Transcriptional regulator (ZCCT) vernalization amino acid change
Genes Identified as Controlling Varietal Differences
37
Process of domestication over the yearshellip
Rachel S Meyer 2012
38
Genetic bottleneck
Cultivated crops undergone with narrowing of diversity problem
But this is not the case in weeds
Weeds are the major threats from the beginning of domestication till
today
Problem arose due to
1 Crop mimicry
2 Genetic assimilation
3 Genetic evolution
39
Super- Domestication Vaughn et al (2007) first used the term super-domestication
The processes that lead to a domesticate with dramatically
increased yield that could not be selected in natural environments
from naturally occurring variation without recourse to new
technologies
Super-domesticates can be constructed with knowledge led
approaches based on current needs using the range of new
technologies now available
Plants exploited for continuous selection introduction hybridization
etc which boosted the process of domestication but now the plant
genetic engineering approach is exploiting plants towards synthetic
biology
40
How to use diversitybull Cross two varieties
bull Genome manipulations
Cell fusion hybrids
bull Chromosome manipulation Backcross a new species
bull Generate recombinants Chromosome recombinations
bull Use a new species wild germplasm
Transgenic approach Modern mutagenesis synthetic gene
construction by utilizing green florescent protein genome editing
NGS GWAS sequencing etchelliphelliphelliphelliphellip
41
Reduce genetic bottlenecks through polyplodization
More vigour
SC
Buffering capacity
Heterotic advantage
Enhaced vegetative
charachers
Enhanced oil
Meiotic stability
42
Approach 1 Use one tetraploid and one diploid as parents (4X ndash 2X) followed by the
chromosome doubling of triploid hybrids (Chrom doubling)
a) Cross between B juncea (AjAjBjBj ) and B oleracea (CoCo) to produce hexaploids
(AjAjBjBjCoCo)
(b) Cross between B napus (AnAnCnCn) and B nigra (BniBni) to produce hexaploids
(AnAnBniBniCnCn)
(c) Cross between B carinata (BcaBcaCcaCca) and B rapa (ArAr) to produce hexaploids
(ArArBcaBcaCcaCca)
Approach 2 Use three tetraploids as parents
(a) Cross between B napus (AnAnCnCn) and B carinata (BcaBcaCcaCca) to produce unbalanced
allotetraploids (AnBcaCnCca ndash unreduced gametes gametes with the somatic chromosome
number and cross with B juncea (AjAjBjBj) to obtain allohexaploids (AnAjBcaBjCnCca
(b) Cross between B napus (AnAnCnCn) and B juncea (AjAjBjBj) to produce unbalanced
allotetraploids (AnAjBjCn ndash unreduced gametes) and cross with B carinata (BcaBcaCcaCca) to
obtain allohexaploids (AnAjBcaBjCnCca)
(c) Cross between B carinata (BcaBcaCcaCca) and B juncea (AjAjBjBj) to produce unbalanced
allotetraploids (AjBcaBjCca - ndash unreduced gametes) and cross with B napus (AnAnCnCn) to obtain
allohexaploids (AnAjBcaBjCnCca)
Approach 3 Use three diploids as parents (2X ndash 2X ndash 2X) Cross between B rapa (ArAr) B
nigra (BniBni) and B oleracea (CoCo) sequentially to obtain hexaploid hybrids (ArArBniBniCoCo)
43
Continuehellip
Guijun Yan 2012
44
Applications of Genomic
tools in Super-Domestication
45
Genome Sequencing
Potential methods of sequencing
1 Clone by clone approach
2 Whole genome shotgun approach
3 Combination of the two methods
Till today no of cultivated plants completely sequenced -85
2016- Arachis duranensis
2015- Solanum cumersonii (Wild potato)
(Ref- NCBI)
46
GWAS
It is a study design in which many markers spread across a genome
are genotyped and test a statistical association with a phenotype are
performed locally along the genome
It is also an examination of many common genetic variants in different
individuals to see if any variant is associated with a trait
Used in successfully studying maize sorghum and barley
Method is efficient for large scale low cost genotyping (even with the
minimum number of SNPs)
Cannot be utilized generally because it needs large population size
GWAS identify rare alleles more precisely
If small population we can opt NAM
47
GWAS and whole genome prediction
Xuehui Huang and Bin Han 2014
48
Five high throuput genotyping methods
Xuehui Huang and Bin Han 2014
49
Role of NGS in domestication Capture of novel genes from wild species will be made easier by
understanding the molecular events associated with crop domestication
Re-sequencing of domesticated species can identify low diversity regions
resulting from selection during domestication
To identify gene-specific sequences to aid the cloning of homologues of key
domestication genes from wild relatives
Candidate genes from wild and domesticated plant populations can define
diversity of target genes in wild populations and lead to the discovery of key
genes for important traits by association analysis
NGS of amplicons of large numbers of candidate genes from wild and
domesticated plant populations can define diversity of target genes in
wild populations and lead to the discovery of key genes for important
traits by association analysis
50
NGS
supports the rapid domestication of new plant species and the efficient
identification and capture of novel genetic variation from related species
Allows whole-genome analysis to determine the genetic basis of
phenotypic differences
NGS allows rapid expansion of genomic analysis to investigation of non-
model species
Made rice study easy by related grass
cost-effective method for plant identification
useful strategy to analysis the chloroplast genome sequence from whole-
genome shot-gun sequencing
facilitates managing this diversity and any changes in crop performance
over time due to genetic drift
Patterns of gene expression have been evaluated in hybrids using NGS
51
Two Approaches to Finding Adaptive Genes
Ross-Ibarra et al2007
52
Techniques to re-wild the plants (transgene free)
1 Introgression
breeding
2 Specific
insertion of lost genes
3 Precision
mutagenesis
Palmgren et al2014
53
Process of rewilding
Palmgren et al2014
54
Synthetic biology projects(i) Modifying cereals including wheat to fix atmospheric nitrogen
(ii) Redesigning metabolic pathways to increase the yield of
secondary metabolites or to generate compounds with enhanced
properties
(iii) Transferring the C4 photosynthesis pathway to rice
(iv) Modifying the glycosylation pathway in plants to accommodate
production of therapeutic proteins
(v) Introducing synthetic signal transduction systems that respond to
external cues
55
Synthetic biology projects
Nicholas J Baltes et al 2015
56
Refactoring the N fixation gene cluster from Klebsiella
Karsten Temme et al 2012
57
Continuehellip
Karsten Temme et al 2012
58
Genome editing
Sequence specific nucleases
1 Meganucleases
2 ZFMs ndashZink finger motifs
3 TALENs- Transcription activator like effector nuclease
4 CRISPR CAS-9 ndash Clustered interspaced short palindromic
repeats
qmp4
59Khaoula Belhaj et al 2015
60
Genome modifications achieved in plants using sequence specific nucleases
Type of DNA
modification
Nuclease Delivery
methods
Plants Targets
Trait stacking Megnucleases Bombardment Cotton Intergenic sequence
ZFN Bombardment Maize Transgene
Rewriting host
DNA
Megnucleases Stable integration Maize Intergenic sequence
ZFN Stable integration Soybean Transgene
TALEN Stable integration Barley PAPhy_a
TALEN Agrobacterium
T-DNA (transient)
Oryza sativa SWEET14
TALEN No Protoplasts Arabidopsis
Tobacco
AtTT4 AtADH NbSurB
TALEN Bombardment Wheat MLO
TALEN Protoplasts
Stable integration
Maize PDS IPK1A IPK MRP4
CRISPRCas Protoplasts
Agrobacterium
T-DNA (transient)
Tobacco
Arabidopsis
Sorghum Oryza
OsSWEET14 transgene
61
Type of DNA
modification
Nuclease Delivery
methods
Plants Targets
Rewriting host
DNA large
deletion
Zinc-finger
nuclease
Stable
integration
Tobacco Transgene
CRISPRCas Protoplasts
Stable
integration
Rice Labdane-related
diterpenoid gene
clusters
on Chr 2 4 and 6
Zinc-finger
nuclease
Agrobacterium
T-DNA
(transient)
Tobacco CHN50 transgene
Zinc-finger
nuclease
Whiskers Maize IPK1
CRISPRCas Protoplasts Rice PDS
Controlling gene
expression
TALE repressor
(SRDX)
Stable
integration
Arabidopsis RD29A transgene
Zinc-finger
activator (VP16)
Stable
integration
Brassica
napus
KasII
Continue
62
Application of genome editing1 Introduction of precise and predictable modifications directly in an elite
background
2 multiple traits can be modified simultaneously
3 NHEJ enables gene knockout and targeted modifications
4 Introduction of transgenes at defined loci that promote high-level
transcription and do not interfere with the activity of endogenous genes
5 Site-specific nucleases also allow targeted molecular trait stacking -low
risk of segregation
6 CRISPR Cas is a transgene free approach ndash No regulatory burdens
7 frequency of off-target mutations is well below that caused by chemical
and physical mutagenesis techniques
8 In future can be utilized for metabolic engineering and molecular farming
63
Six-way Venn diagram showing the distribution of shared genefamilies (sequence clusters) among M acuminata P dactyliferaArabidopsis thaliana Oryza sativa Sorghum bicolor and Brachypodiumdistachyon genomes
A DrsquoHont et al Nature 2012 doi101038nature11241
64
Achieving super-DomesticationINTROGRESS-RECREATE-CREATE-DOMESTICATEREDOMESTICATE
bull Mobilizing left out genetic
variation still available in
land races and wild and
weedy species
bull Replaying the evolutionary
tape( resynthesis in
polyploids)
bull Domesticateredomesticate
65
We can admire and emulate how indigenous people still domesticateplants create biodiversity and manage it to sustain their future Thereis no equivalently dynamic or flexible crop breeding in modernagriculture to promote biodiversity we still have much to learn from
traditional knowledge Jan Salick 2012
32
Centers of Domestication in India
Fuller 2011
33
Indian centers of domestication South India (Deccan centre) Vigna radiate Vigna mungo
Macrotyloma uniflorum (Horse gram) Sataria verticillata S plumila
wheat (macaroni) and two rowed barleybrachiaria ramosa
Orissa (Mahanadi river) Pegion pea Horsegram mung small millets
like Echinichloa Paspalum and Sataria
The Middle Ganges (Harappan civilization) Oryza nivara O rufipogon
and wild sativa Sateria pumia Cannabis sativa even the diffusion of
japonica rice
Saurashtra (Harappan civilization) Eleucine coracana Sateria italica
Paicum spp Brachypodia pearl millet and sorghum Dollicos lablab
The Himalayan foothills of the Punjab region Was a centre of
diversity for Japonica type rice and many temperate fruits and
vegetables
34
DOMESTICATION RELATED TRAITS CONTROLLED BY ONLY FEW GENES
Trait Crops
Plant architecturegrowth habit Ricemaizemilletsbeantomato
Flowering timephotoperiod sensitivity Ricemaizesorghumbeantomato
Fruit size Tomato egg plant
Grain size Ricemaizesorghumbean
Seed dispersal Brassica rice
Inflorescence modification Brassica
Dormancy Bean
35
Genes Crop Moleular and phenotypic function Causative change
Genes identified as controlling domestication traits
tb1 Maize Transcriptional regulator (TCP) plant and
inflorescence structure
regulatory change
tga1 Maize Transcriptional regulator (SBP) seed casing amino acid change
qSH1 Rice Transcriptional regulator (homeodomain)
abscission layer formation shattering
regulatory change
Rc Rice Transcriptional regulator (bHLH) seed color disrupted coding
sequence
sh4 Rice Transcriptional regulator (Myb3) abscission
layer formation shattering
regulatoryamino
acid change
fw22 Tomato Cell signaling fruit weight regulatory change
Q Wheat Transcriptional regulator (AP2) inflorescence
structure
regulatoryamino
acid change
Vrs1 Barley Inflorescence structure Premature stop Amino acid change
Genes of interest in crop domestication and improvement
36
Genes Crop Moleular and phenotypic function Causative change
c1
r1
Maize Transcriptional regulator (MYB) kernel color and
Transcriptional regulator (bHLH) kernel color
regulatory change
sh2 Maize pyrophosphorylase supersweet sweet corn transposon
insertion
su1 Maize isoamylase sweet corn gene amino acid change
brix9-2-5 Tomato Invertase fruit soluble solid content amino acid change
ovate Tomato Unknown fruit shap early stop codon
R Pea Starch branching enzyme seed sugar content transposon
insertion
ehd1 Rice B-type response regulator flowering time amino acid change
hd1 Rice Transcriptional regulator (zinc finger) flowering
time
disrupted coding
sequence
waxy Rice Starch synthase sticky grains intron splicing
defect
rht Wheat Transcriptional regulator (SH2) plant height early stop codon
vrn1 Wheat Transcriptional regulator (MADS) vernalization regulatory change
vrn2 Wheat Transcriptional regulator (ZCCT) vernalization amino acid change
Genes Identified as Controlling Varietal Differences
37
Process of domestication over the yearshellip
Rachel S Meyer 2012
38
Genetic bottleneck
Cultivated crops undergone with narrowing of diversity problem
But this is not the case in weeds
Weeds are the major threats from the beginning of domestication till
today
Problem arose due to
1 Crop mimicry
2 Genetic assimilation
3 Genetic evolution
39
Super- Domestication Vaughn et al (2007) first used the term super-domestication
The processes that lead to a domesticate with dramatically
increased yield that could not be selected in natural environments
from naturally occurring variation without recourse to new
technologies
Super-domesticates can be constructed with knowledge led
approaches based on current needs using the range of new
technologies now available
Plants exploited for continuous selection introduction hybridization
etc which boosted the process of domestication but now the plant
genetic engineering approach is exploiting plants towards synthetic
biology
40
How to use diversitybull Cross two varieties
bull Genome manipulations
Cell fusion hybrids
bull Chromosome manipulation Backcross a new species
bull Generate recombinants Chromosome recombinations
bull Use a new species wild germplasm
Transgenic approach Modern mutagenesis synthetic gene
construction by utilizing green florescent protein genome editing
NGS GWAS sequencing etchelliphelliphelliphelliphellip
41
Reduce genetic bottlenecks through polyplodization
More vigour
SC
Buffering capacity
Heterotic advantage
Enhaced vegetative
charachers
Enhanced oil
Meiotic stability
42
Approach 1 Use one tetraploid and one diploid as parents (4X ndash 2X) followed by the
chromosome doubling of triploid hybrids (Chrom doubling)
a) Cross between B juncea (AjAjBjBj ) and B oleracea (CoCo) to produce hexaploids
(AjAjBjBjCoCo)
(b) Cross between B napus (AnAnCnCn) and B nigra (BniBni) to produce hexaploids
(AnAnBniBniCnCn)
(c) Cross between B carinata (BcaBcaCcaCca) and B rapa (ArAr) to produce hexaploids
(ArArBcaBcaCcaCca)
Approach 2 Use three tetraploids as parents
(a) Cross between B napus (AnAnCnCn) and B carinata (BcaBcaCcaCca) to produce unbalanced
allotetraploids (AnBcaCnCca ndash unreduced gametes gametes with the somatic chromosome
number and cross with B juncea (AjAjBjBj) to obtain allohexaploids (AnAjBcaBjCnCca
(b) Cross between B napus (AnAnCnCn) and B juncea (AjAjBjBj) to produce unbalanced
allotetraploids (AnAjBjCn ndash unreduced gametes) and cross with B carinata (BcaBcaCcaCca) to
obtain allohexaploids (AnAjBcaBjCnCca)
(c) Cross between B carinata (BcaBcaCcaCca) and B juncea (AjAjBjBj) to produce unbalanced
allotetraploids (AjBcaBjCca - ndash unreduced gametes) and cross with B napus (AnAnCnCn) to obtain
allohexaploids (AnAjBcaBjCnCca)
Approach 3 Use three diploids as parents (2X ndash 2X ndash 2X) Cross between B rapa (ArAr) B
nigra (BniBni) and B oleracea (CoCo) sequentially to obtain hexaploid hybrids (ArArBniBniCoCo)
43
Continuehellip
Guijun Yan 2012
44
Applications of Genomic
tools in Super-Domestication
45
Genome Sequencing
Potential methods of sequencing
1 Clone by clone approach
2 Whole genome shotgun approach
3 Combination of the two methods
Till today no of cultivated plants completely sequenced -85
2016- Arachis duranensis
2015- Solanum cumersonii (Wild potato)
(Ref- NCBI)
46
GWAS
It is a study design in which many markers spread across a genome
are genotyped and test a statistical association with a phenotype are
performed locally along the genome
It is also an examination of many common genetic variants in different
individuals to see if any variant is associated with a trait
Used in successfully studying maize sorghum and barley
Method is efficient for large scale low cost genotyping (even with the
minimum number of SNPs)
Cannot be utilized generally because it needs large population size
GWAS identify rare alleles more precisely
If small population we can opt NAM
47
GWAS and whole genome prediction
Xuehui Huang and Bin Han 2014
48
Five high throuput genotyping methods
Xuehui Huang and Bin Han 2014
49
Role of NGS in domestication Capture of novel genes from wild species will be made easier by
understanding the molecular events associated with crop domestication
Re-sequencing of domesticated species can identify low diversity regions
resulting from selection during domestication
To identify gene-specific sequences to aid the cloning of homologues of key
domestication genes from wild relatives
Candidate genes from wild and domesticated plant populations can define
diversity of target genes in wild populations and lead to the discovery of key
genes for important traits by association analysis
NGS of amplicons of large numbers of candidate genes from wild and
domesticated plant populations can define diversity of target genes in
wild populations and lead to the discovery of key genes for important
traits by association analysis
50
NGS
supports the rapid domestication of new plant species and the efficient
identification and capture of novel genetic variation from related species
Allows whole-genome analysis to determine the genetic basis of
phenotypic differences
NGS allows rapid expansion of genomic analysis to investigation of non-
model species
Made rice study easy by related grass
cost-effective method for plant identification
useful strategy to analysis the chloroplast genome sequence from whole-
genome shot-gun sequencing
facilitates managing this diversity and any changes in crop performance
over time due to genetic drift
Patterns of gene expression have been evaluated in hybrids using NGS
51
Two Approaches to Finding Adaptive Genes
Ross-Ibarra et al2007
52
Techniques to re-wild the plants (transgene free)
1 Introgression
breeding
2 Specific
insertion of lost genes
3 Precision
mutagenesis
Palmgren et al2014
53
Process of rewilding
Palmgren et al2014
54
Synthetic biology projects(i) Modifying cereals including wheat to fix atmospheric nitrogen
(ii) Redesigning metabolic pathways to increase the yield of
secondary metabolites or to generate compounds with enhanced
properties
(iii) Transferring the C4 photosynthesis pathway to rice
(iv) Modifying the glycosylation pathway in plants to accommodate
production of therapeutic proteins
(v) Introducing synthetic signal transduction systems that respond to
external cues
55
Synthetic biology projects
Nicholas J Baltes et al 2015
56
Refactoring the N fixation gene cluster from Klebsiella
Karsten Temme et al 2012
57
Continuehellip
Karsten Temme et al 2012
58
Genome editing
Sequence specific nucleases
1 Meganucleases
2 ZFMs ndashZink finger motifs
3 TALENs- Transcription activator like effector nuclease
4 CRISPR CAS-9 ndash Clustered interspaced short palindromic
repeats
qmp4
59Khaoula Belhaj et al 2015
60
Genome modifications achieved in plants using sequence specific nucleases
Type of DNA
modification
Nuclease Delivery
methods
Plants Targets
Trait stacking Megnucleases Bombardment Cotton Intergenic sequence
ZFN Bombardment Maize Transgene
Rewriting host
DNA
Megnucleases Stable integration Maize Intergenic sequence
ZFN Stable integration Soybean Transgene
TALEN Stable integration Barley PAPhy_a
TALEN Agrobacterium
T-DNA (transient)
Oryza sativa SWEET14
TALEN No Protoplasts Arabidopsis
Tobacco
AtTT4 AtADH NbSurB
TALEN Bombardment Wheat MLO
TALEN Protoplasts
Stable integration
Maize PDS IPK1A IPK MRP4
CRISPRCas Protoplasts
Agrobacterium
T-DNA (transient)
Tobacco
Arabidopsis
Sorghum Oryza
OsSWEET14 transgene
61
Type of DNA
modification
Nuclease Delivery
methods
Plants Targets
Rewriting host
DNA large
deletion
Zinc-finger
nuclease
Stable
integration
Tobacco Transgene
CRISPRCas Protoplasts
Stable
integration
Rice Labdane-related
diterpenoid gene
clusters
on Chr 2 4 and 6
Zinc-finger
nuclease
Agrobacterium
T-DNA
(transient)
Tobacco CHN50 transgene
Zinc-finger
nuclease
Whiskers Maize IPK1
CRISPRCas Protoplasts Rice PDS
Controlling gene
expression
TALE repressor
(SRDX)
Stable
integration
Arabidopsis RD29A transgene
Zinc-finger
activator (VP16)
Stable
integration
Brassica
napus
KasII
Continue
62
Application of genome editing1 Introduction of precise and predictable modifications directly in an elite
background
2 multiple traits can be modified simultaneously
3 NHEJ enables gene knockout and targeted modifications
4 Introduction of transgenes at defined loci that promote high-level
transcription and do not interfere with the activity of endogenous genes
5 Site-specific nucleases also allow targeted molecular trait stacking -low
risk of segregation
6 CRISPR Cas is a transgene free approach ndash No regulatory burdens
7 frequency of off-target mutations is well below that caused by chemical
and physical mutagenesis techniques
8 In future can be utilized for metabolic engineering and molecular farming
63
Six-way Venn diagram showing the distribution of shared genefamilies (sequence clusters) among M acuminata P dactyliferaArabidopsis thaliana Oryza sativa Sorghum bicolor and Brachypodiumdistachyon genomes
A DrsquoHont et al Nature 2012 doi101038nature11241
64
Achieving super-DomesticationINTROGRESS-RECREATE-CREATE-DOMESTICATEREDOMESTICATE
bull Mobilizing left out genetic
variation still available in
land races and wild and
weedy species
bull Replaying the evolutionary
tape( resynthesis in
polyploids)
bull Domesticateredomesticate
65
We can admire and emulate how indigenous people still domesticateplants create biodiversity and manage it to sustain their future Thereis no equivalently dynamic or flexible crop breeding in modernagriculture to promote biodiversity we still have much to learn from
traditional knowledge Jan Salick 2012
33
Indian centers of domestication South India (Deccan centre) Vigna radiate Vigna mungo
Macrotyloma uniflorum (Horse gram) Sataria verticillata S plumila
wheat (macaroni) and two rowed barleybrachiaria ramosa
Orissa (Mahanadi river) Pegion pea Horsegram mung small millets
like Echinichloa Paspalum and Sataria
The Middle Ganges (Harappan civilization) Oryza nivara O rufipogon
and wild sativa Sateria pumia Cannabis sativa even the diffusion of
japonica rice
Saurashtra (Harappan civilization) Eleucine coracana Sateria italica
Paicum spp Brachypodia pearl millet and sorghum Dollicos lablab
The Himalayan foothills of the Punjab region Was a centre of
diversity for Japonica type rice and many temperate fruits and
vegetables
34
DOMESTICATION RELATED TRAITS CONTROLLED BY ONLY FEW GENES
Trait Crops
Plant architecturegrowth habit Ricemaizemilletsbeantomato
Flowering timephotoperiod sensitivity Ricemaizesorghumbeantomato
Fruit size Tomato egg plant
Grain size Ricemaizesorghumbean
Seed dispersal Brassica rice
Inflorescence modification Brassica
Dormancy Bean
35
Genes Crop Moleular and phenotypic function Causative change
Genes identified as controlling domestication traits
tb1 Maize Transcriptional regulator (TCP) plant and
inflorescence structure
regulatory change
tga1 Maize Transcriptional regulator (SBP) seed casing amino acid change
qSH1 Rice Transcriptional regulator (homeodomain)
abscission layer formation shattering
regulatory change
Rc Rice Transcriptional regulator (bHLH) seed color disrupted coding
sequence
sh4 Rice Transcriptional regulator (Myb3) abscission
layer formation shattering
regulatoryamino
acid change
fw22 Tomato Cell signaling fruit weight regulatory change
Q Wheat Transcriptional regulator (AP2) inflorescence
structure
regulatoryamino
acid change
Vrs1 Barley Inflorescence structure Premature stop Amino acid change
Genes of interest in crop domestication and improvement
36
Genes Crop Moleular and phenotypic function Causative change
c1
r1
Maize Transcriptional regulator (MYB) kernel color and
Transcriptional regulator (bHLH) kernel color
regulatory change
sh2 Maize pyrophosphorylase supersweet sweet corn transposon
insertion
su1 Maize isoamylase sweet corn gene amino acid change
brix9-2-5 Tomato Invertase fruit soluble solid content amino acid change
ovate Tomato Unknown fruit shap early stop codon
R Pea Starch branching enzyme seed sugar content transposon
insertion
ehd1 Rice B-type response regulator flowering time amino acid change
hd1 Rice Transcriptional regulator (zinc finger) flowering
time
disrupted coding
sequence
waxy Rice Starch synthase sticky grains intron splicing
defect
rht Wheat Transcriptional regulator (SH2) plant height early stop codon
vrn1 Wheat Transcriptional regulator (MADS) vernalization regulatory change
vrn2 Wheat Transcriptional regulator (ZCCT) vernalization amino acid change
Genes Identified as Controlling Varietal Differences
37
Process of domestication over the yearshellip
Rachel S Meyer 2012
38
Genetic bottleneck
Cultivated crops undergone with narrowing of diversity problem
But this is not the case in weeds
Weeds are the major threats from the beginning of domestication till
today
Problem arose due to
1 Crop mimicry
2 Genetic assimilation
3 Genetic evolution
39
Super- Domestication Vaughn et al (2007) first used the term super-domestication
The processes that lead to a domesticate with dramatically
increased yield that could not be selected in natural environments
from naturally occurring variation without recourse to new
technologies
Super-domesticates can be constructed with knowledge led
approaches based on current needs using the range of new
technologies now available
Plants exploited for continuous selection introduction hybridization
etc which boosted the process of domestication but now the plant
genetic engineering approach is exploiting plants towards synthetic
biology
40
How to use diversitybull Cross two varieties
bull Genome manipulations
Cell fusion hybrids
bull Chromosome manipulation Backcross a new species
bull Generate recombinants Chromosome recombinations
bull Use a new species wild germplasm
Transgenic approach Modern mutagenesis synthetic gene
construction by utilizing green florescent protein genome editing
NGS GWAS sequencing etchelliphelliphelliphelliphellip
41
Reduce genetic bottlenecks through polyplodization
More vigour
SC
Buffering capacity
Heterotic advantage
Enhaced vegetative
charachers
Enhanced oil
Meiotic stability
42
Approach 1 Use one tetraploid and one diploid as parents (4X ndash 2X) followed by the
chromosome doubling of triploid hybrids (Chrom doubling)
a) Cross between B juncea (AjAjBjBj ) and B oleracea (CoCo) to produce hexaploids
(AjAjBjBjCoCo)
(b) Cross between B napus (AnAnCnCn) and B nigra (BniBni) to produce hexaploids
(AnAnBniBniCnCn)
(c) Cross between B carinata (BcaBcaCcaCca) and B rapa (ArAr) to produce hexaploids
(ArArBcaBcaCcaCca)
Approach 2 Use three tetraploids as parents
(a) Cross between B napus (AnAnCnCn) and B carinata (BcaBcaCcaCca) to produce unbalanced
allotetraploids (AnBcaCnCca ndash unreduced gametes gametes with the somatic chromosome
number and cross with B juncea (AjAjBjBj) to obtain allohexaploids (AnAjBcaBjCnCca
(b) Cross between B napus (AnAnCnCn) and B juncea (AjAjBjBj) to produce unbalanced
allotetraploids (AnAjBjCn ndash unreduced gametes) and cross with B carinata (BcaBcaCcaCca) to
obtain allohexaploids (AnAjBcaBjCnCca)
(c) Cross between B carinata (BcaBcaCcaCca) and B juncea (AjAjBjBj) to produce unbalanced
allotetraploids (AjBcaBjCca - ndash unreduced gametes) and cross with B napus (AnAnCnCn) to obtain
allohexaploids (AnAjBcaBjCnCca)
Approach 3 Use three diploids as parents (2X ndash 2X ndash 2X) Cross between B rapa (ArAr) B
nigra (BniBni) and B oleracea (CoCo) sequentially to obtain hexaploid hybrids (ArArBniBniCoCo)
43
Continuehellip
Guijun Yan 2012
44
Applications of Genomic
tools in Super-Domestication
45
Genome Sequencing
Potential methods of sequencing
1 Clone by clone approach
2 Whole genome shotgun approach
3 Combination of the two methods
Till today no of cultivated plants completely sequenced -85
2016- Arachis duranensis
2015- Solanum cumersonii (Wild potato)
(Ref- NCBI)
46
GWAS
It is a study design in which many markers spread across a genome
are genotyped and test a statistical association with a phenotype are
performed locally along the genome
It is also an examination of many common genetic variants in different
individuals to see if any variant is associated with a trait
Used in successfully studying maize sorghum and barley
Method is efficient for large scale low cost genotyping (even with the
minimum number of SNPs)
Cannot be utilized generally because it needs large population size
GWAS identify rare alleles more precisely
If small population we can opt NAM
47
GWAS and whole genome prediction
Xuehui Huang and Bin Han 2014
48
Five high throuput genotyping methods
Xuehui Huang and Bin Han 2014
49
Role of NGS in domestication Capture of novel genes from wild species will be made easier by
understanding the molecular events associated with crop domestication
Re-sequencing of domesticated species can identify low diversity regions
resulting from selection during domestication
To identify gene-specific sequences to aid the cloning of homologues of key
domestication genes from wild relatives
Candidate genes from wild and domesticated plant populations can define
diversity of target genes in wild populations and lead to the discovery of key
genes for important traits by association analysis
NGS of amplicons of large numbers of candidate genes from wild and
domesticated plant populations can define diversity of target genes in
wild populations and lead to the discovery of key genes for important
traits by association analysis
50
NGS
supports the rapid domestication of new plant species and the efficient
identification and capture of novel genetic variation from related species
Allows whole-genome analysis to determine the genetic basis of
phenotypic differences
NGS allows rapid expansion of genomic analysis to investigation of non-
model species
Made rice study easy by related grass
cost-effective method for plant identification
useful strategy to analysis the chloroplast genome sequence from whole-
genome shot-gun sequencing
facilitates managing this diversity and any changes in crop performance
over time due to genetic drift
Patterns of gene expression have been evaluated in hybrids using NGS
51
Two Approaches to Finding Adaptive Genes
Ross-Ibarra et al2007
52
Techniques to re-wild the plants (transgene free)
1 Introgression
breeding
2 Specific
insertion of lost genes
3 Precision
mutagenesis
Palmgren et al2014
53
Process of rewilding
Palmgren et al2014
54
Synthetic biology projects(i) Modifying cereals including wheat to fix atmospheric nitrogen
(ii) Redesigning metabolic pathways to increase the yield of
secondary metabolites or to generate compounds with enhanced
properties
(iii) Transferring the C4 photosynthesis pathway to rice
(iv) Modifying the glycosylation pathway in plants to accommodate
production of therapeutic proteins
(v) Introducing synthetic signal transduction systems that respond to
external cues
55
Synthetic biology projects
Nicholas J Baltes et al 2015
56
Refactoring the N fixation gene cluster from Klebsiella
Karsten Temme et al 2012
57
Continuehellip
Karsten Temme et al 2012
58
Genome editing
Sequence specific nucleases
1 Meganucleases
2 ZFMs ndashZink finger motifs
3 TALENs- Transcription activator like effector nuclease
4 CRISPR CAS-9 ndash Clustered interspaced short palindromic
repeats
qmp4
59Khaoula Belhaj et al 2015
60
Genome modifications achieved in plants using sequence specific nucleases
Type of DNA
modification
Nuclease Delivery
methods
Plants Targets
Trait stacking Megnucleases Bombardment Cotton Intergenic sequence
ZFN Bombardment Maize Transgene
Rewriting host
DNA
Megnucleases Stable integration Maize Intergenic sequence
ZFN Stable integration Soybean Transgene
TALEN Stable integration Barley PAPhy_a
TALEN Agrobacterium
T-DNA (transient)
Oryza sativa SWEET14
TALEN No Protoplasts Arabidopsis
Tobacco
AtTT4 AtADH NbSurB
TALEN Bombardment Wheat MLO
TALEN Protoplasts
Stable integration
Maize PDS IPK1A IPK MRP4
CRISPRCas Protoplasts
Agrobacterium
T-DNA (transient)
Tobacco
Arabidopsis
Sorghum Oryza
OsSWEET14 transgene
61
Type of DNA
modification
Nuclease Delivery
methods
Plants Targets
Rewriting host
DNA large
deletion
Zinc-finger
nuclease
Stable
integration
Tobacco Transgene
CRISPRCas Protoplasts
Stable
integration
Rice Labdane-related
diterpenoid gene
clusters
on Chr 2 4 and 6
Zinc-finger
nuclease
Agrobacterium
T-DNA
(transient)
Tobacco CHN50 transgene
Zinc-finger
nuclease
Whiskers Maize IPK1
CRISPRCas Protoplasts Rice PDS
Controlling gene
expression
TALE repressor
(SRDX)
Stable
integration
Arabidopsis RD29A transgene
Zinc-finger
activator (VP16)
Stable
integration
Brassica
napus
KasII
Continue
62
Application of genome editing1 Introduction of precise and predictable modifications directly in an elite
background
2 multiple traits can be modified simultaneously
3 NHEJ enables gene knockout and targeted modifications
4 Introduction of transgenes at defined loci that promote high-level
transcription and do not interfere with the activity of endogenous genes
5 Site-specific nucleases also allow targeted molecular trait stacking -low
risk of segregation
6 CRISPR Cas is a transgene free approach ndash No regulatory burdens
7 frequency of off-target mutations is well below that caused by chemical
and physical mutagenesis techniques
8 In future can be utilized for metabolic engineering and molecular farming
63
Six-way Venn diagram showing the distribution of shared genefamilies (sequence clusters) among M acuminata P dactyliferaArabidopsis thaliana Oryza sativa Sorghum bicolor and Brachypodiumdistachyon genomes
A DrsquoHont et al Nature 2012 doi101038nature11241
64
Achieving super-DomesticationINTROGRESS-RECREATE-CREATE-DOMESTICATEREDOMESTICATE
bull Mobilizing left out genetic
variation still available in
land races and wild and
weedy species
bull Replaying the evolutionary
tape( resynthesis in
polyploids)
bull Domesticateredomesticate
65
We can admire and emulate how indigenous people still domesticateplants create biodiversity and manage it to sustain their future Thereis no equivalently dynamic or flexible crop breeding in modernagriculture to promote biodiversity we still have much to learn from
traditional knowledge Jan Salick 2012
34
DOMESTICATION RELATED TRAITS CONTROLLED BY ONLY FEW GENES
Trait Crops
Plant architecturegrowth habit Ricemaizemilletsbeantomato
Flowering timephotoperiod sensitivity Ricemaizesorghumbeantomato
Fruit size Tomato egg plant
Grain size Ricemaizesorghumbean
Seed dispersal Brassica rice
Inflorescence modification Brassica
Dormancy Bean
35
Genes Crop Moleular and phenotypic function Causative change
Genes identified as controlling domestication traits
tb1 Maize Transcriptional regulator (TCP) plant and
inflorescence structure
regulatory change
tga1 Maize Transcriptional regulator (SBP) seed casing amino acid change
qSH1 Rice Transcriptional regulator (homeodomain)
abscission layer formation shattering
regulatory change
Rc Rice Transcriptional regulator (bHLH) seed color disrupted coding
sequence
sh4 Rice Transcriptional regulator (Myb3) abscission
layer formation shattering
regulatoryamino
acid change
fw22 Tomato Cell signaling fruit weight regulatory change
Q Wheat Transcriptional regulator (AP2) inflorescence
structure
regulatoryamino
acid change
Vrs1 Barley Inflorescence structure Premature stop Amino acid change
Genes of interest in crop domestication and improvement
36
Genes Crop Moleular and phenotypic function Causative change
c1
r1
Maize Transcriptional regulator (MYB) kernel color and
Transcriptional regulator (bHLH) kernel color
regulatory change
sh2 Maize pyrophosphorylase supersweet sweet corn transposon
insertion
su1 Maize isoamylase sweet corn gene amino acid change
brix9-2-5 Tomato Invertase fruit soluble solid content amino acid change
ovate Tomato Unknown fruit shap early stop codon
R Pea Starch branching enzyme seed sugar content transposon
insertion
ehd1 Rice B-type response regulator flowering time amino acid change
hd1 Rice Transcriptional regulator (zinc finger) flowering
time
disrupted coding
sequence
waxy Rice Starch synthase sticky grains intron splicing
defect
rht Wheat Transcriptional regulator (SH2) plant height early stop codon
vrn1 Wheat Transcriptional regulator (MADS) vernalization regulatory change
vrn2 Wheat Transcriptional regulator (ZCCT) vernalization amino acid change
Genes Identified as Controlling Varietal Differences
37
Process of domestication over the yearshellip
Rachel S Meyer 2012
38
Genetic bottleneck
Cultivated crops undergone with narrowing of diversity problem
But this is not the case in weeds
Weeds are the major threats from the beginning of domestication till
today
Problem arose due to
1 Crop mimicry
2 Genetic assimilation
3 Genetic evolution
39
Super- Domestication Vaughn et al (2007) first used the term super-domestication
The processes that lead to a domesticate with dramatically
increased yield that could not be selected in natural environments
from naturally occurring variation without recourse to new
technologies
Super-domesticates can be constructed with knowledge led
approaches based on current needs using the range of new
technologies now available
Plants exploited for continuous selection introduction hybridization
etc which boosted the process of domestication but now the plant
genetic engineering approach is exploiting plants towards synthetic
biology
40
How to use diversitybull Cross two varieties
bull Genome manipulations
Cell fusion hybrids
bull Chromosome manipulation Backcross a new species
bull Generate recombinants Chromosome recombinations
bull Use a new species wild germplasm
Transgenic approach Modern mutagenesis synthetic gene
construction by utilizing green florescent protein genome editing
NGS GWAS sequencing etchelliphelliphelliphelliphellip
41
Reduce genetic bottlenecks through polyplodization
More vigour
SC
Buffering capacity
Heterotic advantage
Enhaced vegetative
charachers
Enhanced oil
Meiotic stability
42
Approach 1 Use one tetraploid and one diploid as parents (4X ndash 2X) followed by the
chromosome doubling of triploid hybrids (Chrom doubling)
a) Cross between B juncea (AjAjBjBj ) and B oleracea (CoCo) to produce hexaploids
(AjAjBjBjCoCo)
(b) Cross between B napus (AnAnCnCn) and B nigra (BniBni) to produce hexaploids
(AnAnBniBniCnCn)
(c) Cross between B carinata (BcaBcaCcaCca) and B rapa (ArAr) to produce hexaploids
(ArArBcaBcaCcaCca)
Approach 2 Use three tetraploids as parents
(a) Cross between B napus (AnAnCnCn) and B carinata (BcaBcaCcaCca) to produce unbalanced
allotetraploids (AnBcaCnCca ndash unreduced gametes gametes with the somatic chromosome
number and cross with B juncea (AjAjBjBj) to obtain allohexaploids (AnAjBcaBjCnCca
(b) Cross between B napus (AnAnCnCn) and B juncea (AjAjBjBj) to produce unbalanced
allotetraploids (AnAjBjCn ndash unreduced gametes) and cross with B carinata (BcaBcaCcaCca) to
obtain allohexaploids (AnAjBcaBjCnCca)
(c) Cross between B carinata (BcaBcaCcaCca) and B juncea (AjAjBjBj) to produce unbalanced
allotetraploids (AjBcaBjCca - ndash unreduced gametes) and cross with B napus (AnAnCnCn) to obtain
allohexaploids (AnAjBcaBjCnCca)
Approach 3 Use three diploids as parents (2X ndash 2X ndash 2X) Cross between B rapa (ArAr) B
nigra (BniBni) and B oleracea (CoCo) sequentially to obtain hexaploid hybrids (ArArBniBniCoCo)
43
Continuehellip
Guijun Yan 2012
44
Applications of Genomic
tools in Super-Domestication
45
Genome Sequencing
Potential methods of sequencing
1 Clone by clone approach
2 Whole genome shotgun approach
3 Combination of the two methods
Till today no of cultivated plants completely sequenced -85
2016- Arachis duranensis
2015- Solanum cumersonii (Wild potato)
(Ref- NCBI)
46
GWAS
It is a study design in which many markers spread across a genome
are genotyped and test a statistical association with a phenotype are
performed locally along the genome
It is also an examination of many common genetic variants in different
individuals to see if any variant is associated with a trait
Used in successfully studying maize sorghum and barley
Method is efficient for large scale low cost genotyping (even with the
minimum number of SNPs)
Cannot be utilized generally because it needs large population size
GWAS identify rare alleles more precisely
If small population we can opt NAM
47
GWAS and whole genome prediction
Xuehui Huang and Bin Han 2014
48
Five high throuput genotyping methods
Xuehui Huang and Bin Han 2014
49
Role of NGS in domestication Capture of novel genes from wild species will be made easier by
understanding the molecular events associated with crop domestication
Re-sequencing of domesticated species can identify low diversity regions
resulting from selection during domestication
To identify gene-specific sequences to aid the cloning of homologues of key
domestication genes from wild relatives
Candidate genes from wild and domesticated plant populations can define
diversity of target genes in wild populations and lead to the discovery of key
genes for important traits by association analysis
NGS of amplicons of large numbers of candidate genes from wild and
domesticated plant populations can define diversity of target genes in
wild populations and lead to the discovery of key genes for important
traits by association analysis
50
NGS
supports the rapid domestication of new plant species and the efficient
identification and capture of novel genetic variation from related species
Allows whole-genome analysis to determine the genetic basis of
phenotypic differences
NGS allows rapid expansion of genomic analysis to investigation of non-
model species
Made rice study easy by related grass
cost-effective method for plant identification
useful strategy to analysis the chloroplast genome sequence from whole-
genome shot-gun sequencing
facilitates managing this diversity and any changes in crop performance
over time due to genetic drift
Patterns of gene expression have been evaluated in hybrids using NGS
51
Two Approaches to Finding Adaptive Genes
Ross-Ibarra et al2007
52
Techniques to re-wild the plants (transgene free)
1 Introgression
breeding
2 Specific
insertion of lost genes
3 Precision
mutagenesis
Palmgren et al2014
53
Process of rewilding
Palmgren et al2014
54
Synthetic biology projects(i) Modifying cereals including wheat to fix atmospheric nitrogen
(ii) Redesigning metabolic pathways to increase the yield of
secondary metabolites or to generate compounds with enhanced
properties
(iii) Transferring the C4 photosynthesis pathway to rice
(iv) Modifying the glycosylation pathway in plants to accommodate
production of therapeutic proteins
(v) Introducing synthetic signal transduction systems that respond to
external cues
55
Synthetic biology projects
Nicholas J Baltes et al 2015
56
Refactoring the N fixation gene cluster from Klebsiella
Karsten Temme et al 2012
57
Continuehellip
Karsten Temme et al 2012
58
Genome editing
Sequence specific nucleases
1 Meganucleases
2 ZFMs ndashZink finger motifs
3 TALENs- Transcription activator like effector nuclease
4 CRISPR CAS-9 ndash Clustered interspaced short palindromic
repeats
qmp4
59Khaoula Belhaj et al 2015
60
Genome modifications achieved in plants using sequence specific nucleases
Type of DNA
modification
Nuclease Delivery
methods
Plants Targets
Trait stacking Megnucleases Bombardment Cotton Intergenic sequence
ZFN Bombardment Maize Transgene
Rewriting host
DNA
Megnucleases Stable integration Maize Intergenic sequence
ZFN Stable integration Soybean Transgene
TALEN Stable integration Barley PAPhy_a
TALEN Agrobacterium
T-DNA (transient)
Oryza sativa SWEET14
TALEN No Protoplasts Arabidopsis
Tobacco
AtTT4 AtADH NbSurB
TALEN Bombardment Wheat MLO
TALEN Protoplasts
Stable integration
Maize PDS IPK1A IPK MRP4
CRISPRCas Protoplasts
Agrobacterium
T-DNA (transient)
Tobacco
Arabidopsis
Sorghum Oryza
OsSWEET14 transgene
61
Type of DNA
modification
Nuclease Delivery
methods
Plants Targets
Rewriting host
DNA large
deletion
Zinc-finger
nuclease
Stable
integration
Tobacco Transgene
CRISPRCas Protoplasts
Stable
integration
Rice Labdane-related
diterpenoid gene
clusters
on Chr 2 4 and 6
Zinc-finger
nuclease
Agrobacterium
T-DNA
(transient)
Tobacco CHN50 transgene
Zinc-finger
nuclease
Whiskers Maize IPK1
CRISPRCas Protoplasts Rice PDS
Controlling gene
expression
TALE repressor
(SRDX)
Stable
integration
Arabidopsis RD29A transgene
Zinc-finger
activator (VP16)
Stable
integration
Brassica
napus
KasII
Continue
62
Application of genome editing1 Introduction of precise and predictable modifications directly in an elite
background
2 multiple traits can be modified simultaneously
3 NHEJ enables gene knockout and targeted modifications
4 Introduction of transgenes at defined loci that promote high-level
transcription and do not interfere with the activity of endogenous genes
5 Site-specific nucleases also allow targeted molecular trait stacking -low
risk of segregation
6 CRISPR Cas is a transgene free approach ndash No regulatory burdens
7 frequency of off-target mutations is well below that caused by chemical
and physical mutagenesis techniques
8 In future can be utilized for metabolic engineering and molecular farming
63
Six-way Venn diagram showing the distribution of shared genefamilies (sequence clusters) among M acuminata P dactyliferaArabidopsis thaliana Oryza sativa Sorghum bicolor and Brachypodiumdistachyon genomes
A DrsquoHont et al Nature 2012 doi101038nature11241
64
Achieving super-DomesticationINTROGRESS-RECREATE-CREATE-DOMESTICATEREDOMESTICATE
bull Mobilizing left out genetic
variation still available in
land races and wild and
weedy species
bull Replaying the evolutionary
tape( resynthesis in
polyploids)
bull Domesticateredomesticate
65
We can admire and emulate how indigenous people still domesticateplants create biodiversity and manage it to sustain their future Thereis no equivalently dynamic or flexible crop breeding in modernagriculture to promote biodiversity we still have much to learn from
traditional knowledge Jan Salick 2012
35
Genes Crop Moleular and phenotypic function Causative change
Genes identified as controlling domestication traits
tb1 Maize Transcriptional regulator (TCP) plant and
inflorescence structure
regulatory change
tga1 Maize Transcriptional regulator (SBP) seed casing amino acid change
qSH1 Rice Transcriptional regulator (homeodomain)
abscission layer formation shattering
regulatory change
Rc Rice Transcriptional regulator (bHLH) seed color disrupted coding
sequence
sh4 Rice Transcriptional regulator (Myb3) abscission
layer formation shattering
regulatoryamino
acid change
fw22 Tomato Cell signaling fruit weight regulatory change
Q Wheat Transcriptional regulator (AP2) inflorescence
structure
regulatoryamino
acid change
Vrs1 Barley Inflorescence structure Premature stop Amino acid change
Genes of interest in crop domestication and improvement
36
Genes Crop Moleular and phenotypic function Causative change
c1
r1
Maize Transcriptional regulator (MYB) kernel color and
Transcriptional regulator (bHLH) kernel color
regulatory change
sh2 Maize pyrophosphorylase supersweet sweet corn transposon
insertion
su1 Maize isoamylase sweet corn gene amino acid change
brix9-2-5 Tomato Invertase fruit soluble solid content amino acid change
ovate Tomato Unknown fruit shap early stop codon
R Pea Starch branching enzyme seed sugar content transposon
insertion
ehd1 Rice B-type response regulator flowering time amino acid change
hd1 Rice Transcriptional regulator (zinc finger) flowering
time
disrupted coding
sequence
waxy Rice Starch synthase sticky grains intron splicing
defect
rht Wheat Transcriptional regulator (SH2) plant height early stop codon
vrn1 Wheat Transcriptional regulator (MADS) vernalization regulatory change
vrn2 Wheat Transcriptional regulator (ZCCT) vernalization amino acid change
Genes Identified as Controlling Varietal Differences
37
Process of domestication over the yearshellip
Rachel S Meyer 2012
38
Genetic bottleneck
Cultivated crops undergone with narrowing of diversity problem
But this is not the case in weeds
Weeds are the major threats from the beginning of domestication till
today
Problem arose due to
1 Crop mimicry
2 Genetic assimilation
3 Genetic evolution
39
Super- Domestication Vaughn et al (2007) first used the term super-domestication
The processes that lead to a domesticate with dramatically
increased yield that could not be selected in natural environments
from naturally occurring variation without recourse to new
technologies
Super-domesticates can be constructed with knowledge led
approaches based on current needs using the range of new
technologies now available
Plants exploited for continuous selection introduction hybridization
etc which boosted the process of domestication but now the plant
genetic engineering approach is exploiting plants towards synthetic
biology
40
How to use diversitybull Cross two varieties
bull Genome manipulations
Cell fusion hybrids
bull Chromosome manipulation Backcross a new species
bull Generate recombinants Chromosome recombinations
bull Use a new species wild germplasm
Transgenic approach Modern mutagenesis synthetic gene
construction by utilizing green florescent protein genome editing
NGS GWAS sequencing etchelliphelliphelliphelliphellip
41
Reduce genetic bottlenecks through polyplodization
More vigour
SC
Buffering capacity
Heterotic advantage
Enhaced vegetative
charachers
Enhanced oil
Meiotic stability
42
Approach 1 Use one tetraploid and one diploid as parents (4X ndash 2X) followed by the
chromosome doubling of triploid hybrids (Chrom doubling)
a) Cross between B juncea (AjAjBjBj ) and B oleracea (CoCo) to produce hexaploids
(AjAjBjBjCoCo)
(b) Cross between B napus (AnAnCnCn) and B nigra (BniBni) to produce hexaploids
(AnAnBniBniCnCn)
(c) Cross between B carinata (BcaBcaCcaCca) and B rapa (ArAr) to produce hexaploids
(ArArBcaBcaCcaCca)
Approach 2 Use three tetraploids as parents
(a) Cross between B napus (AnAnCnCn) and B carinata (BcaBcaCcaCca) to produce unbalanced
allotetraploids (AnBcaCnCca ndash unreduced gametes gametes with the somatic chromosome
number and cross with B juncea (AjAjBjBj) to obtain allohexaploids (AnAjBcaBjCnCca
(b) Cross between B napus (AnAnCnCn) and B juncea (AjAjBjBj) to produce unbalanced
allotetraploids (AnAjBjCn ndash unreduced gametes) and cross with B carinata (BcaBcaCcaCca) to
obtain allohexaploids (AnAjBcaBjCnCca)
(c) Cross between B carinata (BcaBcaCcaCca) and B juncea (AjAjBjBj) to produce unbalanced
allotetraploids (AjBcaBjCca - ndash unreduced gametes) and cross with B napus (AnAnCnCn) to obtain
allohexaploids (AnAjBcaBjCnCca)
Approach 3 Use three diploids as parents (2X ndash 2X ndash 2X) Cross between B rapa (ArAr) B
nigra (BniBni) and B oleracea (CoCo) sequentially to obtain hexaploid hybrids (ArArBniBniCoCo)
43
Continuehellip
Guijun Yan 2012
44
Applications of Genomic
tools in Super-Domestication
45
Genome Sequencing
Potential methods of sequencing
1 Clone by clone approach
2 Whole genome shotgun approach
3 Combination of the two methods
Till today no of cultivated plants completely sequenced -85
2016- Arachis duranensis
2015- Solanum cumersonii (Wild potato)
(Ref- NCBI)
46
GWAS
It is a study design in which many markers spread across a genome
are genotyped and test a statistical association with a phenotype are
performed locally along the genome
It is also an examination of many common genetic variants in different
individuals to see if any variant is associated with a trait
Used in successfully studying maize sorghum and barley
Method is efficient for large scale low cost genotyping (even with the
minimum number of SNPs)
Cannot be utilized generally because it needs large population size
GWAS identify rare alleles more precisely
If small population we can opt NAM
47
GWAS and whole genome prediction
Xuehui Huang and Bin Han 2014
48
Five high throuput genotyping methods
Xuehui Huang and Bin Han 2014
49
Role of NGS in domestication Capture of novel genes from wild species will be made easier by
understanding the molecular events associated with crop domestication
Re-sequencing of domesticated species can identify low diversity regions
resulting from selection during domestication
To identify gene-specific sequences to aid the cloning of homologues of key
domestication genes from wild relatives
Candidate genes from wild and domesticated plant populations can define
diversity of target genes in wild populations and lead to the discovery of key
genes for important traits by association analysis
NGS of amplicons of large numbers of candidate genes from wild and
domesticated plant populations can define diversity of target genes in
wild populations and lead to the discovery of key genes for important
traits by association analysis
50
NGS
supports the rapid domestication of new plant species and the efficient
identification and capture of novel genetic variation from related species
Allows whole-genome analysis to determine the genetic basis of
phenotypic differences
NGS allows rapid expansion of genomic analysis to investigation of non-
model species
Made rice study easy by related grass
cost-effective method for plant identification
useful strategy to analysis the chloroplast genome sequence from whole-
genome shot-gun sequencing
facilitates managing this diversity and any changes in crop performance
over time due to genetic drift
Patterns of gene expression have been evaluated in hybrids using NGS
51
Two Approaches to Finding Adaptive Genes
Ross-Ibarra et al2007
52
Techniques to re-wild the plants (transgene free)
1 Introgression
breeding
2 Specific
insertion of lost genes
3 Precision
mutagenesis
Palmgren et al2014
53
Process of rewilding
Palmgren et al2014
54
Synthetic biology projects(i) Modifying cereals including wheat to fix atmospheric nitrogen
(ii) Redesigning metabolic pathways to increase the yield of
secondary metabolites or to generate compounds with enhanced
properties
(iii) Transferring the C4 photosynthesis pathway to rice
(iv) Modifying the glycosylation pathway in plants to accommodate
production of therapeutic proteins
(v) Introducing synthetic signal transduction systems that respond to
external cues
55
Synthetic biology projects
Nicholas J Baltes et al 2015
56
Refactoring the N fixation gene cluster from Klebsiella
Karsten Temme et al 2012
57
Continuehellip
Karsten Temme et al 2012
58
Genome editing
Sequence specific nucleases
1 Meganucleases
2 ZFMs ndashZink finger motifs
3 TALENs- Transcription activator like effector nuclease
4 CRISPR CAS-9 ndash Clustered interspaced short palindromic
repeats
qmp4
59Khaoula Belhaj et al 2015
60
Genome modifications achieved in plants using sequence specific nucleases
Type of DNA
modification
Nuclease Delivery
methods
Plants Targets
Trait stacking Megnucleases Bombardment Cotton Intergenic sequence
ZFN Bombardment Maize Transgene
Rewriting host
DNA
Megnucleases Stable integration Maize Intergenic sequence
ZFN Stable integration Soybean Transgene
TALEN Stable integration Barley PAPhy_a
TALEN Agrobacterium
T-DNA (transient)
Oryza sativa SWEET14
TALEN No Protoplasts Arabidopsis
Tobacco
AtTT4 AtADH NbSurB
TALEN Bombardment Wheat MLO
TALEN Protoplasts
Stable integration
Maize PDS IPK1A IPK MRP4
CRISPRCas Protoplasts
Agrobacterium
T-DNA (transient)
Tobacco
Arabidopsis
Sorghum Oryza
OsSWEET14 transgene
61
Type of DNA
modification
Nuclease Delivery
methods
Plants Targets
Rewriting host
DNA large
deletion
Zinc-finger
nuclease
Stable
integration
Tobacco Transgene
CRISPRCas Protoplasts
Stable
integration
Rice Labdane-related
diterpenoid gene
clusters
on Chr 2 4 and 6
Zinc-finger
nuclease
Agrobacterium
T-DNA
(transient)
Tobacco CHN50 transgene
Zinc-finger
nuclease
Whiskers Maize IPK1
CRISPRCas Protoplasts Rice PDS
Controlling gene
expression
TALE repressor
(SRDX)
Stable
integration
Arabidopsis RD29A transgene
Zinc-finger
activator (VP16)
Stable
integration
Brassica
napus
KasII
Continue
62
Application of genome editing1 Introduction of precise and predictable modifications directly in an elite
background
2 multiple traits can be modified simultaneously
3 NHEJ enables gene knockout and targeted modifications
4 Introduction of transgenes at defined loci that promote high-level
transcription and do not interfere with the activity of endogenous genes
5 Site-specific nucleases also allow targeted molecular trait stacking -low
risk of segregation
6 CRISPR Cas is a transgene free approach ndash No regulatory burdens
7 frequency of off-target mutations is well below that caused by chemical
and physical mutagenesis techniques
8 In future can be utilized for metabolic engineering and molecular farming
63
Six-way Venn diagram showing the distribution of shared genefamilies (sequence clusters) among M acuminata P dactyliferaArabidopsis thaliana Oryza sativa Sorghum bicolor and Brachypodiumdistachyon genomes
A DrsquoHont et al Nature 2012 doi101038nature11241
64
Achieving super-DomesticationINTROGRESS-RECREATE-CREATE-DOMESTICATEREDOMESTICATE
bull Mobilizing left out genetic
variation still available in
land races and wild and
weedy species
bull Replaying the evolutionary
tape( resynthesis in
polyploids)
bull Domesticateredomesticate
65
We can admire and emulate how indigenous people still domesticateplants create biodiversity and manage it to sustain their future Thereis no equivalently dynamic or flexible crop breeding in modernagriculture to promote biodiversity we still have much to learn from
traditional knowledge Jan Salick 2012
36
Genes Crop Moleular and phenotypic function Causative change
c1
r1
Maize Transcriptional regulator (MYB) kernel color and
Transcriptional regulator (bHLH) kernel color
regulatory change
sh2 Maize pyrophosphorylase supersweet sweet corn transposon
insertion
su1 Maize isoamylase sweet corn gene amino acid change
brix9-2-5 Tomato Invertase fruit soluble solid content amino acid change
ovate Tomato Unknown fruit shap early stop codon
R Pea Starch branching enzyme seed sugar content transposon
insertion
ehd1 Rice B-type response regulator flowering time amino acid change
hd1 Rice Transcriptional regulator (zinc finger) flowering
time
disrupted coding
sequence
waxy Rice Starch synthase sticky grains intron splicing
defect
rht Wheat Transcriptional regulator (SH2) plant height early stop codon
vrn1 Wheat Transcriptional regulator (MADS) vernalization regulatory change
vrn2 Wheat Transcriptional regulator (ZCCT) vernalization amino acid change
Genes Identified as Controlling Varietal Differences
37
Process of domestication over the yearshellip
Rachel S Meyer 2012
38
Genetic bottleneck
Cultivated crops undergone with narrowing of diversity problem
But this is not the case in weeds
Weeds are the major threats from the beginning of domestication till
today
Problem arose due to
1 Crop mimicry
2 Genetic assimilation
3 Genetic evolution
39
Super- Domestication Vaughn et al (2007) first used the term super-domestication
The processes that lead to a domesticate with dramatically
increased yield that could not be selected in natural environments
from naturally occurring variation without recourse to new
technologies
Super-domesticates can be constructed with knowledge led
approaches based on current needs using the range of new
technologies now available
Plants exploited for continuous selection introduction hybridization
etc which boosted the process of domestication but now the plant
genetic engineering approach is exploiting plants towards synthetic
biology
40
How to use diversitybull Cross two varieties
bull Genome manipulations
Cell fusion hybrids
bull Chromosome manipulation Backcross a new species
bull Generate recombinants Chromosome recombinations
bull Use a new species wild germplasm
Transgenic approach Modern mutagenesis synthetic gene
construction by utilizing green florescent protein genome editing
NGS GWAS sequencing etchelliphelliphelliphelliphellip
41
Reduce genetic bottlenecks through polyplodization
More vigour
SC
Buffering capacity
Heterotic advantage
Enhaced vegetative
charachers
Enhanced oil
Meiotic stability
42
Approach 1 Use one tetraploid and one diploid as parents (4X ndash 2X) followed by the
chromosome doubling of triploid hybrids (Chrom doubling)
a) Cross between B juncea (AjAjBjBj ) and B oleracea (CoCo) to produce hexaploids
(AjAjBjBjCoCo)
(b) Cross between B napus (AnAnCnCn) and B nigra (BniBni) to produce hexaploids
(AnAnBniBniCnCn)
(c) Cross between B carinata (BcaBcaCcaCca) and B rapa (ArAr) to produce hexaploids
(ArArBcaBcaCcaCca)
Approach 2 Use three tetraploids as parents
(a) Cross between B napus (AnAnCnCn) and B carinata (BcaBcaCcaCca) to produce unbalanced
allotetraploids (AnBcaCnCca ndash unreduced gametes gametes with the somatic chromosome
number and cross with B juncea (AjAjBjBj) to obtain allohexaploids (AnAjBcaBjCnCca
(b) Cross between B napus (AnAnCnCn) and B juncea (AjAjBjBj) to produce unbalanced
allotetraploids (AnAjBjCn ndash unreduced gametes) and cross with B carinata (BcaBcaCcaCca) to
obtain allohexaploids (AnAjBcaBjCnCca)
(c) Cross between B carinata (BcaBcaCcaCca) and B juncea (AjAjBjBj) to produce unbalanced
allotetraploids (AjBcaBjCca - ndash unreduced gametes) and cross with B napus (AnAnCnCn) to obtain
allohexaploids (AnAjBcaBjCnCca)
Approach 3 Use three diploids as parents (2X ndash 2X ndash 2X) Cross between B rapa (ArAr) B
nigra (BniBni) and B oleracea (CoCo) sequentially to obtain hexaploid hybrids (ArArBniBniCoCo)
43
Continuehellip
Guijun Yan 2012
44
Applications of Genomic
tools in Super-Domestication
45
Genome Sequencing
Potential methods of sequencing
1 Clone by clone approach
2 Whole genome shotgun approach
3 Combination of the two methods
Till today no of cultivated plants completely sequenced -85
2016- Arachis duranensis
2015- Solanum cumersonii (Wild potato)
(Ref- NCBI)
46
GWAS
It is a study design in which many markers spread across a genome
are genotyped and test a statistical association with a phenotype are
performed locally along the genome
It is also an examination of many common genetic variants in different
individuals to see if any variant is associated with a trait
Used in successfully studying maize sorghum and barley
Method is efficient for large scale low cost genotyping (even with the
minimum number of SNPs)
Cannot be utilized generally because it needs large population size
GWAS identify rare alleles more precisely
If small population we can opt NAM
47
GWAS and whole genome prediction
Xuehui Huang and Bin Han 2014
48
Five high throuput genotyping methods
Xuehui Huang and Bin Han 2014
49
Role of NGS in domestication Capture of novel genes from wild species will be made easier by
understanding the molecular events associated with crop domestication
Re-sequencing of domesticated species can identify low diversity regions
resulting from selection during domestication
To identify gene-specific sequences to aid the cloning of homologues of key
domestication genes from wild relatives
Candidate genes from wild and domesticated plant populations can define
diversity of target genes in wild populations and lead to the discovery of key
genes for important traits by association analysis
NGS of amplicons of large numbers of candidate genes from wild and
domesticated plant populations can define diversity of target genes in
wild populations and lead to the discovery of key genes for important
traits by association analysis
50
NGS
supports the rapid domestication of new plant species and the efficient
identification and capture of novel genetic variation from related species
Allows whole-genome analysis to determine the genetic basis of
phenotypic differences
NGS allows rapid expansion of genomic analysis to investigation of non-
model species
Made rice study easy by related grass
cost-effective method for plant identification
useful strategy to analysis the chloroplast genome sequence from whole-
genome shot-gun sequencing
facilitates managing this diversity and any changes in crop performance
over time due to genetic drift
Patterns of gene expression have been evaluated in hybrids using NGS
51
Two Approaches to Finding Adaptive Genes
Ross-Ibarra et al2007
52
Techniques to re-wild the plants (transgene free)
1 Introgression
breeding
2 Specific
insertion of lost genes
3 Precision
mutagenesis
Palmgren et al2014
53
Process of rewilding
Palmgren et al2014
54
Synthetic biology projects(i) Modifying cereals including wheat to fix atmospheric nitrogen
(ii) Redesigning metabolic pathways to increase the yield of
secondary metabolites or to generate compounds with enhanced
properties
(iii) Transferring the C4 photosynthesis pathway to rice
(iv) Modifying the glycosylation pathway in plants to accommodate
production of therapeutic proteins
(v) Introducing synthetic signal transduction systems that respond to
external cues
55
Synthetic biology projects
Nicholas J Baltes et al 2015
56
Refactoring the N fixation gene cluster from Klebsiella
Karsten Temme et al 2012
57
Continuehellip
Karsten Temme et al 2012
58
Genome editing
Sequence specific nucleases
1 Meganucleases
2 ZFMs ndashZink finger motifs
3 TALENs- Transcription activator like effector nuclease
4 CRISPR CAS-9 ndash Clustered interspaced short palindromic
repeats
qmp4
59Khaoula Belhaj et al 2015
60
Genome modifications achieved in plants using sequence specific nucleases
Type of DNA
modification
Nuclease Delivery
methods
Plants Targets
Trait stacking Megnucleases Bombardment Cotton Intergenic sequence
ZFN Bombardment Maize Transgene
Rewriting host
DNA
Megnucleases Stable integration Maize Intergenic sequence
ZFN Stable integration Soybean Transgene
TALEN Stable integration Barley PAPhy_a
TALEN Agrobacterium
T-DNA (transient)
Oryza sativa SWEET14
TALEN No Protoplasts Arabidopsis
Tobacco
AtTT4 AtADH NbSurB
TALEN Bombardment Wheat MLO
TALEN Protoplasts
Stable integration
Maize PDS IPK1A IPK MRP4
CRISPRCas Protoplasts
Agrobacterium
T-DNA (transient)
Tobacco
Arabidopsis
Sorghum Oryza
OsSWEET14 transgene
61
Type of DNA
modification
Nuclease Delivery
methods
Plants Targets
Rewriting host
DNA large
deletion
Zinc-finger
nuclease
Stable
integration
Tobacco Transgene
CRISPRCas Protoplasts
Stable
integration
Rice Labdane-related
diterpenoid gene
clusters
on Chr 2 4 and 6
Zinc-finger
nuclease
Agrobacterium
T-DNA
(transient)
Tobacco CHN50 transgene
Zinc-finger
nuclease
Whiskers Maize IPK1
CRISPRCas Protoplasts Rice PDS
Controlling gene
expression
TALE repressor
(SRDX)
Stable
integration
Arabidopsis RD29A transgene
Zinc-finger
activator (VP16)
Stable
integration
Brassica
napus
KasII
Continue
62
Application of genome editing1 Introduction of precise and predictable modifications directly in an elite
background
2 multiple traits can be modified simultaneously
3 NHEJ enables gene knockout and targeted modifications
4 Introduction of transgenes at defined loci that promote high-level
transcription and do not interfere with the activity of endogenous genes
5 Site-specific nucleases also allow targeted molecular trait stacking -low
risk of segregation
6 CRISPR Cas is a transgene free approach ndash No regulatory burdens
7 frequency of off-target mutations is well below that caused by chemical
and physical mutagenesis techniques
8 In future can be utilized for metabolic engineering and molecular farming
63
Six-way Venn diagram showing the distribution of shared genefamilies (sequence clusters) among M acuminata P dactyliferaArabidopsis thaliana Oryza sativa Sorghum bicolor and Brachypodiumdistachyon genomes
A DrsquoHont et al Nature 2012 doi101038nature11241
64
Achieving super-DomesticationINTROGRESS-RECREATE-CREATE-DOMESTICATEREDOMESTICATE
bull Mobilizing left out genetic
variation still available in
land races and wild and
weedy species
bull Replaying the evolutionary
tape( resynthesis in
polyploids)
bull Domesticateredomesticate
65
We can admire and emulate how indigenous people still domesticateplants create biodiversity and manage it to sustain their future Thereis no equivalently dynamic or flexible crop breeding in modernagriculture to promote biodiversity we still have much to learn from
traditional knowledge Jan Salick 2012
37
Process of domestication over the yearshellip
Rachel S Meyer 2012
38
Genetic bottleneck
Cultivated crops undergone with narrowing of diversity problem
But this is not the case in weeds
Weeds are the major threats from the beginning of domestication till
today
Problem arose due to
1 Crop mimicry
2 Genetic assimilation
3 Genetic evolution
39
Super- Domestication Vaughn et al (2007) first used the term super-domestication
The processes that lead to a domesticate with dramatically
increased yield that could not be selected in natural environments
from naturally occurring variation without recourse to new
technologies
Super-domesticates can be constructed with knowledge led
approaches based on current needs using the range of new
technologies now available
Plants exploited for continuous selection introduction hybridization
etc which boosted the process of domestication but now the plant
genetic engineering approach is exploiting plants towards synthetic
biology
40
How to use diversitybull Cross two varieties
bull Genome manipulations
Cell fusion hybrids
bull Chromosome manipulation Backcross a new species
bull Generate recombinants Chromosome recombinations
bull Use a new species wild germplasm
Transgenic approach Modern mutagenesis synthetic gene
construction by utilizing green florescent protein genome editing
NGS GWAS sequencing etchelliphelliphelliphelliphellip
41
Reduce genetic bottlenecks through polyplodization
More vigour
SC
Buffering capacity
Heterotic advantage
Enhaced vegetative
charachers
Enhanced oil
Meiotic stability
42
Approach 1 Use one tetraploid and one diploid as parents (4X ndash 2X) followed by the
chromosome doubling of triploid hybrids (Chrom doubling)
a) Cross between B juncea (AjAjBjBj ) and B oleracea (CoCo) to produce hexaploids
(AjAjBjBjCoCo)
(b) Cross between B napus (AnAnCnCn) and B nigra (BniBni) to produce hexaploids
(AnAnBniBniCnCn)
(c) Cross between B carinata (BcaBcaCcaCca) and B rapa (ArAr) to produce hexaploids
(ArArBcaBcaCcaCca)
Approach 2 Use three tetraploids as parents
(a) Cross between B napus (AnAnCnCn) and B carinata (BcaBcaCcaCca) to produce unbalanced
allotetraploids (AnBcaCnCca ndash unreduced gametes gametes with the somatic chromosome
number and cross with B juncea (AjAjBjBj) to obtain allohexaploids (AnAjBcaBjCnCca
(b) Cross between B napus (AnAnCnCn) and B juncea (AjAjBjBj) to produce unbalanced
allotetraploids (AnAjBjCn ndash unreduced gametes) and cross with B carinata (BcaBcaCcaCca) to
obtain allohexaploids (AnAjBcaBjCnCca)
(c) Cross between B carinata (BcaBcaCcaCca) and B juncea (AjAjBjBj) to produce unbalanced
allotetraploids (AjBcaBjCca - ndash unreduced gametes) and cross with B napus (AnAnCnCn) to obtain
allohexaploids (AnAjBcaBjCnCca)
Approach 3 Use three diploids as parents (2X ndash 2X ndash 2X) Cross between B rapa (ArAr) B
nigra (BniBni) and B oleracea (CoCo) sequentially to obtain hexaploid hybrids (ArArBniBniCoCo)
43
Continuehellip
Guijun Yan 2012
44
Applications of Genomic
tools in Super-Domestication
45
Genome Sequencing
Potential methods of sequencing
1 Clone by clone approach
2 Whole genome shotgun approach
3 Combination of the two methods
Till today no of cultivated plants completely sequenced -85
2016- Arachis duranensis
2015- Solanum cumersonii (Wild potato)
(Ref- NCBI)
46
GWAS
It is a study design in which many markers spread across a genome
are genotyped and test a statistical association with a phenotype are
performed locally along the genome
It is also an examination of many common genetic variants in different
individuals to see if any variant is associated with a trait
Used in successfully studying maize sorghum and barley
Method is efficient for large scale low cost genotyping (even with the
minimum number of SNPs)
Cannot be utilized generally because it needs large population size
GWAS identify rare alleles more precisely
If small population we can opt NAM
47
GWAS and whole genome prediction
Xuehui Huang and Bin Han 2014
48
Five high throuput genotyping methods
Xuehui Huang and Bin Han 2014
49
Role of NGS in domestication Capture of novel genes from wild species will be made easier by
understanding the molecular events associated with crop domestication
Re-sequencing of domesticated species can identify low diversity regions
resulting from selection during domestication
To identify gene-specific sequences to aid the cloning of homologues of key
domestication genes from wild relatives
Candidate genes from wild and domesticated plant populations can define
diversity of target genes in wild populations and lead to the discovery of key
genes for important traits by association analysis
NGS of amplicons of large numbers of candidate genes from wild and
domesticated plant populations can define diversity of target genes in
wild populations and lead to the discovery of key genes for important
traits by association analysis
50
NGS
supports the rapid domestication of new plant species and the efficient
identification and capture of novel genetic variation from related species
Allows whole-genome analysis to determine the genetic basis of
phenotypic differences
NGS allows rapid expansion of genomic analysis to investigation of non-
model species
Made rice study easy by related grass
cost-effective method for plant identification
useful strategy to analysis the chloroplast genome sequence from whole-
genome shot-gun sequencing
facilitates managing this diversity and any changes in crop performance
over time due to genetic drift
Patterns of gene expression have been evaluated in hybrids using NGS
51
Two Approaches to Finding Adaptive Genes
Ross-Ibarra et al2007
52
Techniques to re-wild the plants (transgene free)
1 Introgression
breeding
2 Specific
insertion of lost genes
3 Precision
mutagenesis
Palmgren et al2014
53
Process of rewilding
Palmgren et al2014
54
Synthetic biology projects(i) Modifying cereals including wheat to fix atmospheric nitrogen
(ii) Redesigning metabolic pathways to increase the yield of
secondary metabolites or to generate compounds with enhanced
properties
(iii) Transferring the C4 photosynthesis pathway to rice
(iv) Modifying the glycosylation pathway in plants to accommodate
production of therapeutic proteins
(v) Introducing synthetic signal transduction systems that respond to
external cues
55
Synthetic biology projects
Nicholas J Baltes et al 2015
56
Refactoring the N fixation gene cluster from Klebsiella
Karsten Temme et al 2012
57
Continuehellip
Karsten Temme et al 2012
58
Genome editing
Sequence specific nucleases
1 Meganucleases
2 ZFMs ndashZink finger motifs
3 TALENs- Transcription activator like effector nuclease
4 CRISPR CAS-9 ndash Clustered interspaced short palindromic
repeats
qmp4
59Khaoula Belhaj et al 2015
60
Genome modifications achieved in plants using sequence specific nucleases
Type of DNA
modification
Nuclease Delivery
methods
Plants Targets
Trait stacking Megnucleases Bombardment Cotton Intergenic sequence
ZFN Bombardment Maize Transgene
Rewriting host
DNA
Megnucleases Stable integration Maize Intergenic sequence
ZFN Stable integration Soybean Transgene
TALEN Stable integration Barley PAPhy_a
TALEN Agrobacterium
T-DNA (transient)
Oryza sativa SWEET14
TALEN No Protoplasts Arabidopsis
Tobacco
AtTT4 AtADH NbSurB
TALEN Bombardment Wheat MLO
TALEN Protoplasts
Stable integration
Maize PDS IPK1A IPK MRP4
CRISPRCas Protoplasts
Agrobacterium
T-DNA (transient)
Tobacco
Arabidopsis
Sorghum Oryza
OsSWEET14 transgene
61
Type of DNA
modification
Nuclease Delivery
methods
Plants Targets
Rewriting host
DNA large
deletion
Zinc-finger
nuclease
Stable
integration
Tobacco Transgene
CRISPRCas Protoplasts
Stable
integration
Rice Labdane-related
diterpenoid gene
clusters
on Chr 2 4 and 6
Zinc-finger
nuclease
Agrobacterium
T-DNA
(transient)
Tobacco CHN50 transgene
Zinc-finger
nuclease
Whiskers Maize IPK1
CRISPRCas Protoplasts Rice PDS
Controlling gene
expression
TALE repressor
(SRDX)
Stable
integration
Arabidopsis RD29A transgene
Zinc-finger
activator (VP16)
Stable
integration
Brassica
napus
KasII
Continue
62
Application of genome editing1 Introduction of precise and predictable modifications directly in an elite
background
2 multiple traits can be modified simultaneously
3 NHEJ enables gene knockout and targeted modifications
4 Introduction of transgenes at defined loci that promote high-level
transcription and do not interfere with the activity of endogenous genes
5 Site-specific nucleases also allow targeted molecular trait stacking -low
risk of segregation
6 CRISPR Cas is a transgene free approach ndash No regulatory burdens
7 frequency of off-target mutations is well below that caused by chemical
and physical mutagenesis techniques
8 In future can be utilized for metabolic engineering and molecular farming
63
Six-way Venn diagram showing the distribution of shared genefamilies (sequence clusters) among M acuminata P dactyliferaArabidopsis thaliana Oryza sativa Sorghum bicolor and Brachypodiumdistachyon genomes
A DrsquoHont et al Nature 2012 doi101038nature11241
64
Achieving super-DomesticationINTROGRESS-RECREATE-CREATE-DOMESTICATEREDOMESTICATE
bull Mobilizing left out genetic
variation still available in
land races and wild and
weedy species
bull Replaying the evolutionary
tape( resynthesis in
polyploids)
bull Domesticateredomesticate
65
We can admire and emulate how indigenous people still domesticateplants create biodiversity and manage it to sustain their future Thereis no equivalently dynamic or flexible crop breeding in modernagriculture to promote biodiversity we still have much to learn from
traditional knowledge Jan Salick 2012
38
Genetic bottleneck
Cultivated crops undergone with narrowing of diversity problem
But this is not the case in weeds
Weeds are the major threats from the beginning of domestication till
today
Problem arose due to
1 Crop mimicry
2 Genetic assimilation
3 Genetic evolution
39
Super- Domestication Vaughn et al (2007) first used the term super-domestication
The processes that lead to a domesticate with dramatically
increased yield that could not be selected in natural environments
from naturally occurring variation without recourse to new
technologies
Super-domesticates can be constructed with knowledge led
approaches based on current needs using the range of new
technologies now available
Plants exploited for continuous selection introduction hybridization
etc which boosted the process of domestication but now the plant
genetic engineering approach is exploiting plants towards synthetic
biology
40
How to use diversitybull Cross two varieties
bull Genome manipulations
Cell fusion hybrids
bull Chromosome manipulation Backcross a new species
bull Generate recombinants Chromosome recombinations
bull Use a new species wild germplasm
Transgenic approach Modern mutagenesis synthetic gene
construction by utilizing green florescent protein genome editing
NGS GWAS sequencing etchelliphelliphelliphelliphellip
41
Reduce genetic bottlenecks through polyplodization
More vigour
SC
Buffering capacity
Heterotic advantage
Enhaced vegetative
charachers
Enhanced oil
Meiotic stability
42
Approach 1 Use one tetraploid and one diploid as parents (4X ndash 2X) followed by the
chromosome doubling of triploid hybrids (Chrom doubling)
a) Cross between B juncea (AjAjBjBj ) and B oleracea (CoCo) to produce hexaploids
(AjAjBjBjCoCo)
(b) Cross between B napus (AnAnCnCn) and B nigra (BniBni) to produce hexaploids
(AnAnBniBniCnCn)
(c) Cross between B carinata (BcaBcaCcaCca) and B rapa (ArAr) to produce hexaploids
(ArArBcaBcaCcaCca)
Approach 2 Use three tetraploids as parents
(a) Cross between B napus (AnAnCnCn) and B carinata (BcaBcaCcaCca) to produce unbalanced
allotetraploids (AnBcaCnCca ndash unreduced gametes gametes with the somatic chromosome
number and cross with B juncea (AjAjBjBj) to obtain allohexaploids (AnAjBcaBjCnCca
(b) Cross between B napus (AnAnCnCn) and B juncea (AjAjBjBj) to produce unbalanced
allotetraploids (AnAjBjCn ndash unreduced gametes) and cross with B carinata (BcaBcaCcaCca) to
obtain allohexaploids (AnAjBcaBjCnCca)
(c) Cross between B carinata (BcaBcaCcaCca) and B juncea (AjAjBjBj) to produce unbalanced
allotetraploids (AjBcaBjCca - ndash unreduced gametes) and cross with B napus (AnAnCnCn) to obtain
allohexaploids (AnAjBcaBjCnCca)
Approach 3 Use three diploids as parents (2X ndash 2X ndash 2X) Cross between B rapa (ArAr) B
nigra (BniBni) and B oleracea (CoCo) sequentially to obtain hexaploid hybrids (ArArBniBniCoCo)
43
Continuehellip
Guijun Yan 2012
44
Applications of Genomic
tools in Super-Domestication
45
Genome Sequencing
Potential methods of sequencing
1 Clone by clone approach
2 Whole genome shotgun approach
3 Combination of the two methods
Till today no of cultivated plants completely sequenced -85
2016- Arachis duranensis
2015- Solanum cumersonii (Wild potato)
(Ref- NCBI)
46
GWAS
It is a study design in which many markers spread across a genome
are genotyped and test a statistical association with a phenotype are
performed locally along the genome
It is also an examination of many common genetic variants in different
individuals to see if any variant is associated with a trait
Used in successfully studying maize sorghum and barley
Method is efficient for large scale low cost genotyping (even with the
minimum number of SNPs)
Cannot be utilized generally because it needs large population size
GWAS identify rare alleles more precisely
If small population we can opt NAM
47
GWAS and whole genome prediction
Xuehui Huang and Bin Han 2014
48
Five high throuput genotyping methods
Xuehui Huang and Bin Han 2014
49
Role of NGS in domestication Capture of novel genes from wild species will be made easier by
understanding the molecular events associated with crop domestication
Re-sequencing of domesticated species can identify low diversity regions
resulting from selection during domestication
To identify gene-specific sequences to aid the cloning of homologues of key
domestication genes from wild relatives
Candidate genes from wild and domesticated plant populations can define
diversity of target genes in wild populations and lead to the discovery of key
genes for important traits by association analysis
NGS of amplicons of large numbers of candidate genes from wild and
domesticated plant populations can define diversity of target genes in
wild populations and lead to the discovery of key genes for important
traits by association analysis
50
NGS
supports the rapid domestication of new plant species and the efficient
identification and capture of novel genetic variation from related species
Allows whole-genome analysis to determine the genetic basis of
phenotypic differences
NGS allows rapid expansion of genomic analysis to investigation of non-
model species
Made rice study easy by related grass
cost-effective method for plant identification
useful strategy to analysis the chloroplast genome sequence from whole-
genome shot-gun sequencing
facilitates managing this diversity and any changes in crop performance
over time due to genetic drift
Patterns of gene expression have been evaluated in hybrids using NGS
51
Two Approaches to Finding Adaptive Genes
Ross-Ibarra et al2007
52
Techniques to re-wild the plants (transgene free)
1 Introgression
breeding
2 Specific
insertion of lost genes
3 Precision
mutagenesis
Palmgren et al2014
53
Process of rewilding
Palmgren et al2014
54
Synthetic biology projects(i) Modifying cereals including wheat to fix atmospheric nitrogen
(ii) Redesigning metabolic pathways to increase the yield of
secondary metabolites or to generate compounds with enhanced
properties
(iii) Transferring the C4 photosynthesis pathway to rice
(iv) Modifying the glycosylation pathway in plants to accommodate
production of therapeutic proteins
(v) Introducing synthetic signal transduction systems that respond to
external cues
55
Synthetic biology projects
Nicholas J Baltes et al 2015
56
Refactoring the N fixation gene cluster from Klebsiella
Karsten Temme et al 2012
57
Continuehellip
Karsten Temme et al 2012
58
Genome editing
Sequence specific nucleases
1 Meganucleases
2 ZFMs ndashZink finger motifs
3 TALENs- Transcription activator like effector nuclease
4 CRISPR CAS-9 ndash Clustered interspaced short palindromic
repeats
qmp4
59Khaoula Belhaj et al 2015
60
Genome modifications achieved in plants using sequence specific nucleases
Type of DNA
modification
Nuclease Delivery
methods
Plants Targets
Trait stacking Megnucleases Bombardment Cotton Intergenic sequence
ZFN Bombardment Maize Transgene
Rewriting host
DNA
Megnucleases Stable integration Maize Intergenic sequence
ZFN Stable integration Soybean Transgene
TALEN Stable integration Barley PAPhy_a
TALEN Agrobacterium
T-DNA (transient)
Oryza sativa SWEET14
TALEN No Protoplasts Arabidopsis
Tobacco
AtTT4 AtADH NbSurB
TALEN Bombardment Wheat MLO
TALEN Protoplasts
Stable integration
Maize PDS IPK1A IPK MRP4
CRISPRCas Protoplasts
Agrobacterium
T-DNA (transient)
Tobacco
Arabidopsis
Sorghum Oryza
OsSWEET14 transgene
61
Type of DNA
modification
Nuclease Delivery
methods
Plants Targets
Rewriting host
DNA large
deletion
Zinc-finger
nuclease
Stable
integration
Tobacco Transgene
CRISPRCas Protoplasts
Stable
integration
Rice Labdane-related
diterpenoid gene
clusters
on Chr 2 4 and 6
Zinc-finger
nuclease
Agrobacterium
T-DNA
(transient)
Tobacco CHN50 transgene
Zinc-finger
nuclease
Whiskers Maize IPK1
CRISPRCas Protoplasts Rice PDS
Controlling gene
expression
TALE repressor
(SRDX)
Stable
integration
Arabidopsis RD29A transgene
Zinc-finger
activator (VP16)
Stable
integration
Brassica
napus
KasII
Continue
62
Application of genome editing1 Introduction of precise and predictable modifications directly in an elite
background
2 multiple traits can be modified simultaneously
3 NHEJ enables gene knockout and targeted modifications
4 Introduction of transgenes at defined loci that promote high-level
transcription and do not interfere with the activity of endogenous genes
5 Site-specific nucleases also allow targeted molecular trait stacking -low
risk of segregation
6 CRISPR Cas is a transgene free approach ndash No regulatory burdens
7 frequency of off-target mutations is well below that caused by chemical
and physical mutagenesis techniques
8 In future can be utilized for metabolic engineering and molecular farming
63
Six-way Venn diagram showing the distribution of shared genefamilies (sequence clusters) among M acuminata P dactyliferaArabidopsis thaliana Oryza sativa Sorghum bicolor and Brachypodiumdistachyon genomes
A DrsquoHont et al Nature 2012 doi101038nature11241
64
Achieving super-DomesticationINTROGRESS-RECREATE-CREATE-DOMESTICATEREDOMESTICATE
bull Mobilizing left out genetic
variation still available in
land races and wild and
weedy species
bull Replaying the evolutionary
tape( resynthesis in
polyploids)
bull Domesticateredomesticate
65
We can admire and emulate how indigenous people still domesticateplants create biodiversity and manage it to sustain their future Thereis no equivalently dynamic or flexible crop breeding in modernagriculture to promote biodiversity we still have much to learn from
traditional knowledge Jan Salick 2012
39
Super- Domestication Vaughn et al (2007) first used the term super-domestication
The processes that lead to a domesticate with dramatically
increased yield that could not be selected in natural environments
from naturally occurring variation without recourse to new
technologies
Super-domesticates can be constructed with knowledge led
approaches based on current needs using the range of new
technologies now available
Plants exploited for continuous selection introduction hybridization
etc which boosted the process of domestication but now the plant
genetic engineering approach is exploiting plants towards synthetic
biology
40
How to use diversitybull Cross two varieties
bull Genome manipulations
Cell fusion hybrids
bull Chromosome manipulation Backcross a new species
bull Generate recombinants Chromosome recombinations
bull Use a new species wild germplasm
Transgenic approach Modern mutagenesis synthetic gene
construction by utilizing green florescent protein genome editing
NGS GWAS sequencing etchelliphelliphelliphelliphellip
41
Reduce genetic bottlenecks through polyplodization
More vigour
SC
Buffering capacity
Heterotic advantage
Enhaced vegetative
charachers
Enhanced oil
Meiotic stability
42
Approach 1 Use one tetraploid and one diploid as parents (4X ndash 2X) followed by the
chromosome doubling of triploid hybrids (Chrom doubling)
a) Cross between B juncea (AjAjBjBj ) and B oleracea (CoCo) to produce hexaploids
(AjAjBjBjCoCo)
(b) Cross between B napus (AnAnCnCn) and B nigra (BniBni) to produce hexaploids
(AnAnBniBniCnCn)
(c) Cross between B carinata (BcaBcaCcaCca) and B rapa (ArAr) to produce hexaploids
(ArArBcaBcaCcaCca)
Approach 2 Use three tetraploids as parents
(a) Cross between B napus (AnAnCnCn) and B carinata (BcaBcaCcaCca) to produce unbalanced
allotetraploids (AnBcaCnCca ndash unreduced gametes gametes with the somatic chromosome
number and cross with B juncea (AjAjBjBj) to obtain allohexaploids (AnAjBcaBjCnCca
(b) Cross between B napus (AnAnCnCn) and B juncea (AjAjBjBj) to produce unbalanced
allotetraploids (AnAjBjCn ndash unreduced gametes) and cross with B carinata (BcaBcaCcaCca) to
obtain allohexaploids (AnAjBcaBjCnCca)
(c) Cross between B carinata (BcaBcaCcaCca) and B juncea (AjAjBjBj) to produce unbalanced
allotetraploids (AjBcaBjCca - ndash unreduced gametes) and cross with B napus (AnAnCnCn) to obtain
allohexaploids (AnAjBcaBjCnCca)
Approach 3 Use three diploids as parents (2X ndash 2X ndash 2X) Cross between B rapa (ArAr) B
nigra (BniBni) and B oleracea (CoCo) sequentially to obtain hexaploid hybrids (ArArBniBniCoCo)
43
Continuehellip
Guijun Yan 2012
44
Applications of Genomic
tools in Super-Domestication
45
Genome Sequencing
Potential methods of sequencing
1 Clone by clone approach
2 Whole genome shotgun approach
3 Combination of the two methods
Till today no of cultivated plants completely sequenced -85
2016- Arachis duranensis
2015- Solanum cumersonii (Wild potato)
(Ref- NCBI)
46
GWAS
It is a study design in which many markers spread across a genome
are genotyped and test a statistical association with a phenotype are
performed locally along the genome
It is also an examination of many common genetic variants in different
individuals to see if any variant is associated with a trait
Used in successfully studying maize sorghum and barley
Method is efficient for large scale low cost genotyping (even with the
minimum number of SNPs)
Cannot be utilized generally because it needs large population size
GWAS identify rare alleles more precisely
If small population we can opt NAM
47
GWAS and whole genome prediction
Xuehui Huang and Bin Han 2014
48
Five high throuput genotyping methods
Xuehui Huang and Bin Han 2014
49
Role of NGS in domestication Capture of novel genes from wild species will be made easier by
understanding the molecular events associated with crop domestication
Re-sequencing of domesticated species can identify low diversity regions
resulting from selection during domestication
To identify gene-specific sequences to aid the cloning of homologues of key
domestication genes from wild relatives
Candidate genes from wild and domesticated plant populations can define
diversity of target genes in wild populations and lead to the discovery of key
genes for important traits by association analysis
NGS of amplicons of large numbers of candidate genes from wild and
domesticated plant populations can define diversity of target genes in
wild populations and lead to the discovery of key genes for important
traits by association analysis
50
NGS
supports the rapid domestication of new plant species and the efficient
identification and capture of novel genetic variation from related species
Allows whole-genome analysis to determine the genetic basis of
phenotypic differences
NGS allows rapid expansion of genomic analysis to investigation of non-
model species
Made rice study easy by related grass
cost-effective method for plant identification
useful strategy to analysis the chloroplast genome sequence from whole-
genome shot-gun sequencing
facilitates managing this diversity and any changes in crop performance
over time due to genetic drift
Patterns of gene expression have been evaluated in hybrids using NGS
51
Two Approaches to Finding Adaptive Genes
Ross-Ibarra et al2007
52
Techniques to re-wild the plants (transgene free)
1 Introgression
breeding
2 Specific
insertion of lost genes
3 Precision
mutagenesis
Palmgren et al2014
53
Process of rewilding
Palmgren et al2014
54
Synthetic biology projects(i) Modifying cereals including wheat to fix atmospheric nitrogen
(ii) Redesigning metabolic pathways to increase the yield of
secondary metabolites or to generate compounds with enhanced
properties
(iii) Transferring the C4 photosynthesis pathway to rice
(iv) Modifying the glycosylation pathway in plants to accommodate
production of therapeutic proteins
(v) Introducing synthetic signal transduction systems that respond to
external cues
55
Synthetic biology projects
Nicholas J Baltes et al 2015
56
Refactoring the N fixation gene cluster from Klebsiella
Karsten Temme et al 2012
57
Continuehellip
Karsten Temme et al 2012
58
Genome editing
Sequence specific nucleases
1 Meganucleases
2 ZFMs ndashZink finger motifs
3 TALENs- Transcription activator like effector nuclease
4 CRISPR CAS-9 ndash Clustered interspaced short palindromic
repeats
qmp4
59Khaoula Belhaj et al 2015
60
Genome modifications achieved in plants using sequence specific nucleases
Type of DNA
modification
Nuclease Delivery
methods
Plants Targets
Trait stacking Megnucleases Bombardment Cotton Intergenic sequence
ZFN Bombardment Maize Transgene
Rewriting host
DNA
Megnucleases Stable integration Maize Intergenic sequence
ZFN Stable integration Soybean Transgene
TALEN Stable integration Barley PAPhy_a
TALEN Agrobacterium
T-DNA (transient)
Oryza sativa SWEET14
TALEN No Protoplasts Arabidopsis
Tobacco
AtTT4 AtADH NbSurB
TALEN Bombardment Wheat MLO
TALEN Protoplasts
Stable integration
Maize PDS IPK1A IPK MRP4
CRISPRCas Protoplasts
Agrobacterium
T-DNA (transient)
Tobacco
Arabidopsis
Sorghum Oryza
OsSWEET14 transgene
61
Type of DNA
modification
Nuclease Delivery
methods
Plants Targets
Rewriting host
DNA large
deletion
Zinc-finger
nuclease
Stable
integration
Tobacco Transgene
CRISPRCas Protoplasts
Stable
integration
Rice Labdane-related
diterpenoid gene
clusters
on Chr 2 4 and 6
Zinc-finger
nuclease
Agrobacterium
T-DNA
(transient)
Tobacco CHN50 transgene
Zinc-finger
nuclease
Whiskers Maize IPK1
CRISPRCas Protoplasts Rice PDS
Controlling gene
expression
TALE repressor
(SRDX)
Stable
integration
Arabidopsis RD29A transgene
Zinc-finger
activator (VP16)
Stable
integration
Brassica
napus
KasII
Continue
62
Application of genome editing1 Introduction of precise and predictable modifications directly in an elite
background
2 multiple traits can be modified simultaneously
3 NHEJ enables gene knockout and targeted modifications
4 Introduction of transgenes at defined loci that promote high-level
transcription and do not interfere with the activity of endogenous genes
5 Site-specific nucleases also allow targeted molecular trait stacking -low
risk of segregation
6 CRISPR Cas is a transgene free approach ndash No regulatory burdens
7 frequency of off-target mutations is well below that caused by chemical
and physical mutagenesis techniques
8 In future can be utilized for metabolic engineering and molecular farming
63
Six-way Venn diagram showing the distribution of shared genefamilies (sequence clusters) among M acuminata P dactyliferaArabidopsis thaliana Oryza sativa Sorghum bicolor and Brachypodiumdistachyon genomes
A DrsquoHont et al Nature 2012 doi101038nature11241
64
Achieving super-DomesticationINTROGRESS-RECREATE-CREATE-DOMESTICATEREDOMESTICATE
bull Mobilizing left out genetic
variation still available in
land races and wild and
weedy species
bull Replaying the evolutionary
tape( resynthesis in
polyploids)
bull Domesticateredomesticate
65
We can admire and emulate how indigenous people still domesticateplants create biodiversity and manage it to sustain their future Thereis no equivalently dynamic or flexible crop breeding in modernagriculture to promote biodiversity we still have much to learn from
traditional knowledge Jan Salick 2012
40
How to use diversitybull Cross two varieties
bull Genome manipulations
Cell fusion hybrids
bull Chromosome manipulation Backcross a new species
bull Generate recombinants Chromosome recombinations
bull Use a new species wild germplasm
Transgenic approach Modern mutagenesis synthetic gene
construction by utilizing green florescent protein genome editing
NGS GWAS sequencing etchelliphelliphelliphelliphellip
41
Reduce genetic bottlenecks through polyplodization
More vigour
SC
Buffering capacity
Heterotic advantage
Enhaced vegetative
charachers
Enhanced oil
Meiotic stability
42
Approach 1 Use one tetraploid and one diploid as parents (4X ndash 2X) followed by the
chromosome doubling of triploid hybrids (Chrom doubling)
a) Cross between B juncea (AjAjBjBj ) and B oleracea (CoCo) to produce hexaploids
(AjAjBjBjCoCo)
(b) Cross between B napus (AnAnCnCn) and B nigra (BniBni) to produce hexaploids
(AnAnBniBniCnCn)
(c) Cross between B carinata (BcaBcaCcaCca) and B rapa (ArAr) to produce hexaploids
(ArArBcaBcaCcaCca)
Approach 2 Use three tetraploids as parents
(a) Cross between B napus (AnAnCnCn) and B carinata (BcaBcaCcaCca) to produce unbalanced
allotetraploids (AnBcaCnCca ndash unreduced gametes gametes with the somatic chromosome
number and cross with B juncea (AjAjBjBj) to obtain allohexaploids (AnAjBcaBjCnCca
(b) Cross between B napus (AnAnCnCn) and B juncea (AjAjBjBj) to produce unbalanced
allotetraploids (AnAjBjCn ndash unreduced gametes) and cross with B carinata (BcaBcaCcaCca) to
obtain allohexaploids (AnAjBcaBjCnCca)
(c) Cross between B carinata (BcaBcaCcaCca) and B juncea (AjAjBjBj) to produce unbalanced
allotetraploids (AjBcaBjCca - ndash unreduced gametes) and cross with B napus (AnAnCnCn) to obtain
allohexaploids (AnAjBcaBjCnCca)
Approach 3 Use three diploids as parents (2X ndash 2X ndash 2X) Cross between B rapa (ArAr) B
nigra (BniBni) and B oleracea (CoCo) sequentially to obtain hexaploid hybrids (ArArBniBniCoCo)
43
Continuehellip
Guijun Yan 2012
44
Applications of Genomic
tools in Super-Domestication
45
Genome Sequencing
Potential methods of sequencing
1 Clone by clone approach
2 Whole genome shotgun approach
3 Combination of the two methods
Till today no of cultivated plants completely sequenced -85
2016- Arachis duranensis
2015- Solanum cumersonii (Wild potato)
(Ref- NCBI)
46
GWAS
It is a study design in which many markers spread across a genome
are genotyped and test a statistical association with a phenotype are
performed locally along the genome
It is also an examination of many common genetic variants in different
individuals to see if any variant is associated with a trait
Used in successfully studying maize sorghum and barley
Method is efficient for large scale low cost genotyping (even with the
minimum number of SNPs)
Cannot be utilized generally because it needs large population size
GWAS identify rare alleles more precisely
If small population we can opt NAM
47
GWAS and whole genome prediction
Xuehui Huang and Bin Han 2014
48
Five high throuput genotyping methods
Xuehui Huang and Bin Han 2014
49
Role of NGS in domestication Capture of novel genes from wild species will be made easier by
understanding the molecular events associated with crop domestication
Re-sequencing of domesticated species can identify low diversity regions
resulting from selection during domestication
To identify gene-specific sequences to aid the cloning of homologues of key
domestication genes from wild relatives
Candidate genes from wild and domesticated plant populations can define
diversity of target genes in wild populations and lead to the discovery of key
genes for important traits by association analysis
NGS of amplicons of large numbers of candidate genes from wild and
domesticated plant populations can define diversity of target genes in
wild populations and lead to the discovery of key genes for important
traits by association analysis
50
NGS
supports the rapid domestication of new plant species and the efficient
identification and capture of novel genetic variation from related species
Allows whole-genome analysis to determine the genetic basis of
phenotypic differences
NGS allows rapid expansion of genomic analysis to investigation of non-
model species
Made rice study easy by related grass
cost-effective method for plant identification
useful strategy to analysis the chloroplast genome sequence from whole-
genome shot-gun sequencing
facilitates managing this diversity and any changes in crop performance
over time due to genetic drift
Patterns of gene expression have been evaluated in hybrids using NGS
51
Two Approaches to Finding Adaptive Genes
Ross-Ibarra et al2007
52
Techniques to re-wild the plants (transgene free)
1 Introgression
breeding
2 Specific
insertion of lost genes
3 Precision
mutagenesis
Palmgren et al2014
53
Process of rewilding
Palmgren et al2014
54
Synthetic biology projects(i) Modifying cereals including wheat to fix atmospheric nitrogen
(ii) Redesigning metabolic pathways to increase the yield of
secondary metabolites or to generate compounds with enhanced
properties
(iii) Transferring the C4 photosynthesis pathway to rice
(iv) Modifying the glycosylation pathway in plants to accommodate
production of therapeutic proteins
(v) Introducing synthetic signal transduction systems that respond to
external cues
55
Synthetic biology projects
Nicholas J Baltes et al 2015
56
Refactoring the N fixation gene cluster from Klebsiella
Karsten Temme et al 2012
57
Continuehellip
Karsten Temme et al 2012
58
Genome editing
Sequence specific nucleases
1 Meganucleases
2 ZFMs ndashZink finger motifs
3 TALENs- Transcription activator like effector nuclease
4 CRISPR CAS-9 ndash Clustered interspaced short palindromic
repeats
qmp4
59Khaoula Belhaj et al 2015
60
Genome modifications achieved in plants using sequence specific nucleases
Type of DNA
modification
Nuclease Delivery
methods
Plants Targets
Trait stacking Megnucleases Bombardment Cotton Intergenic sequence
ZFN Bombardment Maize Transgene
Rewriting host
DNA
Megnucleases Stable integration Maize Intergenic sequence
ZFN Stable integration Soybean Transgene
TALEN Stable integration Barley PAPhy_a
TALEN Agrobacterium
T-DNA (transient)
Oryza sativa SWEET14
TALEN No Protoplasts Arabidopsis
Tobacco
AtTT4 AtADH NbSurB
TALEN Bombardment Wheat MLO
TALEN Protoplasts
Stable integration
Maize PDS IPK1A IPK MRP4
CRISPRCas Protoplasts
Agrobacterium
T-DNA (transient)
Tobacco
Arabidopsis
Sorghum Oryza
OsSWEET14 transgene
61
Type of DNA
modification
Nuclease Delivery
methods
Plants Targets
Rewriting host
DNA large
deletion
Zinc-finger
nuclease
Stable
integration
Tobacco Transgene
CRISPRCas Protoplasts
Stable
integration
Rice Labdane-related
diterpenoid gene
clusters
on Chr 2 4 and 6
Zinc-finger
nuclease
Agrobacterium
T-DNA
(transient)
Tobacco CHN50 transgene
Zinc-finger
nuclease
Whiskers Maize IPK1
CRISPRCas Protoplasts Rice PDS
Controlling gene
expression
TALE repressor
(SRDX)
Stable
integration
Arabidopsis RD29A transgene
Zinc-finger
activator (VP16)
Stable
integration
Brassica
napus
KasII
Continue
62
Application of genome editing1 Introduction of precise and predictable modifications directly in an elite
background
2 multiple traits can be modified simultaneously
3 NHEJ enables gene knockout and targeted modifications
4 Introduction of transgenes at defined loci that promote high-level
transcription and do not interfere with the activity of endogenous genes
5 Site-specific nucleases also allow targeted molecular trait stacking -low
risk of segregation
6 CRISPR Cas is a transgene free approach ndash No regulatory burdens
7 frequency of off-target mutations is well below that caused by chemical
and physical mutagenesis techniques
8 In future can be utilized for metabolic engineering and molecular farming
63
Six-way Venn diagram showing the distribution of shared genefamilies (sequence clusters) among M acuminata P dactyliferaArabidopsis thaliana Oryza sativa Sorghum bicolor and Brachypodiumdistachyon genomes
A DrsquoHont et al Nature 2012 doi101038nature11241
64
Achieving super-DomesticationINTROGRESS-RECREATE-CREATE-DOMESTICATEREDOMESTICATE
bull Mobilizing left out genetic
variation still available in
land races and wild and
weedy species
bull Replaying the evolutionary
tape( resynthesis in
polyploids)
bull Domesticateredomesticate
65
We can admire and emulate how indigenous people still domesticateplants create biodiversity and manage it to sustain their future Thereis no equivalently dynamic or flexible crop breeding in modernagriculture to promote biodiversity we still have much to learn from
traditional knowledge Jan Salick 2012
41
Reduce genetic bottlenecks through polyplodization
More vigour
SC
Buffering capacity
Heterotic advantage
Enhaced vegetative
charachers
Enhanced oil
Meiotic stability
42
Approach 1 Use one tetraploid and one diploid as parents (4X ndash 2X) followed by the
chromosome doubling of triploid hybrids (Chrom doubling)
a) Cross between B juncea (AjAjBjBj ) and B oleracea (CoCo) to produce hexaploids
(AjAjBjBjCoCo)
(b) Cross between B napus (AnAnCnCn) and B nigra (BniBni) to produce hexaploids
(AnAnBniBniCnCn)
(c) Cross between B carinata (BcaBcaCcaCca) and B rapa (ArAr) to produce hexaploids
(ArArBcaBcaCcaCca)
Approach 2 Use three tetraploids as parents
(a) Cross between B napus (AnAnCnCn) and B carinata (BcaBcaCcaCca) to produce unbalanced
allotetraploids (AnBcaCnCca ndash unreduced gametes gametes with the somatic chromosome
number and cross with B juncea (AjAjBjBj) to obtain allohexaploids (AnAjBcaBjCnCca
(b) Cross between B napus (AnAnCnCn) and B juncea (AjAjBjBj) to produce unbalanced
allotetraploids (AnAjBjCn ndash unreduced gametes) and cross with B carinata (BcaBcaCcaCca) to
obtain allohexaploids (AnAjBcaBjCnCca)
(c) Cross between B carinata (BcaBcaCcaCca) and B juncea (AjAjBjBj) to produce unbalanced
allotetraploids (AjBcaBjCca - ndash unreduced gametes) and cross with B napus (AnAnCnCn) to obtain
allohexaploids (AnAjBcaBjCnCca)
Approach 3 Use three diploids as parents (2X ndash 2X ndash 2X) Cross between B rapa (ArAr) B
nigra (BniBni) and B oleracea (CoCo) sequentially to obtain hexaploid hybrids (ArArBniBniCoCo)
43
Continuehellip
Guijun Yan 2012
44
Applications of Genomic
tools in Super-Domestication
45
Genome Sequencing
Potential methods of sequencing
1 Clone by clone approach
2 Whole genome shotgun approach
3 Combination of the two methods
Till today no of cultivated plants completely sequenced -85
2016- Arachis duranensis
2015- Solanum cumersonii (Wild potato)
(Ref- NCBI)
46
GWAS
It is a study design in which many markers spread across a genome
are genotyped and test a statistical association with a phenotype are
performed locally along the genome
It is also an examination of many common genetic variants in different
individuals to see if any variant is associated with a trait
Used in successfully studying maize sorghum and barley
Method is efficient for large scale low cost genotyping (even with the
minimum number of SNPs)
Cannot be utilized generally because it needs large population size
GWAS identify rare alleles more precisely
If small population we can opt NAM
47
GWAS and whole genome prediction
Xuehui Huang and Bin Han 2014
48
Five high throuput genotyping methods
Xuehui Huang and Bin Han 2014
49
Role of NGS in domestication Capture of novel genes from wild species will be made easier by
understanding the molecular events associated with crop domestication
Re-sequencing of domesticated species can identify low diversity regions
resulting from selection during domestication
To identify gene-specific sequences to aid the cloning of homologues of key
domestication genes from wild relatives
Candidate genes from wild and domesticated plant populations can define
diversity of target genes in wild populations and lead to the discovery of key
genes for important traits by association analysis
NGS of amplicons of large numbers of candidate genes from wild and
domesticated plant populations can define diversity of target genes in
wild populations and lead to the discovery of key genes for important
traits by association analysis
50
NGS
supports the rapid domestication of new plant species and the efficient
identification and capture of novel genetic variation from related species
Allows whole-genome analysis to determine the genetic basis of
phenotypic differences
NGS allows rapid expansion of genomic analysis to investigation of non-
model species
Made rice study easy by related grass
cost-effective method for plant identification
useful strategy to analysis the chloroplast genome sequence from whole-
genome shot-gun sequencing
facilitates managing this diversity and any changes in crop performance
over time due to genetic drift
Patterns of gene expression have been evaluated in hybrids using NGS
51
Two Approaches to Finding Adaptive Genes
Ross-Ibarra et al2007
52
Techniques to re-wild the plants (transgene free)
1 Introgression
breeding
2 Specific
insertion of lost genes
3 Precision
mutagenesis
Palmgren et al2014
53
Process of rewilding
Palmgren et al2014
54
Synthetic biology projects(i) Modifying cereals including wheat to fix atmospheric nitrogen
(ii) Redesigning metabolic pathways to increase the yield of
secondary metabolites or to generate compounds with enhanced
properties
(iii) Transferring the C4 photosynthesis pathway to rice
(iv) Modifying the glycosylation pathway in plants to accommodate
production of therapeutic proteins
(v) Introducing synthetic signal transduction systems that respond to
external cues
55
Synthetic biology projects
Nicholas J Baltes et al 2015
56
Refactoring the N fixation gene cluster from Klebsiella
Karsten Temme et al 2012
57
Continuehellip
Karsten Temme et al 2012
58
Genome editing
Sequence specific nucleases
1 Meganucleases
2 ZFMs ndashZink finger motifs
3 TALENs- Transcription activator like effector nuclease
4 CRISPR CAS-9 ndash Clustered interspaced short palindromic
repeats
qmp4
59Khaoula Belhaj et al 2015
60
Genome modifications achieved in plants using sequence specific nucleases
Type of DNA
modification
Nuclease Delivery
methods
Plants Targets
Trait stacking Megnucleases Bombardment Cotton Intergenic sequence
ZFN Bombardment Maize Transgene
Rewriting host
DNA
Megnucleases Stable integration Maize Intergenic sequence
ZFN Stable integration Soybean Transgene
TALEN Stable integration Barley PAPhy_a
TALEN Agrobacterium
T-DNA (transient)
Oryza sativa SWEET14
TALEN No Protoplasts Arabidopsis
Tobacco
AtTT4 AtADH NbSurB
TALEN Bombardment Wheat MLO
TALEN Protoplasts
Stable integration
Maize PDS IPK1A IPK MRP4
CRISPRCas Protoplasts
Agrobacterium
T-DNA (transient)
Tobacco
Arabidopsis
Sorghum Oryza
OsSWEET14 transgene
61
Type of DNA
modification
Nuclease Delivery
methods
Plants Targets
Rewriting host
DNA large
deletion
Zinc-finger
nuclease
Stable
integration
Tobacco Transgene
CRISPRCas Protoplasts
Stable
integration
Rice Labdane-related
diterpenoid gene
clusters
on Chr 2 4 and 6
Zinc-finger
nuclease
Agrobacterium
T-DNA
(transient)
Tobacco CHN50 transgene
Zinc-finger
nuclease
Whiskers Maize IPK1
CRISPRCas Protoplasts Rice PDS
Controlling gene
expression
TALE repressor
(SRDX)
Stable
integration
Arabidopsis RD29A transgene
Zinc-finger
activator (VP16)
Stable
integration
Brassica
napus
KasII
Continue
62
Application of genome editing1 Introduction of precise and predictable modifications directly in an elite
background
2 multiple traits can be modified simultaneously
3 NHEJ enables gene knockout and targeted modifications
4 Introduction of transgenes at defined loci that promote high-level
transcription and do not interfere with the activity of endogenous genes
5 Site-specific nucleases also allow targeted molecular trait stacking -low
risk of segregation
6 CRISPR Cas is a transgene free approach ndash No regulatory burdens
7 frequency of off-target mutations is well below that caused by chemical
and physical mutagenesis techniques
8 In future can be utilized for metabolic engineering and molecular farming
63
Six-way Venn diagram showing the distribution of shared genefamilies (sequence clusters) among M acuminata P dactyliferaArabidopsis thaliana Oryza sativa Sorghum bicolor and Brachypodiumdistachyon genomes
A DrsquoHont et al Nature 2012 doi101038nature11241
64
Achieving super-DomesticationINTROGRESS-RECREATE-CREATE-DOMESTICATEREDOMESTICATE
bull Mobilizing left out genetic
variation still available in
land races and wild and
weedy species
bull Replaying the evolutionary
tape( resynthesis in
polyploids)
bull Domesticateredomesticate
65
We can admire and emulate how indigenous people still domesticateplants create biodiversity and manage it to sustain their future Thereis no equivalently dynamic or flexible crop breeding in modernagriculture to promote biodiversity we still have much to learn from
traditional knowledge Jan Salick 2012
42
Approach 1 Use one tetraploid and one diploid as parents (4X ndash 2X) followed by the
chromosome doubling of triploid hybrids (Chrom doubling)
a) Cross between B juncea (AjAjBjBj ) and B oleracea (CoCo) to produce hexaploids
(AjAjBjBjCoCo)
(b) Cross between B napus (AnAnCnCn) and B nigra (BniBni) to produce hexaploids
(AnAnBniBniCnCn)
(c) Cross between B carinata (BcaBcaCcaCca) and B rapa (ArAr) to produce hexaploids
(ArArBcaBcaCcaCca)
Approach 2 Use three tetraploids as parents
(a) Cross between B napus (AnAnCnCn) and B carinata (BcaBcaCcaCca) to produce unbalanced
allotetraploids (AnBcaCnCca ndash unreduced gametes gametes with the somatic chromosome
number and cross with B juncea (AjAjBjBj) to obtain allohexaploids (AnAjBcaBjCnCca
(b) Cross between B napus (AnAnCnCn) and B juncea (AjAjBjBj) to produce unbalanced
allotetraploids (AnAjBjCn ndash unreduced gametes) and cross with B carinata (BcaBcaCcaCca) to
obtain allohexaploids (AnAjBcaBjCnCca)
(c) Cross between B carinata (BcaBcaCcaCca) and B juncea (AjAjBjBj) to produce unbalanced
allotetraploids (AjBcaBjCca - ndash unreduced gametes) and cross with B napus (AnAnCnCn) to obtain
allohexaploids (AnAjBcaBjCnCca)
Approach 3 Use three diploids as parents (2X ndash 2X ndash 2X) Cross between B rapa (ArAr) B
nigra (BniBni) and B oleracea (CoCo) sequentially to obtain hexaploid hybrids (ArArBniBniCoCo)
43
Continuehellip
Guijun Yan 2012
44
Applications of Genomic
tools in Super-Domestication
45
Genome Sequencing
Potential methods of sequencing
1 Clone by clone approach
2 Whole genome shotgun approach
3 Combination of the two methods
Till today no of cultivated plants completely sequenced -85
2016- Arachis duranensis
2015- Solanum cumersonii (Wild potato)
(Ref- NCBI)
46
GWAS
It is a study design in which many markers spread across a genome
are genotyped and test a statistical association with a phenotype are
performed locally along the genome
It is also an examination of many common genetic variants in different
individuals to see if any variant is associated with a trait
Used in successfully studying maize sorghum and barley
Method is efficient for large scale low cost genotyping (even with the
minimum number of SNPs)
Cannot be utilized generally because it needs large population size
GWAS identify rare alleles more precisely
If small population we can opt NAM
47
GWAS and whole genome prediction
Xuehui Huang and Bin Han 2014
48
Five high throuput genotyping methods
Xuehui Huang and Bin Han 2014
49
Role of NGS in domestication Capture of novel genes from wild species will be made easier by
understanding the molecular events associated with crop domestication
Re-sequencing of domesticated species can identify low diversity regions
resulting from selection during domestication
To identify gene-specific sequences to aid the cloning of homologues of key
domestication genes from wild relatives
Candidate genes from wild and domesticated plant populations can define
diversity of target genes in wild populations and lead to the discovery of key
genes for important traits by association analysis
NGS of amplicons of large numbers of candidate genes from wild and
domesticated plant populations can define diversity of target genes in
wild populations and lead to the discovery of key genes for important
traits by association analysis
50
NGS
supports the rapid domestication of new plant species and the efficient
identification and capture of novel genetic variation from related species
Allows whole-genome analysis to determine the genetic basis of
phenotypic differences
NGS allows rapid expansion of genomic analysis to investigation of non-
model species
Made rice study easy by related grass
cost-effective method for plant identification
useful strategy to analysis the chloroplast genome sequence from whole-
genome shot-gun sequencing
facilitates managing this diversity and any changes in crop performance
over time due to genetic drift
Patterns of gene expression have been evaluated in hybrids using NGS
51
Two Approaches to Finding Adaptive Genes
Ross-Ibarra et al2007
52
Techniques to re-wild the plants (transgene free)
1 Introgression
breeding
2 Specific
insertion of lost genes
3 Precision
mutagenesis
Palmgren et al2014
53
Process of rewilding
Palmgren et al2014
54
Synthetic biology projects(i) Modifying cereals including wheat to fix atmospheric nitrogen
(ii) Redesigning metabolic pathways to increase the yield of
secondary metabolites or to generate compounds with enhanced
properties
(iii) Transferring the C4 photosynthesis pathway to rice
(iv) Modifying the glycosylation pathway in plants to accommodate
production of therapeutic proteins
(v) Introducing synthetic signal transduction systems that respond to
external cues
55
Synthetic biology projects
Nicholas J Baltes et al 2015
56
Refactoring the N fixation gene cluster from Klebsiella
Karsten Temme et al 2012
57
Continuehellip
Karsten Temme et al 2012
58
Genome editing
Sequence specific nucleases
1 Meganucleases
2 ZFMs ndashZink finger motifs
3 TALENs- Transcription activator like effector nuclease
4 CRISPR CAS-9 ndash Clustered interspaced short palindromic
repeats
qmp4
59Khaoula Belhaj et al 2015
60
Genome modifications achieved in plants using sequence specific nucleases
Type of DNA
modification
Nuclease Delivery
methods
Plants Targets
Trait stacking Megnucleases Bombardment Cotton Intergenic sequence
ZFN Bombardment Maize Transgene
Rewriting host
DNA
Megnucleases Stable integration Maize Intergenic sequence
ZFN Stable integration Soybean Transgene
TALEN Stable integration Barley PAPhy_a
TALEN Agrobacterium
T-DNA (transient)
Oryza sativa SWEET14
TALEN No Protoplasts Arabidopsis
Tobacco
AtTT4 AtADH NbSurB
TALEN Bombardment Wheat MLO
TALEN Protoplasts
Stable integration
Maize PDS IPK1A IPK MRP4
CRISPRCas Protoplasts
Agrobacterium
T-DNA (transient)
Tobacco
Arabidopsis
Sorghum Oryza
OsSWEET14 transgene
61
Type of DNA
modification
Nuclease Delivery
methods
Plants Targets
Rewriting host
DNA large
deletion
Zinc-finger
nuclease
Stable
integration
Tobacco Transgene
CRISPRCas Protoplasts
Stable
integration
Rice Labdane-related
diterpenoid gene
clusters
on Chr 2 4 and 6
Zinc-finger
nuclease
Agrobacterium
T-DNA
(transient)
Tobacco CHN50 transgene
Zinc-finger
nuclease
Whiskers Maize IPK1
CRISPRCas Protoplasts Rice PDS
Controlling gene
expression
TALE repressor
(SRDX)
Stable
integration
Arabidopsis RD29A transgene
Zinc-finger
activator (VP16)
Stable
integration
Brassica
napus
KasII
Continue
62
Application of genome editing1 Introduction of precise and predictable modifications directly in an elite
background
2 multiple traits can be modified simultaneously
3 NHEJ enables gene knockout and targeted modifications
4 Introduction of transgenes at defined loci that promote high-level
transcription and do not interfere with the activity of endogenous genes
5 Site-specific nucleases also allow targeted molecular trait stacking -low
risk of segregation
6 CRISPR Cas is a transgene free approach ndash No regulatory burdens
7 frequency of off-target mutations is well below that caused by chemical
and physical mutagenesis techniques
8 In future can be utilized for metabolic engineering and molecular farming
63
Six-way Venn diagram showing the distribution of shared genefamilies (sequence clusters) among M acuminata P dactyliferaArabidopsis thaliana Oryza sativa Sorghum bicolor and Brachypodiumdistachyon genomes
A DrsquoHont et al Nature 2012 doi101038nature11241
64
Achieving super-DomesticationINTROGRESS-RECREATE-CREATE-DOMESTICATEREDOMESTICATE
bull Mobilizing left out genetic
variation still available in
land races and wild and
weedy species
bull Replaying the evolutionary
tape( resynthesis in
polyploids)
bull Domesticateredomesticate
65
We can admire and emulate how indigenous people still domesticateplants create biodiversity and manage it to sustain their future Thereis no equivalently dynamic or flexible crop breeding in modernagriculture to promote biodiversity we still have much to learn from
traditional knowledge Jan Salick 2012
43
Continuehellip
Guijun Yan 2012
44
Applications of Genomic
tools in Super-Domestication
45
Genome Sequencing
Potential methods of sequencing
1 Clone by clone approach
2 Whole genome shotgun approach
3 Combination of the two methods
Till today no of cultivated plants completely sequenced -85
2016- Arachis duranensis
2015- Solanum cumersonii (Wild potato)
(Ref- NCBI)
46
GWAS
It is a study design in which many markers spread across a genome
are genotyped and test a statistical association with a phenotype are
performed locally along the genome
It is also an examination of many common genetic variants in different
individuals to see if any variant is associated with a trait
Used in successfully studying maize sorghum and barley
Method is efficient for large scale low cost genotyping (even with the
minimum number of SNPs)
Cannot be utilized generally because it needs large population size
GWAS identify rare alleles more precisely
If small population we can opt NAM
47
GWAS and whole genome prediction
Xuehui Huang and Bin Han 2014
48
Five high throuput genotyping methods
Xuehui Huang and Bin Han 2014
49
Role of NGS in domestication Capture of novel genes from wild species will be made easier by
understanding the molecular events associated with crop domestication
Re-sequencing of domesticated species can identify low diversity regions
resulting from selection during domestication
To identify gene-specific sequences to aid the cloning of homologues of key
domestication genes from wild relatives
Candidate genes from wild and domesticated plant populations can define
diversity of target genes in wild populations and lead to the discovery of key
genes for important traits by association analysis
NGS of amplicons of large numbers of candidate genes from wild and
domesticated plant populations can define diversity of target genes in
wild populations and lead to the discovery of key genes for important
traits by association analysis
50
NGS
supports the rapid domestication of new plant species and the efficient
identification and capture of novel genetic variation from related species
Allows whole-genome analysis to determine the genetic basis of
phenotypic differences
NGS allows rapid expansion of genomic analysis to investigation of non-
model species
Made rice study easy by related grass
cost-effective method for plant identification
useful strategy to analysis the chloroplast genome sequence from whole-
genome shot-gun sequencing
facilitates managing this diversity and any changes in crop performance
over time due to genetic drift
Patterns of gene expression have been evaluated in hybrids using NGS
51
Two Approaches to Finding Adaptive Genes
Ross-Ibarra et al2007
52
Techniques to re-wild the plants (transgene free)
1 Introgression
breeding
2 Specific
insertion of lost genes
3 Precision
mutagenesis
Palmgren et al2014
53
Process of rewilding
Palmgren et al2014
54
Synthetic biology projects(i) Modifying cereals including wheat to fix atmospheric nitrogen
(ii) Redesigning metabolic pathways to increase the yield of
secondary metabolites or to generate compounds with enhanced
properties
(iii) Transferring the C4 photosynthesis pathway to rice
(iv) Modifying the glycosylation pathway in plants to accommodate
production of therapeutic proteins
(v) Introducing synthetic signal transduction systems that respond to
external cues
55
Synthetic biology projects
Nicholas J Baltes et al 2015
56
Refactoring the N fixation gene cluster from Klebsiella
Karsten Temme et al 2012
57
Continuehellip
Karsten Temme et al 2012
58
Genome editing
Sequence specific nucleases
1 Meganucleases
2 ZFMs ndashZink finger motifs
3 TALENs- Transcription activator like effector nuclease
4 CRISPR CAS-9 ndash Clustered interspaced short palindromic
repeats
qmp4
59Khaoula Belhaj et al 2015
60
Genome modifications achieved in plants using sequence specific nucleases
Type of DNA
modification
Nuclease Delivery
methods
Plants Targets
Trait stacking Megnucleases Bombardment Cotton Intergenic sequence
ZFN Bombardment Maize Transgene
Rewriting host
DNA
Megnucleases Stable integration Maize Intergenic sequence
ZFN Stable integration Soybean Transgene
TALEN Stable integration Barley PAPhy_a
TALEN Agrobacterium
T-DNA (transient)
Oryza sativa SWEET14
TALEN No Protoplasts Arabidopsis
Tobacco
AtTT4 AtADH NbSurB
TALEN Bombardment Wheat MLO
TALEN Protoplasts
Stable integration
Maize PDS IPK1A IPK MRP4
CRISPRCas Protoplasts
Agrobacterium
T-DNA (transient)
Tobacco
Arabidopsis
Sorghum Oryza
OsSWEET14 transgene
61
Type of DNA
modification
Nuclease Delivery
methods
Plants Targets
Rewriting host
DNA large
deletion
Zinc-finger
nuclease
Stable
integration
Tobacco Transgene
CRISPRCas Protoplasts
Stable
integration
Rice Labdane-related
diterpenoid gene
clusters
on Chr 2 4 and 6
Zinc-finger
nuclease
Agrobacterium
T-DNA
(transient)
Tobacco CHN50 transgene
Zinc-finger
nuclease
Whiskers Maize IPK1
CRISPRCas Protoplasts Rice PDS
Controlling gene
expression
TALE repressor
(SRDX)
Stable
integration
Arabidopsis RD29A transgene
Zinc-finger
activator (VP16)
Stable
integration
Brassica
napus
KasII
Continue
62
Application of genome editing1 Introduction of precise and predictable modifications directly in an elite
background
2 multiple traits can be modified simultaneously
3 NHEJ enables gene knockout and targeted modifications
4 Introduction of transgenes at defined loci that promote high-level
transcription and do not interfere with the activity of endogenous genes
5 Site-specific nucleases also allow targeted molecular trait stacking -low
risk of segregation
6 CRISPR Cas is a transgene free approach ndash No regulatory burdens
7 frequency of off-target mutations is well below that caused by chemical
and physical mutagenesis techniques
8 In future can be utilized for metabolic engineering and molecular farming
63
Six-way Venn diagram showing the distribution of shared genefamilies (sequence clusters) among M acuminata P dactyliferaArabidopsis thaliana Oryza sativa Sorghum bicolor and Brachypodiumdistachyon genomes
A DrsquoHont et al Nature 2012 doi101038nature11241
64
Achieving super-DomesticationINTROGRESS-RECREATE-CREATE-DOMESTICATEREDOMESTICATE
bull Mobilizing left out genetic
variation still available in
land races and wild and
weedy species
bull Replaying the evolutionary
tape( resynthesis in
polyploids)
bull Domesticateredomesticate
65
We can admire and emulate how indigenous people still domesticateplants create biodiversity and manage it to sustain their future Thereis no equivalently dynamic or flexible crop breeding in modernagriculture to promote biodiversity we still have much to learn from
traditional knowledge Jan Salick 2012
44
Applications of Genomic
tools in Super-Domestication
45
Genome Sequencing
Potential methods of sequencing
1 Clone by clone approach
2 Whole genome shotgun approach
3 Combination of the two methods
Till today no of cultivated plants completely sequenced -85
2016- Arachis duranensis
2015- Solanum cumersonii (Wild potato)
(Ref- NCBI)
46
GWAS
It is a study design in which many markers spread across a genome
are genotyped and test a statistical association with a phenotype are
performed locally along the genome
It is also an examination of many common genetic variants in different
individuals to see if any variant is associated with a trait
Used in successfully studying maize sorghum and barley
Method is efficient for large scale low cost genotyping (even with the
minimum number of SNPs)
Cannot be utilized generally because it needs large population size
GWAS identify rare alleles more precisely
If small population we can opt NAM
47
GWAS and whole genome prediction
Xuehui Huang and Bin Han 2014
48
Five high throuput genotyping methods
Xuehui Huang and Bin Han 2014
49
Role of NGS in domestication Capture of novel genes from wild species will be made easier by
understanding the molecular events associated with crop domestication
Re-sequencing of domesticated species can identify low diversity regions
resulting from selection during domestication
To identify gene-specific sequences to aid the cloning of homologues of key
domestication genes from wild relatives
Candidate genes from wild and domesticated plant populations can define
diversity of target genes in wild populations and lead to the discovery of key
genes for important traits by association analysis
NGS of amplicons of large numbers of candidate genes from wild and
domesticated plant populations can define diversity of target genes in
wild populations and lead to the discovery of key genes for important
traits by association analysis
50
NGS
supports the rapid domestication of new plant species and the efficient
identification and capture of novel genetic variation from related species
Allows whole-genome analysis to determine the genetic basis of
phenotypic differences
NGS allows rapid expansion of genomic analysis to investigation of non-
model species
Made rice study easy by related grass
cost-effective method for plant identification
useful strategy to analysis the chloroplast genome sequence from whole-
genome shot-gun sequencing
facilitates managing this diversity and any changes in crop performance
over time due to genetic drift
Patterns of gene expression have been evaluated in hybrids using NGS
51
Two Approaches to Finding Adaptive Genes
Ross-Ibarra et al2007
52
Techniques to re-wild the plants (transgene free)
1 Introgression
breeding
2 Specific
insertion of lost genes
3 Precision
mutagenesis
Palmgren et al2014
53
Process of rewilding
Palmgren et al2014
54
Synthetic biology projects(i) Modifying cereals including wheat to fix atmospheric nitrogen
(ii) Redesigning metabolic pathways to increase the yield of
secondary metabolites or to generate compounds with enhanced
properties
(iii) Transferring the C4 photosynthesis pathway to rice
(iv) Modifying the glycosylation pathway in plants to accommodate
production of therapeutic proteins
(v) Introducing synthetic signal transduction systems that respond to
external cues
55
Synthetic biology projects
Nicholas J Baltes et al 2015
56
Refactoring the N fixation gene cluster from Klebsiella
Karsten Temme et al 2012
57
Continuehellip
Karsten Temme et al 2012
58
Genome editing
Sequence specific nucleases
1 Meganucleases
2 ZFMs ndashZink finger motifs
3 TALENs- Transcription activator like effector nuclease
4 CRISPR CAS-9 ndash Clustered interspaced short palindromic
repeats
qmp4
59Khaoula Belhaj et al 2015
60
Genome modifications achieved in plants using sequence specific nucleases
Type of DNA
modification
Nuclease Delivery
methods
Plants Targets
Trait stacking Megnucleases Bombardment Cotton Intergenic sequence
ZFN Bombardment Maize Transgene
Rewriting host
DNA
Megnucleases Stable integration Maize Intergenic sequence
ZFN Stable integration Soybean Transgene
TALEN Stable integration Barley PAPhy_a
TALEN Agrobacterium
T-DNA (transient)
Oryza sativa SWEET14
TALEN No Protoplasts Arabidopsis
Tobacco
AtTT4 AtADH NbSurB
TALEN Bombardment Wheat MLO
TALEN Protoplasts
Stable integration
Maize PDS IPK1A IPK MRP4
CRISPRCas Protoplasts
Agrobacterium
T-DNA (transient)
Tobacco
Arabidopsis
Sorghum Oryza
OsSWEET14 transgene
61
Type of DNA
modification
Nuclease Delivery
methods
Plants Targets
Rewriting host
DNA large
deletion
Zinc-finger
nuclease
Stable
integration
Tobacco Transgene
CRISPRCas Protoplasts
Stable
integration
Rice Labdane-related
diterpenoid gene
clusters
on Chr 2 4 and 6
Zinc-finger
nuclease
Agrobacterium
T-DNA
(transient)
Tobacco CHN50 transgene
Zinc-finger
nuclease
Whiskers Maize IPK1
CRISPRCas Protoplasts Rice PDS
Controlling gene
expression
TALE repressor
(SRDX)
Stable
integration
Arabidopsis RD29A transgene
Zinc-finger
activator (VP16)
Stable
integration
Brassica
napus
KasII
Continue
62
Application of genome editing1 Introduction of precise and predictable modifications directly in an elite
background
2 multiple traits can be modified simultaneously
3 NHEJ enables gene knockout and targeted modifications
4 Introduction of transgenes at defined loci that promote high-level
transcription and do not interfere with the activity of endogenous genes
5 Site-specific nucleases also allow targeted molecular trait stacking -low
risk of segregation
6 CRISPR Cas is a transgene free approach ndash No regulatory burdens
7 frequency of off-target mutations is well below that caused by chemical
and physical mutagenesis techniques
8 In future can be utilized for metabolic engineering and molecular farming
63
Six-way Venn diagram showing the distribution of shared genefamilies (sequence clusters) among M acuminata P dactyliferaArabidopsis thaliana Oryza sativa Sorghum bicolor and Brachypodiumdistachyon genomes
A DrsquoHont et al Nature 2012 doi101038nature11241
64
Achieving super-DomesticationINTROGRESS-RECREATE-CREATE-DOMESTICATEREDOMESTICATE
bull Mobilizing left out genetic
variation still available in
land races and wild and
weedy species
bull Replaying the evolutionary
tape( resynthesis in
polyploids)
bull Domesticateredomesticate
65
We can admire and emulate how indigenous people still domesticateplants create biodiversity and manage it to sustain their future Thereis no equivalently dynamic or flexible crop breeding in modernagriculture to promote biodiversity we still have much to learn from
traditional knowledge Jan Salick 2012
45
Genome Sequencing
Potential methods of sequencing
1 Clone by clone approach
2 Whole genome shotgun approach
3 Combination of the two methods
Till today no of cultivated plants completely sequenced -85
2016- Arachis duranensis
2015- Solanum cumersonii (Wild potato)
(Ref- NCBI)
46
GWAS
It is a study design in which many markers spread across a genome
are genotyped and test a statistical association with a phenotype are
performed locally along the genome
It is also an examination of many common genetic variants in different
individuals to see if any variant is associated with a trait
Used in successfully studying maize sorghum and barley
Method is efficient for large scale low cost genotyping (even with the
minimum number of SNPs)
Cannot be utilized generally because it needs large population size
GWAS identify rare alleles more precisely
If small population we can opt NAM
47
GWAS and whole genome prediction
Xuehui Huang and Bin Han 2014
48
Five high throuput genotyping methods
Xuehui Huang and Bin Han 2014
49
Role of NGS in domestication Capture of novel genes from wild species will be made easier by
understanding the molecular events associated with crop domestication
Re-sequencing of domesticated species can identify low diversity regions
resulting from selection during domestication
To identify gene-specific sequences to aid the cloning of homologues of key
domestication genes from wild relatives
Candidate genes from wild and domesticated plant populations can define
diversity of target genes in wild populations and lead to the discovery of key
genes for important traits by association analysis
NGS of amplicons of large numbers of candidate genes from wild and
domesticated plant populations can define diversity of target genes in
wild populations and lead to the discovery of key genes for important
traits by association analysis
50
NGS
supports the rapid domestication of new plant species and the efficient
identification and capture of novel genetic variation from related species
Allows whole-genome analysis to determine the genetic basis of
phenotypic differences
NGS allows rapid expansion of genomic analysis to investigation of non-
model species
Made rice study easy by related grass
cost-effective method for plant identification
useful strategy to analysis the chloroplast genome sequence from whole-
genome shot-gun sequencing
facilitates managing this diversity and any changes in crop performance
over time due to genetic drift
Patterns of gene expression have been evaluated in hybrids using NGS
51
Two Approaches to Finding Adaptive Genes
Ross-Ibarra et al2007
52
Techniques to re-wild the plants (transgene free)
1 Introgression
breeding
2 Specific
insertion of lost genes
3 Precision
mutagenesis
Palmgren et al2014
53
Process of rewilding
Palmgren et al2014
54
Synthetic biology projects(i) Modifying cereals including wheat to fix atmospheric nitrogen
(ii) Redesigning metabolic pathways to increase the yield of
secondary metabolites or to generate compounds with enhanced
properties
(iii) Transferring the C4 photosynthesis pathway to rice
(iv) Modifying the glycosylation pathway in plants to accommodate
production of therapeutic proteins
(v) Introducing synthetic signal transduction systems that respond to
external cues
55
Synthetic biology projects
Nicholas J Baltes et al 2015
56
Refactoring the N fixation gene cluster from Klebsiella
Karsten Temme et al 2012
57
Continuehellip
Karsten Temme et al 2012
58
Genome editing
Sequence specific nucleases
1 Meganucleases
2 ZFMs ndashZink finger motifs
3 TALENs- Transcription activator like effector nuclease
4 CRISPR CAS-9 ndash Clustered interspaced short palindromic
repeats
qmp4
59Khaoula Belhaj et al 2015
60
Genome modifications achieved in plants using sequence specific nucleases
Type of DNA
modification
Nuclease Delivery
methods
Plants Targets
Trait stacking Megnucleases Bombardment Cotton Intergenic sequence
ZFN Bombardment Maize Transgene
Rewriting host
DNA
Megnucleases Stable integration Maize Intergenic sequence
ZFN Stable integration Soybean Transgene
TALEN Stable integration Barley PAPhy_a
TALEN Agrobacterium
T-DNA (transient)
Oryza sativa SWEET14
TALEN No Protoplasts Arabidopsis
Tobacco
AtTT4 AtADH NbSurB
TALEN Bombardment Wheat MLO
TALEN Protoplasts
Stable integration
Maize PDS IPK1A IPK MRP4
CRISPRCas Protoplasts
Agrobacterium
T-DNA (transient)
Tobacco
Arabidopsis
Sorghum Oryza
OsSWEET14 transgene
61
Type of DNA
modification
Nuclease Delivery
methods
Plants Targets
Rewriting host
DNA large
deletion
Zinc-finger
nuclease
Stable
integration
Tobacco Transgene
CRISPRCas Protoplasts
Stable
integration
Rice Labdane-related
diterpenoid gene
clusters
on Chr 2 4 and 6
Zinc-finger
nuclease
Agrobacterium
T-DNA
(transient)
Tobacco CHN50 transgene
Zinc-finger
nuclease
Whiskers Maize IPK1
CRISPRCas Protoplasts Rice PDS
Controlling gene
expression
TALE repressor
(SRDX)
Stable
integration
Arabidopsis RD29A transgene
Zinc-finger
activator (VP16)
Stable
integration
Brassica
napus
KasII
Continue
62
Application of genome editing1 Introduction of precise and predictable modifications directly in an elite
background
2 multiple traits can be modified simultaneously
3 NHEJ enables gene knockout and targeted modifications
4 Introduction of transgenes at defined loci that promote high-level
transcription and do not interfere with the activity of endogenous genes
5 Site-specific nucleases also allow targeted molecular trait stacking -low
risk of segregation
6 CRISPR Cas is a transgene free approach ndash No regulatory burdens
7 frequency of off-target mutations is well below that caused by chemical
and physical mutagenesis techniques
8 In future can be utilized for metabolic engineering and molecular farming
63
Six-way Venn diagram showing the distribution of shared genefamilies (sequence clusters) among M acuminata P dactyliferaArabidopsis thaliana Oryza sativa Sorghum bicolor and Brachypodiumdistachyon genomes
A DrsquoHont et al Nature 2012 doi101038nature11241
64
Achieving super-DomesticationINTROGRESS-RECREATE-CREATE-DOMESTICATEREDOMESTICATE
bull Mobilizing left out genetic
variation still available in
land races and wild and
weedy species
bull Replaying the evolutionary
tape( resynthesis in
polyploids)
bull Domesticateredomesticate
65
We can admire and emulate how indigenous people still domesticateplants create biodiversity and manage it to sustain their future Thereis no equivalently dynamic or flexible crop breeding in modernagriculture to promote biodiversity we still have much to learn from
traditional knowledge Jan Salick 2012
46
GWAS
It is a study design in which many markers spread across a genome
are genotyped and test a statistical association with a phenotype are
performed locally along the genome
It is also an examination of many common genetic variants in different
individuals to see if any variant is associated with a trait
Used in successfully studying maize sorghum and barley
Method is efficient for large scale low cost genotyping (even with the
minimum number of SNPs)
Cannot be utilized generally because it needs large population size
GWAS identify rare alleles more precisely
If small population we can opt NAM
47
GWAS and whole genome prediction
Xuehui Huang and Bin Han 2014
48
Five high throuput genotyping methods
Xuehui Huang and Bin Han 2014
49
Role of NGS in domestication Capture of novel genes from wild species will be made easier by
understanding the molecular events associated with crop domestication
Re-sequencing of domesticated species can identify low diversity regions
resulting from selection during domestication
To identify gene-specific sequences to aid the cloning of homologues of key
domestication genes from wild relatives
Candidate genes from wild and domesticated plant populations can define
diversity of target genes in wild populations and lead to the discovery of key
genes for important traits by association analysis
NGS of amplicons of large numbers of candidate genes from wild and
domesticated plant populations can define diversity of target genes in
wild populations and lead to the discovery of key genes for important
traits by association analysis
50
NGS
supports the rapid domestication of new plant species and the efficient
identification and capture of novel genetic variation from related species
Allows whole-genome analysis to determine the genetic basis of
phenotypic differences
NGS allows rapid expansion of genomic analysis to investigation of non-
model species
Made rice study easy by related grass
cost-effective method for plant identification
useful strategy to analysis the chloroplast genome sequence from whole-
genome shot-gun sequencing
facilitates managing this diversity and any changes in crop performance
over time due to genetic drift
Patterns of gene expression have been evaluated in hybrids using NGS
51
Two Approaches to Finding Adaptive Genes
Ross-Ibarra et al2007
52
Techniques to re-wild the plants (transgene free)
1 Introgression
breeding
2 Specific
insertion of lost genes
3 Precision
mutagenesis
Palmgren et al2014
53
Process of rewilding
Palmgren et al2014
54
Synthetic biology projects(i) Modifying cereals including wheat to fix atmospheric nitrogen
(ii) Redesigning metabolic pathways to increase the yield of
secondary metabolites or to generate compounds with enhanced
properties
(iii) Transferring the C4 photosynthesis pathway to rice
(iv) Modifying the glycosylation pathway in plants to accommodate
production of therapeutic proteins
(v) Introducing synthetic signal transduction systems that respond to
external cues
55
Synthetic biology projects
Nicholas J Baltes et al 2015
56
Refactoring the N fixation gene cluster from Klebsiella
Karsten Temme et al 2012
57
Continuehellip
Karsten Temme et al 2012
58
Genome editing
Sequence specific nucleases
1 Meganucleases
2 ZFMs ndashZink finger motifs
3 TALENs- Transcription activator like effector nuclease
4 CRISPR CAS-9 ndash Clustered interspaced short palindromic
repeats
qmp4
59Khaoula Belhaj et al 2015
60
Genome modifications achieved in plants using sequence specific nucleases
Type of DNA
modification
Nuclease Delivery
methods
Plants Targets
Trait stacking Megnucleases Bombardment Cotton Intergenic sequence
ZFN Bombardment Maize Transgene
Rewriting host
DNA
Megnucleases Stable integration Maize Intergenic sequence
ZFN Stable integration Soybean Transgene
TALEN Stable integration Barley PAPhy_a
TALEN Agrobacterium
T-DNA (transient)
Oryza sativa SWEET14
TALEN No Protoplasts Arabidopsis
Tobacco
AtTT4 AtADH NbSurB
TALEN Bombardment Wheat MLO
TALEN Protoplasts
Stable integration
Maize PDS IPK1A IPK MRP4
CRISPRCas Protoplasts
Agrobacterium
T-DNA (transient)
Tobacco
Arabidopsis
Sorghum Oryza
OsSWEET14 transgene
61
Type of DNA
modification
Nuclease Delivery
methods
Plants Targets
Rewriting host
DNA large
deletion
Zinc-finger
nuclease
Stable
integration
Tobacco Transgene
CRISPRCas Protoplasts
Stable
integration
Rice Labdane-related
diterpenoid gene
clusters
on Chr 2 4 and 6
Zinc-finger
nuclease
Agrobacterium
T-DNA
(transient)
Tobacco CHN50 transgene
Zinc-finger
nuclease
Whiskers Maize IPK1
CRISPRCas Protoplasts Rice PDS
Controlling gene
expression
TALE repressor
(SRDX)
Stable
integration
Arabidopsis RD29A transgene
Zinc-finger
activator (VP16)
Stable
integration
Brassica
napus
KasII
Continue
62
Application of genome editing1 Introduction of precise and predictable modifications directly in an elite
background
2 multiple traits can be modified simultaneously
3 NHEJ enables gene knockout and targeted modifications
4 Introduction of transgenes at defined loci that promote high-level
transcription and do not interfere with the activity of endogenous genes
5 Site-specific nucleases also allow targeted molecular trait stacking -low
risk of segregation
6 CRISPR Cas is a transgene free approach ndash No regulatory burdens
7 frequency of off-target mutations is well below that caused by chemical
and physical mutagenesis techniques
8 In future can be utilized for metabolic engineering and molecular farming
63
Six-way Venn diagram showing the distribution of shared genefamilies (sequence clusters) among M acuminata P dactyliferaArabidopsis thaliana Oryza sativa Sorghum bicolor and Brachypodiumdistachyon genomes
A DrsquoHont et al Nature 2012 doi101038nature11241
64
Achieving super-DomesticationINTROGRESS-RECREATE-CREATE-DOMESTICATEREDOMESTICATE
bull Mobilizing left out genetic
variation still available in
land races and wild and
weedy species
bull Replaying the evolutionary
tape( resynthesis in
polyploids)
bull Domesticateredomesticate
65
We can admire and emulate how indigenous people still domesticateplants create biodiversity and manage it to sustain their future Thereis no equivalently dynamic or flexible crop breeding in modernagriculture to promote biodiversity we still have much to learn from
traditional knowledge Jan Salick 2012
47
GWAS and whole genome prediction
Xuehui Huang and Bin Han 2014
48
Five high throuput genotyping methods
Xuehui Huang and Bin Han 2014
49
Role of NGS in domestication Capture of novel genes from wild species will be made easier by
understanding the molecular events associated with crop domestication
Re-sequencing of domesticated species can identify low diversity regions
resulting from selection during domestication
To identify gene-specific sequences to aid the cloning of homologues of key
domestication genes from wild relatives
Candidate genes from wild and domesticated plant populations can define
diversity of target genes in wild populations and lead to the discovery of key
genes for important traits by association analysis
NGS of amplicons of large numbers of candidate genes from wild and
domesticated plant populations can define diversity of target genes in
wild populations and lead to the discovery of key genes for important
traits by association analysis
50
NGS
supports the rapid domestication of new plant species and the efficient
identification and capture of novel genetic variation from related species
Allows whole-genome analysis to determine the genetic basis of
phenotypic differences
NGS allows rapid expansion of genomic analysis to investigation of non-
model species
Made rice study easy by related grass
cost-effective method for plant identification
useful strategy to analysis the chloroplast genome sequence from whole-
genome shot-gun sequencing
facilitates managing this diversity and any changes in crop performance
over time due to genetic drift
Patterns of gene expression have been evaluated in hybrids using NGS
51
Two Approaches to Finding Adaptive Genes
Ross-Ibarra et al2007
52
Techniques to re-wild the plants (transgene free)
1 Introgression
breeding
2 Specific
insertion of lost genes
3 Precision
mutagenesis
Palmgren et al2014
53
Process of rewilding
Palmgren et al2014
54
Synthetic biology projects(i) Modifying cereals including wheat to fix atmospheric nitrogen
(ii) Redesigning metabolic pathways to increase the yield of
secondary metabolites or to generate compounds with enhanced
properties
(iii) Transferring the C4 photosynthesis pathway to rice
(iv) Modifying the glycosylation pathway in plants to accommodate
production of therapeutic proteins
(v) Introducing synthetic signal transduction systems that respond to
external cues
55
Synthetic biology projects
Nicholas J Baltes et al 2015
56
Refactoring the N fixation gene cluster from Klebsiella
Karsten Temme et al 2012
57
Continuehellip
Karsten Temme et al 2012
58
Genome editing
Sequence specific nucleases
1 Meganucleases
2 ZFMs ndashZink finger motifs
3 TALENs- Transcription activator like effector nuclease
4 CRISPR CAS-9 ndash Clustered interspaced short palindromic
repeats
qmp4
59Khaoula Belhaj et al 2015
60
Genome modifications achieved in plants using sequence specific nucleases
Type of DNA
modification
Nuclease Delivery
methods
Plants Targets
Trait stacking Megnucleases Bombardment Cotton Intergenic sequence
ZFN Bombardment Maize Transgene
Rewriting host
DNA
Megnucleases Stable integration Maize Intergenic sequence
ZFN Stable integration Soybean Transgene
TALEN Stable integration Barley PAPhy_a
TALEN Agrobacterium
T-DNA (transient)
Oryza sativa SWEET14
TALEN No Protoplasts Arabidopsis
Tobacco
AtTT4 AtADH NbSurB
TALEN Bombardment Wheat MLO
TALEN Protoplasts
Stable integration
Maize PDS IPK1A IPK MRP4
CRISPRCas Protoplasts
Agrobacterium
T-DNA (transient)
Tobacco
Arabidopsis
Sorghum Oryza
OsSWEET14 transgene
61
Type of DNA
modification
Nuclease Delivery
methods
Plants Targets
Rewriting host
DNA large
deletion
Zinc-finger
nuclease
Stable
integration
Tobacco Transgene
CRISPRCas Protoplasts
Stable
integration
Rice Labdane-related
diterpenoid gene
clusters
on Chr 2 4 and 6
Zinc-finger
nuclease
Agrobacterium
T-DNA
(transient)
Tobacco CHN50 transgene
Zinc-finger
nuclease
Whiskers Maize IPK1
CRISPRCas Protoplasts Rice PDS
Controlling gene
expression
TALE repressor
(SRDX)
Stable
integration
Arabidopsis RD29A transgene
Zinc-finger
activator (VP16)
Stable
integration
Brassica
napus
KasII
Continue
62
Application of genome editing1 Introduction of precise and predictable modifications directly in an elite
background
2 multiple traits can be modified simultaneously
3 NHEJ enables gene knockout and targeted modifications
4 Introduction of transgenes at defined loci that promote high-level
transcription and do not interfere with the activity of endogenous genes
5 Site-specific nucleases also allow targeted molecular trait stacking -low
risk of segregation
6 CRISPR Cas is a transgene free approach ndash No regulatory burdens
7 frequency of off-target mutations is well below that caused by chemical
and physical mutagenesis techniques
8 In future can be utilized for metabolic engineering and molecular farming
63
Six-way Venn diagram showing the distribution of shared genefamilies (sequence clusters) among M acuminata P dactyliferaArabidopsis thaliana Oryza sativa Sorghum bicolor and Brachypodiumdistachyon genomes
A DrsquoHont et al Nature 2012 doi101038nature11241
64
Achieving super-DomesticationINTROGRESS-RECREATE-CREATE-DOMESTICATEREDOMESTICATE
bull Mobilizing left out genetic
variation still available in
land races and wild and
weedy species
bull Replaying the evolutionary
tape( resynthesis in
polyploids)
bull Domesticateredomesticate
65
We can admire and emulate how indigenous people still domesticateplants create biodiversity and manage it to sustain their future Thereis no equivalently dynamic or flexible crop breeding in modernagriculture to promote biodiversity we still have much to learn from
traditional knowledge Jan Salick 2012
48
Five high throuput genotyping methods
Xuehui Huang and Bin Han 2014
49
Role of NGS in domestication Capture of novel genes from wild species will be made easier by
understanding the molecular events associated with crop domestication
Re-sequencing of domesticated species can identify low diversity regions
resulting from selection during domestication
To identify gene-specific sequences to aid the cloning of homologues of key
domestication genes from wild relatives
Candidate genes from wild and domesticated plant populations can define
diversity of target genes in wild populations and lead to the discovery of key
genes for important traits by association analysis
NGS of amplicons of large numbers of candidate genes from wild and
domesticated plant populations can define diversity of target genes in
wild populations and lead to the discovery of key genes for important
traits by association analysis
50
NGS
supports the rapid domestication of new plant species and the efficient
identification and capture of novel genetic variation from related species
Allows whole-genome analysis to determine the genetic basis of
phenotypic differences
NGS allows rapid expansion of genomic analysis to investigation of non-
model species
Made rice study easy by related grass
cost-effective method for plant identification
useful strategy to analysis the chloroplast genome sequence from whole-
genome shot-gun sequencing
facilitates managing this diversity and any changes in crop performance
over time due to genetic drift
Patterns of gene expression have been evaluated in hybrids using NGS
51
Two Approaches to Finding Adaptive Genes
Ross-Ibarra et al2007
52
Techniques to re-wild the plants (transgene free)
1 Introgression
breeding
2 Specific
insertion of lost genes
3 Precision
mutagenesis
Palmgren et al2014
53
Process of rewilding
Palmgren et al2014
54
Synthetic biology projects(i) Modifying cereals including wheat to fix atmospheric nitrogen
(ii) Redesigning metabolic pathways to increase the yield of
secondary metabolites or to generate compounds with enhanced
properties
(iii) Transferring the C4 photosynthesis pathway to rice
(iv) Modifying the glycosylation pathway in plants to accommodate
production of therapeutic proteins
(v) Introducing synthetic signal transduction systems that respond to
external cues
55
Synthetic biology projects
Nicholas J Baltes et al 2015
56
Refactoring the N fixation gene cluster from Klebsiella
Karsten Temme et al 2012
57
Continuehellip
Karsten Temme et al 2012
58
Genome editing
Sequence specific nucleases
1 Meganucleases
2 ZFMs ndashZink finger motifs
3 TALENs- Transcription activator like effector nuclease
4 CRISPR CAS-9 ndash Clustered interspaced short palindromic
repeats
qmp4
59Khaoula Belhaj et al 2015
60
Genome modifications achieved in plants using sequence specific nucleases
Type of DNA
modification
Nuclease Delivery
methods
Plants Targets
Trait stacking Megnucleases Bombardment Cotton Intergenic sequence
ZFN Bombardment Maize Transgene
Rewriting host
DNA
Megnucleases Stable integration Maize Intergenic sequence
ZFN Stable integration Soybean Transgene
TALEN Stable integration Barley PAPhy_a
TALEN Agrobacterium
T-DNA (transient)
Oryza sativa SWEET14
TALEN No Protoplasts Arabidopsis
Tobacco
AtTT4 AtADH NbSurB
TALEN Bombardment Wheat MLO
TALEN Protoplasts
Stable integration
Maize PDS IPK1A IPK MRP4
CRISPRCas Protoplasts
Agrobacterium
T-DNA (transient)
Tobacco
Arabidopsis
Sorghum Oryza
OsSWEET14 transgene
61
Type of DNA
modification
Nuclease Delivery
methods
Plants Targets
Rewriting host
DNA large
deletion
Zinc-finger
nuclease
Stable
integration
Tobacco Transgene
CRISPRCas Protoplasts
Stable
integration
Rice Labdane-related
diterpenoid gene
clusters
on Chr 2 4 and 6
Zinc-finger
nuclease
Agrobacterium
T-DNA
(transient)
Tobacco CHN50 transgene
Zinc-finger
nuclease
Whiskers Maize IPK1
CRISPRCas Protoplasts Rice PDS
Controlling gene
expression
TALE repressor
(SRDX)
Stable
integration
Arabidopsis RD29A transgene
Zinc-finger
activator (VP16)
Stable
integration
Brassica
napus
KasII
Continue
62
Application of genome editing1 Introduction of precise and predictable modifications directly in an elite
background
2 multiple traits can be modified simultaneously
3 NHEJ enables gene knockout and targeted modifications
4 Introduction of transgenes at defined loci that promote high-level
transcription and do not interfere with the activity of endogenous genes
5 Site-specific nucleases also allow targeted molecular trait stacking -low
risk of segregation
6 CRISPR Cas is a transgene free approach ndash No regulatory burdens
7 frequency of off-target mutations is well below that caused by chemical
and physical mutagenesis techniques
8 In future can be utilized for metabolic engineering and molecular farming
63
Six-way Venn diagram showing the distribution of shared genefamilies (sequence clusters) among M acuminata P dactyliferaArabidopsis thaliana Oryza sativa Sorghum bicolor and Brachypodiumdistachyon genomes
A DrsquoHont et al Nature 2012 doi101038nature11241
64
Achieving super-DomesticationINTROGRESS-RECREATE-CREATE-DOMESTICATEREDOMESTICATE
bull Mobilizing left out genetic
variation still available in
land races and wild and
weedy species
bull Replaying the evolutionary
tape( resynthesis in
polyploids)
bull Domesticateredomesticate
65
We can admire and emulate how indigenous people still domesticateplants create biodiversity and manage it to sustain their future Thereis no equivalently dynamic or flexible crop breeding in modernagriculture to promote biodiversity we still have much to learn from
traditional knowledge Jan Salick 2012
49
Role of NGS in domestication Capture of novel genes from wild species will be made easier by
understanding the molecular events associated with crop domestication
Re-sequencing of domesticated species can identify low diversity regions
resulting from selection during domestication
To identify gene-specific sequences to aid the cloning of homologues of key
domestication genes from wild relatives
Candidate genes from wild and domesticated plant populations can define
diversity of target genes in wild populations and lead to the discovery of key
genes for important traits by association analysis
NGS of amplicons of large numbers of candidate genes from wild and
domesticated plant populations can define diversity of target genes in
wild populations and lead to the discovery of key genes for important
traits by association analysis
50
NGS
supports the rapid domestication of new plant species and the efficient
identification and capture of novel genetic variation from related species
Allows whole-genome analysis to determine the genetic basis of
phenotypic differences
NGS allows rapid expansion of genomic analysis to investigation of non-
model species
Made rice study easy by related grass
cost-effective method for plant identification
useful strategy to analysis the chloroplast genome sequence from whole-
genome shot-gun sequencing
facilitates managing this diversity and any changes in crop performance
over time due to genetic drift
Patterns of gene expression have been evaluated in hybrids using NGS
51
Two Approaches to Finding Adaptive Genes
Ross-Ibarra et al2007
52
Techniques to re-wild the plants (transgene free)
1 Introgression
breeding
2 Specific
insertion of lost genes
3 Precision
mutagenesis
Palmgren et al2014
53
Process of rewilding
Palmgren et al2014
54
Synthetic biology projects(i) Modifying cereals including wheat to fix atmospheric nitrogen
(ii) Redesigning metabolic pathways to increase the yield of
secondary metabolites or to generate compounds with enhanced
properties
(iii) Transferring the C4 photosynthesis pathway to rice
(iv) Modifying the glycosylation pathway in plants to accommodate
production of therapeutic proteins
(v) Introducing synthetic signal transduction systems that respond to
external cues
55
Synthetic biology projects
Nicholas J Baltes et al 2015
56
Refactoring the N fixation gene cluster from Klebsiella
Karsten Temme et al 2012
57
Continuehellip
Karsten Temme et al 2012
58
Genome editing
Sequence specific nucleases
1 Meganucleases
2 ZFMs ndashZink finger motifs
3 TALENs- Transcription activator like effector nuclease
4 CRISPR CAS-9 ndash Clustered interspaced short palindromic
repeats
qmp4
59Khaoula Belhaj et al 2015
60
Genome modifications achieved in plants using sequence specific nucleases
Type of DNA
modification
Nuclease Delivery
methods
Plants Targets
Trait stacking Megnucleases Bombardment Cotton Intergenic sequence
ZFN Bombardment Maize Transgene
Rewriting host
DNA
Megnucleases Stable integration Maize Intergenic sequence
ZFN Stable integration Soybean Transgene
TALEN Stable integration Barley PAPhy_a
TALEN Agrobacterium
T-DNA (transient)
Oryza sativa SWEET14
TALEN No Protoplasts Arabidopsis
Tobacco
AtTT4 AtADH NbSurB
TALEN Bombardment Wheat MLO
TALEN Protoplasts
Stable integration
Maize PDS IPK1A IPK MRP4
CRISPRCas Protoplasts
Agrobacterium
T-DNA (transient)
Tobacco
Arabidopsis
Sorghum Oryza
OsSWEET14 transgene
61
Type of DNA
modification
Nuclease Delivery
methods
Plants Targets
Rewriting host
DNA large
deletion
Zinc-finger
nuclease
Stable
integration
Tobacco Transgene
CRISPRCas Protoplasts
Stable
integration
Rice Labdane-related
diterpenoid gene
clusters
on Chr 2 4 and 6
Zinc-finger
nuclease
Agrobacterium
T-DNA
(transient)
Tobacco CHN50 transgene
Zinc-finger
nuclease
Whiskers Maize IPK1
CRISPRCas Protoplasts Rice PDS
Controlling gene
expression
TALE repressor
(SRDX)
Stable
integration
Arabidopsis RD29A transgene
Zinc-finger
activator (VP16)
Stable
integration
Brassica
napus
KasII
Continue
62
Application of genome editing1 Introduction of precise and predictable modifications directly in an elite
background
2 multiple traits can be modified simultaneously
3 NHEJ enables gene knockout and targeted modifications
4 Introduction of transgenes at defined loci that promote high-level
transcription and do not interfere with the activity of endogenous genes
5 Site-specific nucleases also allow targeted molecular trait stacking -low
risk of segregation
6 CRISPR Cas is a transgene free approach ndash No regulatory burdens
7 frequency of off-target mutations is well below that caused by chemical
and physical mutagenesis techniques
8 In future can be utilized for metabolic engineering and molecular farming
63
Six-way Venn diagram showing the distribution of shared genefamilies (sequence clusters) among M acuminata P dactyliferaArabidopsis thaliana Oryza sativa Sorghum bicolor and Brachypodiumdistachyon genomes
A DrsquoHont et al Nature 2012 doi101038nature11241
64
Achieving super-DomesticationINTROGRESS-RECREATE-CREATE-DOMESTICATEREDOMESTICATE
bull Mobilizing left out genetic
variation still available in
land races and wild and
weedy species
bull Replaying the evolutionary
tape( resynthesis in
polyploids)
bull Domesticateredomesticate
65
We can admire and emulate how indigenous people still domesticateplants create biodiversity and manage it to sustain their future Thereis no equivalently dynamic or flexible crop breeding in modernagriculture to promote biodiversity we still have much to learn from
traditional knowledge Jan Salick 2012
50
NGS
supports the rapid domestication of new plant species and the efficient
identification and capture of novel genetic variation from related species
Allows whole-genome analysis to determine the genetic basis of
phenotypic differences
NGS allows rapid expansion of genomic analysis to investigation of non-
model species
Made rice study easy by related grass
cost-effective method for plant identification
useful strategy to analysis the chloroplast genome sequence from whole-
genome shot-gun sequencing
facilitates managing this diversity and any changes in crop performance
over time due to genetic drift
Patterns of gene expression have been evaluated in hybrids using NGS
51
Two Approaches to Finding Adaptive Genes
Ross-Ibarra et al2007
52
Techniques to re-wild the plants (transgene free)
1 Introgression
breeding
2 Specific
insertion of lost genes
3 Precision
mutagenesis
Palmgren et al2014
53
Process of rewilding
Palmgren et al2014
54
Synthetic biology projects(i) Modifying cereals including wheat to fix atmospheric nitrogen
(ii) Redesigning metabolic pathways to increase the yield of
secondary metabolites or to generate compounds with enhanced
properties
(iii) Transferring the C4 photosynthesis pathway to rice
(iv) Modifying the glycosylation pathway in plants to accommodate
production of therapeutic proteins
(v) Introducing synthetic signal transduction systems that respond to
external cues
55
Synthetic biology projects
Nicholas J Baltes et al 2015
56
Refactoring the N fixation gene cluster from Klebsiella
Karsten Temme et al 2012
57
Continuehellip
Karsten Temme et al 2012
58
Genome editing
Sequence specific nucleases
1 Meganucleases
2 ZFMs ndashZink finger motifs
3 TALENs- Transcription activator like effector nuclease
4 CRISPR CAS-9 ndash Clustered interspaced short palindromic
repeats
qmp4
59Khaoula Belhaj et al 2015
60
Genome modifications achieved in plants using sequence specific nucleases
Type of DNA
modification
Nuclease Delivery
methods
Plants Targets
Trait stacking Megnucleases Bombardment Cotton Intergenic sequence
ZFN Bombardment Maize Transgene
Rewriting host
DNA
Megnucleases Stable integration Maize Intergenic sequence
ZFN Stable integration Soybean Transgene
TALEN Stable integration Barley PAPhy_a
TALEN Agrobacterium
T-DNA (transient)
Oryza sativa SWEET14
TALEN No Protoplasts Arabidopsis
Tobacco
AtTT4 AtADH NbSurB
TALEN Bombardment Wheat MLO
TALEN Protoplasts
Stable integration
Maize PDS IPK1A IPK MRP4
CRISPRCas Protoplasts
Agrobacterium
T-DNA (transient)
Tobacco
Arabidopsis
Sorghum Oryza
OsSWEET14 transgene
61
Type of DNA
modification
Nuclease Delivery
methods
Plants Targets
Rewriting host
DNA large
deletion
Zinc-finger
nuclease
Stable
integration
Tobacco Transgene
CRISPRCas Protoplasts
Stable
integration
Rice Labdane-related
diterpenoid gene
clusters
on Chr 2 4 and 6
Zinc-finger
nuclease
Agrobacterium
T-DNA
(transient)
Tobacco CHN50 transgene
Zinc-finger
nuclease
Whiskers Maize IPK1
CRISPRCas Protoplasts Rice PDS
Controlling gene
expression
TALE repressor
(SRDX)
Stable
integration
Arabidopsis RD29A transgene
Zinc-finger
activator (VP16)
Stable
integration
Brassica
napus
KasII
Continue
62
Application of genome editing1 Introduction of precise and predictable modifications directly in an elite
background
2 multiple traits can be modified simultaneously
3 NHEJ enables gene knockout and targeted modifications
4 Introduction of transgenes at defined loci that promote high-level
transcription and do not interfere with the activity of endogenous genes
5 Site-specific nucleases also allow targeted molecular trait stacking -low
risk of segregation
6 CRISPR Cas is a transgene free approach ndash No regulatory burdens
7 frequency of off-target mutations is well below that caused by chemical
and physical mutagenesis techniques
8 In future can be utilized for metabolic engineering and molecular farming
63
Six-way Venn diagram showing the distribution of shared genefamilies (sequence clusters) among M acuminata P dactyliferaArabidopsis thaliana Oryza sativa Sorghum bicolor and Brachypodiumdistachyon genomes
A DrsquoHont et al Nature 2012 doi101038nature11241
64
Achieving super-DomesticationINTROGRESS-RECREATE-CREATE-DOMESTICATEREDOMESTICATE
bull Mobilizing left out genetic
variation still available in
land races and wild and
weedy species
bull Replaying the evolutionary
tape( resynthesis in
polyploids)
bull Domesticateredomesticate
65
We can admire and emulate how indigenous people still domesticateplants create biodiversity and manage it to sustain their future Thereis no equivalently dynamic or flexible crop breeding in modernagriculture to promote biodiversity we still have much to learn from
traditional knowledge Jan Salick 2012
51
Two Approaches to Finding Adaptive Genes
Ross-Ibarra et al2007
52
Techniques to re-wild the plants (transgene free)
1 Introgression
breeding
2 Specific
insertion of lost genes
3 Precision
mutagenesis
Palmgren et al2014
53
Process of rewilding
Palmgren et al2014
54
Synthetic biology projects(i) Modifying cereals including wheat to fix atmospheric nitrogen
(ii) Redesigning metabolic pathways to increase the yield of
secondary metabolites or to generate compounds with enhanced
properties
(iii) Transferring the C4 photosynthesis pathway to rice
(iv) Modifying the glycosylation pathway in plants to accommodate
production of therapeutic proteins
(v) Introducing synthetic signal transduction systems that respond to
external cues
55
Synthetic biology projects
Nicholas J Baltes et al 2015
56
Refactoring the N fixation gene cluster from Klebsiella
Karsten Temme et al 2012
57
Continuehellip
Karsten Temme et al 2012
58
Genome editing
Sequence specific nucleases
1 Meganucleases
2 ZFMs ndashZink finger motifs
3 TALENs- Transcription activator like effector nuclease
4 CRISPR CAS-9 ndash Clustered interspaced short palindromic
repeats
qmp4
59Khaoula Belhaj et al 2015
60
Genome modifications achieved in plants using sequence specific nucleases
Type of DNA
modification
Nuclease Delivery
methods
Plants Targets
Trait stacking Megnucleases Bombardment Cotton Intergenic sequence
ZFN Bombardment Maize Transgene
Rewriting host
DNA
Megnucleases Stable integration Maize Intergenic sequence
ZFN Stable integration Soybean Transgene
TALEN Stable integration Barley PAPhy_a
TALEN Agrobacterium
T-DNA (transient)
Oryza sativa SWEET14
TALEN No Protoplasts Arabidopsis
Tobacco
AtTT4 AtADH NbSurB
TALEN Bombardment Wheat MLO
TALEN Protoplasts
Stable integration
Maize PDS IPK1A IPK MRP4
CRISPRCas Protoplasts
Agrobacterium
T-DNA (transient)
Tobacco
Arabidopsis
Sorghum Oryza
OsSWEET14 transgene
61
Type of DNA
modification
Nuclease Delivery
methods
Plants Targets
Rewriting host
DNA large
deletion
Zinc-finger
nuclease
Stable
integration
Tobacco Transgene
CRISPRCas Protoplasts
Stable
integration
Rice Labdane-related
diterpenoid gene
clusters
on Chr 2 4 and 6
Zinc-finger
nuclease
Agrobacterium
T-DNA
(transient)
Tobacco CHN50 transgene
Zinc-finger
nuclease
Whiskers Maize IPK1
CRISPRCas Protoplasts Rice PDS
Controlling gene
expression
TALE repressor
(SRDX)
Stable
integration
Arabidopsis RD29A transgene
Zinc-finger
activator (VP16)
Stable
integration
Brassica
napus
KasII
Continue
62
Application of genome editing1 Introduction of precise and predictable modifications directly in an elite
background
2 multiple traits can be modified simultaneously
3 NHEJ enables gene knockout and targeted modifications
4 Introduction of transgenes at defined loci that promote high-level
transcription and do not interfere with the activity of endogenous genes
5 Site-specific nucleases also allow targeted molecular trait stacking -low
risk of segregation
6 CRISPR Cas is a transgene free approach ndash No regulatory burdens
7 frequency of off-target mutations is well below that caused by chemical
and physical mutagenesis techniques
8 In future can be utilized for metabolic engineering and molecular farming
63
Six-way Venn diagram showing the distribution of shared genefamilies (sequence clusters) among M acuminata P dactyliferaArabidopsis thaliana Oryza sativa Sorghum bicolor and Brachypodiumdistachyon genomes
A DrsquoHont et al Nature 2012 doi101038nature11241
64
Achieving super-DomesticationINTROGRESS-RECREATE-CREATE-DOMESTICATEREDOMESTICATE
bull Mobilizing left out genetic
variation still available in
land races and wild and
weedy species
bull Replaying the evolutionary
tape( resynthesis in
polyploids)
bull Domesticateredomesticate
65
We can admire and emulate how indigenous people still domesticateplants create biodiversity and manage it to sustain their future Thereis no equivalently dynamic or flexible crop breeding in modernagriculture to promote biodiversity we still have much to learn from
traditional knowledge Jan Salick 2012
52
Techniques to re-wild the plants (transgene free)
1 Introgression
breeding
2 Specific
insertion of lost genes
3 Precision
mutagenesis
Palmgren et al2014
53
Process of rewilding
Palmgren et al2014
54
Synthetic biology projects(i) Modifying cereals including wheat to fix atmospheric nitrogen
(ii) Redesigning metabolic pathways to increase the yield of
secondary metabolites or to generate compounds with enhanced
properties
(iii) Transferring the C4 photosynthesis pathway to rice
(iv) Modifying the glycosylation pathway in plants to accommodate
production of therapeutic proteins
(v) Introducing synthetic signal transduction systems that respond to
external cues
55
Synthetic biology projects
Nicholas J Baltes et al 2015
56
Refactoring the N fixation gene cluster from Klebsiella
Karsten Temme et al 2012
57
Continuehellip
Karsten Temme et al 2012
58
Genome editing
Sequence specific nucleases
1 Meganucleases
2 ZFMs ndashZink finger motifs
3 TALENs- Transcription activator like effector nuclease
4 CRISPR CAS-9 ndash Clustered interspaced short palindromic
repeats
qmp4
59Khaoula Belhaj et al 2015
60
Genome modifications achieved in plants using sequence specific nucleases
Type of DNA
modification
Nuclease Delivery
methods
Plants Targets
Trait stacking Megnucleases Bombardment Cotton Intergenic sequence
ZFN Bombardment Maize Transgene
Rewriting host
DNA
Megnucleases Stable integration Maize Intergenic sequence
ZFN Stable integration Soybean Transgene
TALEN Stable integration Barley PAPhy_a
TALEN Agrobacterium
T-DNA (transient)
Oryza sativa SWEET14
TALEN No Protoplasts Arabidopsis
Tobacco
AtTT4 AtADH NbSurB
TALEN Bombardment Wheat MLO
TALEN Protoplasts
Stable integration
Maize PDS IPK1A IPK MRP4
CRISPRCas Protoplasts
Agrobacterium
T-DNA (transient)
Tobacco
Arabidopsis
Sorghum Oryza
OsSWEET14 transgene
61
Type of DNA
modification
Nuclease Delivery
methods
Plants Targets
Rewriting host
DNA large
deletion
Zinc-finger
nuclease
Stable
integration
Tobacco Transgene
CRISPRCas Protoplasts
Stable
integration
Rice Labdane-related
diterpenoid gene
clusters
on Chr 2 4 and 6
Zinc-finger
nuclease
Agrobacterium
T-DNA
(transient)
Tobacco CHN50 transgene
Zinc-finger
nuclease
Whiskers Maize IPK1
CRISPRCas Protoplasts Rice PDS
Controlling gene
expression
TALE repressor
(SRDX)
Stable
integration
Arabidopsis RD29A transgene
Zinc-finger
activator (VP16)
Stable
integration
Brassica
napus
KasII
Continue
62
Application of genome editing1 Introduction of precise and predictable modifications directly in an elite
background
2 multiple traits can be modified simultaneously
3 NHEJ enables gene knockout and targeted modifications
4 Introduction of transgenes at defined loci that promote high-level
transcription and do not interfere with the activity of endogenous genes
5 Site-specific nucleases also allow targeted molecular trait stacking -low
risk of segregation
6 CRISPR Cas is a transgene free approach ndash No regulatory burdens
7 frequency of off-target mutations is well below that caused by chemical
and physical mutagenesis techniques
8 In future can be utilized for metabolic engineering and molecular farming
63
Six-way Venn diagram showing the distribution of shared genefamilies (sequence clusters) among M acuminata P dactyliferaArabidopsis thaliana Oryza sativa Sorghum bicolor and Brachypodiumdistachyon genomes
A DrsquoHont et al Nature 2012 doi101038nature11241
64
Achieving super-DomesticationINTROGRESS-RECREATE-CREATE-DOMESTICATEREDOMESTICATE
bull Mobilizing left out genetic
variation still available in
land races and wild and
weedy species
bull Replaying the evolutionary
tape( resynthesis in
polyploids)
bull Domesticateredomesticate
65
We can admire and emulate how indigenous people still domesticateplants create biodiversity and manage it to sustain their future Thereis no equivalently dynamic or flexible crop breeding in modernagriculture to promote biodiversity we still have much to learn from
traditional knowledge Jan Salick 2012
53
Process of rewilding
Palmgren et al2014
54
Synthetic biology projects(i) Modifying cereals including wheat to fix atmospheric nitrogen
(ii) Redesigning metabolic pathways to increase the yield of
secondary metabolites or to generate compounds with enhanced
properties
(iii) Transferring the C4 photosynthesis pathway to rice
(iv) Modifying the glycosylation pathway in plants to accommodate
production of therapeutic proteins
(v) Introducing synthetic signal transduction systems that respond to
external cues
55
Synthetic biology projects
Nicholas J Baltes et al 2015
56
Refactoring the N fixation gene cluster from Klebsiella
Karsten Temme et al 2012
57
Continuehellip
Karsten Temme et al 2012
58
Genome editing
Sequence specific nucleases
1 Meganucleases
2 ZFMs ndashZink finger motifs
3 TALENs- Transcription activator like effector nuclease
4 CRISPR CAS-9 ndash Clustered interspaced short palindromic
repeats
qmp4
59Khaoula Belhaj et al 2015
60
Genome modifications achieved in plants using sequence specific nucleases
Type of DNA
modification
Nuclease Delivery
methods
Plants Targets
Trait stacking Megnucleases Bombardment Cotton Intergenic sequence
ZFN Bombardment Maize Transgene
Rewriting host
DNA
Megnucleases Stable integration Maize Intergenic sequence
ZFN Stable integration Soybean Transgene
TALEN Stable integration Barley PAPhy_a
TALEN Agrobacterium
T-DNA (transient)
Oryza sativa SWEET14
TALEN No Protoplasts Arabidopsis
Tobacco
AtTT4 AtADH NbSurB
TALEN Bombardment Wheat MLO
TALEN Protoplasts
Stable integration
Maize PDS IPK1A IPK MRP4
CRISPRCas Protoplasts
Agrobacterium
T-DNA (transient)
Tobacco
Arabidopsis
Sorghum Oryza
OsSWEET14 transgene
61
Type of DNA
modification
Nuclease Delivery
methods
Plants Targets
Rewriting host
DNA large
deletion
Zinc-finger
nuclease
Stable
integration
Tobacco Transgene
CRISPRCas Protoplasts
Stable
integration
Rice Labdane-related
diterpenoid gene
clusters
on Chr 2 4 and 6
Zinc-finger
nuclease
Agrobacterium
T-DNA
(transient)
Tobacco CHN50 transgene
Zinc-finger
nuclease
Whiskers Maize IPK1
CRISPRCas Protoplasts Rice PDS
Controlling gene
expression
TALE repressor
(SRDX)
Stable
integration
Arabidopsis RD29A transgene
Zinc-finger
activator (VP16)
Stable
integration
Brassica
napus
KasII
Continue
62
Application of genome editing1 Introduction of precise and predictable modifications directly in an elite
background
2 multiple traits can be modified simultaneously
3 NHEJ enables gene knockout and targeted modifications
4 Introduction of transgenes at defined loci that promote high-level
transcription and do not interfere with the activity of endogenous genes
5 Site-specific nucleases also allow targeted molecular trait stacking -low
risk of segregation
6 CRISPR Cas is a transgene free approach ndash No regulatory burdens
7 frequency of off-target mutations is well below that caused by chemical
and physical mutagenesis techniques
8 In future can be utilized for metabolic engineering and molecular farming
63
Six-way Venn diagram showing the distribution of shared genefamilies (sequence clusters) among M acuminata P dactyliferaArabidopsis thaliana Oryza sativa Sorghum bicolor and Brachypodiumdistachyon genomes
A DrsquoHont et al Nature 2012 doi101038nature11241
64
Achieving super-DomesticationINTROGRESS-RECREATE-CREATE-DOMESTICATEREDOMESTICATE
bull Mobilizing left out genetic
variation still available in
land races and wild and
weedy species
bull Replaying the evolutionary
tape( resynthesis in
polyploids)
bull Domesticateredomesticate
65
We can admire and emulate how indigenous people still domesticateplants create biodiversity and manage it to sustain their future Thereis no equivalently dynamic or flexible crop breeding in modernagriculture to promote biodiversity we still have much to learn from
traditional knowledge Jan Salick 2012
54
Synthetic biology projects(i) Modifying cereals including wheat to fix atmospheric nitrogen
(ii) Redesigning metabolic pathways to increase the yield of
secondary metabolites or to generate compounds with enhanced
properties
(iii) Transferring the C4 photosynthesis pathway to rice
(iv) Modifying the glycosylation pathway in plants to accommodate
production of therapeutic proteins
(v) Introducing synthetic signal transduction systems that respond to
external cues
55
Synthetic biology projects
Nicholas J Baltes et al 2015
56
Refactoring the N fixation gene cluster from Klebsiella
Karsten Temme et al 2012
57
Continuehellip
Karsten Temme et al 2012
58
Genome editing
Sequence specific nucleases
1 Meganucleases
2 ZFMs ndashZink finger motifs
3 TALENs- Transcription activator like effector nuclease
4 CRISPR CAS-9 ndash Clustered interspaced short palindromic
repeats
qmp4
59Khaoula Belhaj et al 2015
60
Genome modifications achieved in plants using sequence specific nucleases
Type of DNA
modification
Nuclease Delivery
methods
Plants Targets
Trait stacking Megnucleases Bombardment Cotton Intergenic sequence
ZFN Bombardment Maize Transgene
Rewriting host
DNA
Megnucleases Stable integration Maize Intergenic sequence
ZFN Stable integration Soybean Transgene
TALEN Stable integration Barley PAPhy_a
TALEN Agrobacterium
T-DNA (transient)
Oryza sativa SWEET14
TALEN No Protoplasts Arabidopsis
Tobacco
AtTT4 AtADH NbSurB
TALEN Bombardment Wheat MLO
TALEN Protoplasts
Stable integration
Maize PDS IPK1A IPK MRP4
CRISPRCas Protoplasts
Agrobacterium
T-DNA (transient)
Tobacco
Arabidopsis
Sorghum Oryza
OsSWEET14 transgene
61
Type of DNA
modification
Nuclease Delivery
methods
Plants Targets
Rewriting host
DNA large
deletion
Zinc-finger
nuclease
Stable
integration
Tobacco Transgene
CRISPRCas Protoplasts
Stable
integration
Rice Labdane-related
diterpenoid gene
clusters
on Chr 2 4 and 6
Zinc-finger
nuclease
Agrobacterium
T-DNA
(transient)
Tobacco CHN50 transgene
Zinc-finger
nuclease
Whiskers Maize IPK1
CRISPRCas Protoplasts Rice PDS
Controlling gene
expression
TALE repressor
(SRDX)
Stable
integration
Arabidopsis RD29A transgene
Zinc-finger
activator (VP16)
Stable
integration
Brassica
napus
KasII
Continue
62
Application of genome editing1 Introduction of precise and predictable modifications directly in an elite
background
2 multiple traits can be modified simultaneously
3 NHEJ enables gene knockout and targeted modifications
4 Introduction of transgenes at defined loci that promote high-level
transcription and do not interfere with the activity of endogenous genes
5 Site-specific nucleases also allow targeted molecular trait stacking -low
risk of segregation
6 CRISPR Cas is a transgene free approach ndash No regulatory burdens
7 frequency of off-target mutations is well below that caused by chemical
and physical mutagenesis techniques
8 In future can be utilized for metabolic engineering and molecular farming
63
Six-way Venn diagram showing the distribution of shared genefamilies (sequence clusters) among M acuminata P dactyliferaArabidopsis thaliana Oryza sativa Sorghum bicolor and Brachypodiumdistachyon genomes
A DrsquoHont et al Nature 2012 doi101038nature11241
64
Achieving super-DomesticationINTROGRESS-RECREATE-CREATE-DOMESTICATEREDOMESTICATE
bull Mobilizing left out genetic
variation still available in
land races and wild and
weedy species
bull Replaying the evolutionary
tape( resynthesis in
polyploids)
bull Domesticateredomesticate
65
We can admire and emulate how indigenous people still domesticateplants create biodiversity and manage it to sustain their future Thereis no equivalently dynamic or flexible crop breeding in modernagriculture to promote biodiversity we still have much to learn from
traditional knowledge Jan Salick 2012
55
Synthetic biology projects
Nicholas J Baltes et al 2015
56
Refactoring the N fixation gene cluster from Klebsiella
Karsten Temme et al 2012
57
Continuehellip
Karsten Temme et al 2012
58
Genome editing
Sequence specific nucleases
1 Meganucleases
2 ZFMs ndashZink finger motifs
3 TALENs- Transcription activator like effector nuclease
4 CRISPR CAS-9 ndash Clustered interspaced short palindromic
repeats
qmp4
59Khaoula Belhaj et al 2015
60
Genome modifications achieved in plants using sequence specific nucleases
Type of DNA
modification
Nuclease Delivery
methods
Plants Targets
Trait stacking Megnucleases Bombardment Cotton Intergenic sequence
ZFN Bombardment Maize Transgene
Rewriting host
DNA
Megnucleases Stable integration Maize Intergenic sequence
ZFN Stable integration Soybean Transgene
TALEN Stable integration Barley PAPhy_a
TALEN Agrobacterium
T-DNA (transient)
Oryza sativa SWEET14
TALEN No Protoplasts Arabidopsis
Tobacco
AtTT4 AtADH NbSurB
TALEN Bombardment Wheat MLO
TALEN Protoplasts
Stable integration
Maize PDS IPK1A IPK MRP4
CRISPRCas Protoplasts
Agrobacterium
T-DNA (transient)
Tobacco
Arabidopsis
Sorghum Oryza
OsSWEET14 transgene
61
Type of DNA
modification
Nuclease Delivery
methods
Plants Targets
Rewriting host
DNA large
deletion
Zinc-finger
nuclease
Stable
integration
Tobacco Transgene
CRISPRCas Protoplasts
Stable
integration
Rice Labdane-related
diterpenoid gene
clusters
on Chr 2 4 and 6
Zinc-finger
nuclease
Agrobacterium
T-DNA
(transient)
Tobacco CHN50 transgene
Zinc-finger
nuclease
Whiskers Maize IPK1
CRISPRCas Protoplasts Rice PDS
Controlling gene
expression
TALE repressor
(SRDX)
Stable
integration
Arabidopsis RD29A transgene
Zinc-finger
activator (VP16)
Stable
integration
Brassica
napus
KasII
Continue
62
Application of genome editing1 Introduction of precise and predictable modifications directly in an elite
background
2 multiple traits can be modified simultaneously
3 NHEJ enables gene knockout and targeted modifications
4 Introduction of transgenes at defined loci that promote high-level
transcription and do not interfere with the activity of endogenous genes
5 Site-specific nucleases also allow targeted molecular trait stacking -low
risk of segregation
6 CRISPR Cas is a transgene free approach ndash No regulatory burdens
7 frequency of off-target mutations is well below that caused by chemical
and physical mutagenesis techniques
8 In future can be utilized for metabolic engineering and molecular farming
63
Six-way Venn diagram showing the distribution of shared genefamilies (sequence clusters) among M acuminata P dactyliferaArabidopsis thaliana Oryza sativa Sorghum bicolor and Brachypodiumdistachyon genomes
A DrsquoHont et al Nature 2012 doi101038nature11241
64
Achieving super-DomesticationINTROGRESS-RECREATE-CREATE-DOMESTICATEREDOMESTICATE
bull Mobilizing left out genetic
variation still available in
land races and wild and
weedy species
bull Replaying the evolutionary
tape( resynthesis in
polyploids)
bull Domesticateredomesticate
65
We can admire and emulate how indigenous people still domesticateplants create biodiversity and manage it to sustain their future Thereis no equivalently dynamic or flexible crop breeding in modernagriculture to promote biodiversity we still have much to learn from
traditional knowledge Jan Salick 2012
56
Refactoring the N fixation gene cluster from Klebsiella
Karsten Temme et al 2012
57
Continuehellip
Karsten Temme et al 2012
58
Genome editing
Sequence specific nucleases
1 Meganucleases
2 ZFMs ndashZink finger motifs
3 TALENs- Transcription activator like effector nuclease
4 CRISPR CAS-9 ndash Clustered interspaced short palindromic
repeats
qmp4
59Khaoula Belhaj et al 2015
60
Genome modifications achieved in plants using sequence specific nucleases
Type of DNA
modification
Nuclease Delivery
methods
Plants Targets
Trait stacking Megnucleases Bombardment Cotton Intergenic sequence
ZFN Bombardment Maize Transgene
Rewriting host
DNA
Megnucleases Stable integration Maize Intergenic sequence
ZFN Stable integration Soybean Transgene
TALEN Stable integration Barley PAPhy_a
TALEN Agrobacterium
T-DNA (transient)
Oryza sativa SWEET14
TALEN No Protoplasts Arabidopsis
Tobacco
AtTT4 AtADH NbSurB
TALEN Bombardment Wheat MLO
TALEN Protoplasts
Stable integration
Maize PDS IPK1A IPK MRP4
CRISPRCas Protoplasts
Agrobacterium
T-DNA (transient)
Tobacco
Arabidopsis
Sorghum Oryza
OsSWEET14 transgene
61
Type of DNA
modification
Nuclease Delivery
methods
Plants Targets
Rewriting host
DNA large
deletion
Zinc-finger
nuclease
Stable
integration
Tobacco Transgene
CRISPRCas Protoplasts
Stable
integration
Rice Labdane-related
diterpenoid gene
clusters
on Chr 2 4 and 6
Zinc-finger
nuclease
Agrobacterium
T-DNA
(transient)
Tobacco CHN50 transgene
Zinc-finger
nuclease
Whiskers Maize IPK1
CRISPRCas Protoplasts Rice PDS
Controlling gene
expression
TALE repressor
(SRDX)
Stable
integration
Arabidopsis RD29A transgene
Zinc-finger
activator (VP16)
Stable
integration
Brassica
napus
KasII
Continue
62
Application of genome editing1 Introduction of precise and predictable modifications directly in an elite
background
2 multiple traits can be modified simultaneously
3 NHEJ enables gene knockout and targeted modifications
4 Introduction of transgenes at defined loci that promote high-level
transcription and do not interfere with the activity of endogenous genes
5 Site-specific nucleases also allow targeted molecular trait stacking -low
risk of segregation
6 CRISPR Cas is a transgene free approach ndash No regulatory burdens
7 frequency of off-target mutations is well below that caused by chemical
and physical mutagenesis techniques
8 In future can be utilized for metabolic engineering and molecular farming
63
Six-way Venn diagram showing the distribution of shared genefamilies (sequence clusters) among M acuminata P dactyliferaArabidopsis thaliana Oryza sativa Sorghum bicolor and Brachypodiumdistachyon genomes
A DrsquoHont et al Nature 2012 doi101038nature11241
64
Achieving super-DomesticationINTROGRESS-RECREATE-CREATE-DOMESTICATEREDOMESTICATE
bull Mobilizing left out genetic
variation still available in
land races and wild and
weedy species
bull Replaying the evolutionary
tape( resynthesis in
polyploids)
bull Domesticateredomesticate
65
We can admire and emulate how indigenous people still domesticateplants create biodiversity and manage it to sustain their future Thereis no equivalently dynamic or flexible crop breeding in modernagriculture to promote biodiversity we still have much to learn from
traditional knowledge Jan Salick 2012
57
Continuehellip
Karsten Temme et al 2012
58
Genome editing
Sequence specific nucleases
1 Meganucleases
2 ZFMs ndashZink finger motifs
3 TALENs- Transcription activator like effector nuclease
4 CRISPR CAS-9 ndash Clustered interspaced short palindromic
repeats
qmp4
59Khaoula Belhaj et al 2015
60
Genome modifications achieved in plants using sequence specific nucleases
Type of DNA
modification
Nuclease Delivery
methods
Plants Targets
Trait stacking Megnucleases Bombardment Cotton Intergenic sequence
ZFN Bombardment Maize Transgene
Rewriting host
DNA
Megnucleases Stable integration Maize Intergenic sequence
ZFN Stable integration Soybean Transgene
TALEN Stable integration Barley PAPhy_a
TALEN Agrobacterium
T-DNA (transient)
Oryza sativa SWEET14
TALEN No Protoplasts Arabidopsis
Tobacco
AtTT4 AtADH NbSurB
TALEN Bombardment Wheat MLO
TALEN Protoplasts
Stable integration
Maize PDS IPK1A IPK MRP4
CRISPRCas Protoplasts
Agrobacterium
T-DNA (transient)
Tobacco
Arabidopsis
Sorghum Oryza
OsSWEET14 transgene
61
Type of DNA
modification
Nuclease Delivery
methods
Plants Targets
Rewriting host
DNA large
deletion
Zinc-finger
nuclease
Stable
integration
Tobacco Transgene
CRISPRCas Protoplasts
Stable
integration
Rice Labdane-related
diterpenoid gene
clusters
on Chr 2 4 and 6
Zinc-finger
nuclease
Agrobacterium
T-DNA
(transient)
Tobacco CHN50 transgene
Zinc-finger
nuclease
Whiskers Maize IPK1
CRISPRCas Protoplasts Rice PDS
Controlling gene
expression
TALE repressor
(SRDX)
Stable
integration
Arabidopsis RD29A transgene
Zinc-finger
activator (VP16)
Stable
integration
Brassica
napus
KasII
Continue
62
Application of genome editing1 Introduction of precise and predictable modifications directly in an elite
background
2 multiple traits can be modified simultaneously
3 NHEJ enables gene knockout and targeted modifications
4 Introduction of transgenes at defined loci that promote high-level
transcription and do not interfere with the activity of endogenous genes
5 Site-specific nucleases also allow targeted molecular trait stacking -low
risk of segregation
6 CRISPR Cas is a transgene free approach ndash No regulatory burdens
7 frequency of off-target mutations is well below that caused by chemical
and physical mutagenesis techniques
8 In future can be utilized for metabolic engineering and molecular farming
63
Six-way Venn diagram showing the distribution of shared genefamilies (sequence clusters) among M acuminata P dactyliferaArabidopsis thaliana Oryza sativa Sorghum bicolor and Brachypodiumdistachyon genomes
A DrsquoHont et al Nature 2012 doi101038nature11241
64
Achieving super-DomesticationINTROGRESS-RECREATE-CREATE-DOMESTICATEREDOMESTICATE
bull Mobilizing left out genetic
variation still available in
land races and wild and
weedy species
bull Replaying the evolutionary
tape( resynthesis in
polyploids)
bull Domesticateredomesticate
65
We can admire and emulate how indigenous people still domesticateplants create biodiversity and manage it to sustain their future Thereis no equivalently dynamic or flexible crop breeding in modernagriculture to promote biodiversity we still have much to learn from
traditional knowledge Jan Salick 2012
58
Genome editing
Sequence specific nucleases
1 Meganucleases
2 ZFMs ndashZink finger motifs
3 TALENs- Transcription activator like effector nuclease
4 CRISPR CAS-9 ndash Clustered interspaced short palindromic
repeats
qmp4
59Khaoula Belhaj et al 2015
60
Genome modifications achieved in plants using sequence specific nucleases
Type of DNA
modification
Nuclease Delivery
methods
Plants Targets
Trait stacking Megnucleases Bombardment Cotton Intergenic sequence
ZFN Bombardment Maize Transgene
Rewriting host
DNA
Megnucleases Stable integration Maize Intergenic sequence
ZFN Stable integration Soybean Transgene
TALEN Stable integration Barley PAPhy_a
TALEN Agrobacterium
T-DNA (transient)
Oryza sativa SWEET14
TALEN No Protoplasts Arabidopsis
Tobacco
AtTT4 AtADH NbSurB
TALEN Bombardment Wheat MLO
TALEN Protoplasts
Stable integration
Maize PDS IPK1A IPK MRP4
CRISPRCas Protoplasts
Agrobacterium
T-DNA (transient)
Tobacco
Arabidopsis
Sorghum Oryza
OsSWEET14 transgene
61
Type of DNA
modification
Nuclease Delivery
methods
Plants Targets
Rewriting host
DNA large
deletion
Zinc-finger
nuclease
Stable
integration
Tobacco Transgene
CRISPRCas Protoplasts
Stable
integration
Rice Labdane-related
diterpenoid gene
clusters
on Chr 2 4 and 6
Zinc-finger
nuclease
Agrobacterium
T-DNA
(transient)
Tobacco CHN50 transgene
Zinc-finger
nuclease
Whiskers Maize IPK1
CRISPRCas Protoplasts Rice PDS
Controlling gene
expression
TALE repressor
(SRDX)
Stable
integration
Arabidopsis RD29A transgene
Zinc-finger
activator (VP16)
Stable
integration
Brassica
napus
KasII
Continue
62
Application of genome editing1 Introduction of precise and predictable modifications directly in an elite
background
2 multiple traits can be modified simultaneously
3 NHEJ enables gene knockout and targeted modifications
4 Introduction of transgenes at defined loci that promote high-level
transcription and do not interfere with the activity of endogenous genes
5 Site-specific nucleases also allow targeted molecular trait stacking -low
risk of segregation
6 CRISPR Cas is a transgene free approach ndash No regulatory burdens
7 frequency of off-target mutations is well below that caused by chemical
and physical mutagenesis techniques
8 In future can be utilized for metabolic engineering and molecular farming
63
Six-way Venn diagram showing the distribution of shared genefamilies (sequence clusters) among M acuminata P dactyliferaArabidopsis thaliana Oryza sativa Sorghum bicolor and Brachypodiumdistachyon genomes
A DrsquoHont et al Nature 2012 doi101038nature11241
64
Achieving super-DomesticationINTROGRESS-RECREATE-CREATE-DOMESTICATEREDOMESTICATE
bull Mobilizing left out genetic
variation still available in
land races and wild and
weedy species
bull Replaying the evolutionary
tape( resynthesis in
polyploids)
bull Domesticateredomesticate
65
We can admire and emulate how indigenous people still domesticateplants create biodiversity and manage it to sustain their future Thereis no equivalently dynamic or flexible crop breeding in modernagriculture to promote biodiversity we still have much to learn from
traditional knowledge Jan Salick 2012
59Khaoula Belhaj et al 2015
60
Genome modifications achieved in plants using sequence specific nucleases
Type of DNA
modification
Nuclease Delivery
methods
Plants Targets
Trait stacking Megnucleases Bombardment Cotton Intergenic sequence
ZFN Bombardment Maize Transgene
Rewriting host
DNA
Megnucleases Stable integration Maize Intergenic sequence
ZFN Stable integration Soybean Transgene
TALEN Stable integration Barley PAPhy_a
TALEN Agrobacterium
T-DNA (transient)
Oryza sativa SWEET14
TALEN No Protoplasts Arabidopsis
Tobacco
AtTT4 AtADH NbSurB
TALEN Bombardment Wheat MLO
TALEN Protoplasts
Stable integration
Maize PDS IPK1A IPK MRP4
CRISPRCas Protoplasts
Agrobacterium
T-DNA (transient)
Tobacco
Arabidopsis
Sorghum Oryza
OsSWEET14 transgene
61
Type of DNA
modification
Nuclease Delivery
methods
Plants Targets
Rewriting host
DNA large
deletion
Zinc-finger
nuclease
Stable
integration
Tobacco Transgene
CRISPRCas Protoplasts
Stable
integration
Rice Labdane-related
diterpenoid gene
clusters
on Chr 2 4 and 6
Zinc-finger
nuclease
Agrobacterium
T-DNA
(transient)
Tobacco CHN50 transgene
Zinc-finger
nuclease
Whiskers Maize IPK1
CRISPRCas Protoplasts Rice PDS
Controlling gene
expression
TALE repressor
(SRDX)
Stable
integration
Arabidopsis RD29A transgene
Zinc-finger
activator (VP16)
Stable
integration
Brassica
napus
KasII
Continue
62
Application of genome editing1 Introduction of precise and predictable modifications directly in an elite
background
2 multiple traits can be modified simultaneously
3 NHEJ enables gene knockout and targeted modifications
4 Introduction of transgenes at defined loci that promote high-level
transcription and do not interfere with the activity of endogenous genes
5 Site-specific nucleases also allow targeted molecular trait stacking -low
risk of segregation
6 CRISPR Cas is a transgene free approach ndash No regulatory burdens
7 frequency of off-target mutations is well below that caused by chemical
and physical mutagenesis techniques
8 In future can be utilized for metabolic engineering and molecular farming
63
Six-way Venn diagram showing the distribution of shared genefamilies (sequence clusters) among M acuminata P dactyliferaArabidopsis thaliana Oryza sativa Sorghum bicolor and Brachypodiumdistachyon genomes
A DrsquoHont et al Nature 2012 doi101038nature11241
64
Achieving super-DomesticationINTROGRESS-RECREATE-CREATE-DOMESTICATEREDOMESTICATE
bull Mobilizing left out genetic
variation still available in
land races and wild and
weedy species
bull Replaying the evolutionary
tape( resynthesis in
polyploids)
bull Domesticateredomesticate
65
We can admire and emulate how indigenous people still domesticateplants create biodiversity and manage it to sustain their future Thereis no equivalently dynamic or flexible crop breeding in modernagriculture to promote biodiversity we still have much to learn from
traditional knowledge Jan Salick 2012
60
Genome modifications achieved in plants using sequence specific nucleases
Type of DNA
modification
Nuclease Delivery
methods
Plants Targets
Trait stacking Megnucleases Bombardment Cotton Intergenic sequence
ZFN Bombardment Maize Transgene
Rewriting host
DNA
Megnucleases Stable integration Maize Intergenic sequence
ZFN Stable integration Soybean Transgene
TALEN Stable integration Barley PAPhy_a
TALEN Agrobacterium
T-DNA (transient)
Oryza sativa SWEET14
TALEN No Protoplasts Arabidopsis
Tobacco
AtTT4 AtADH NbSurB
TALEN Bombardment Wheat MLO
TALEN Protoplasts
Stable integration
Maize PDS IPK1A IPK MRP4
CRISPRCas Protoplasts
Agrobacterium
T-DNA (transient)
Tobacco
Arabidopsis
Sorghum Oryza
OsSWEET14 transgene
61
Type of DNA
modification
Nuclease Delivery
methods
Plants Targets
Rewriting host
DNA large
deletion
Zinc-finger
nuclease
Stable
integration
Tobacco Transgene
CRISPRCas Protoplasts
Stable
integration
Rice Labdane-related
diterpenoid gene
clusters
on Chr 2 4 and 6
Zinc-finger
nuclease
Agrobacterium
T-DNA
(transient)
Tobacco CHN50 transgene
Zinc-finger
nuclease
Whiskers Maize IPK1
CRISPRCas Protoplasts Rice PDS
Controlling gene
expression
TALE repressor
(SRDX)
Stable
integration
Arabidopsis RD29A transgene
Zinc-finger
activator (VP16)
Stable
integration
Brassica
napus
KasII
Continue
62
Application of genome editing1 Introduction of precise and predictable modifications directly in an elite
background
2 multiple traits can be modified simultaneously
3 NHEJ enables gene knockout and targeted modifications
4 Introduction of transgenes at defined loci that promote high-level
transcription and do not interfere with the activity of endogenous genes
5 Site-specific nucleases also allow targeted molecular trait stacking -low
risk of segregation
6 CRISPR Cas is a transgene free approach ndash No regulatory burdens
7 frequency of off-target mutations is well below that caused by chemical
and physical mutagenesis techniques
8 In future can be utilized for metabolic engineering and molecular farming
63
Six-way Venn diagram showing the distribution of shared genefamilies (sequence clusters) among M acuminata P dactyliferaArabidopsis thaliana Oryza sativa Sorghum bicolor and Brachypodiumdistachyon genomes
A DrsquoHont et al Nature 2012 doi101038nature11241
64
Achieving super-DomesticationINTROGRESS-RECREATE-CREATE-DOMESTICATEREDOMESTICATE
bull Mobilizing left out genetic
variation still available in
land races and wild and
weedy species
bull Replaying the evolutionary
tape( resynthesis in
polyploids)
bull Domesticateredomesticate
65
We can admire and emulate how indigenous people still domesticateplants create biodiversity and manage it to sustain their future Thereis no equivalently dynamic or flexible crop breeding in modernagriculture to promote biodiversity we still have much to learn from
traditional knowledge Jan Salick 2012
61
Type of DNA
modification
Nuclease Delivery
methods
Plants Targets
Rewriting host
DNA large
deletion
Zinc-finger
nuclease
Stable
integration
Tobacco Transgene
CRISPRCas Protoplasts
Stable
integration
Rice Labdane-related
diterpenoid gene
clusters
on Chr 2 4 and 6
Zinc-finger
nuclease
Agrobacterium
T-DNA
(transient)
Tobacco CHN50 transgene
Zinc-finger
nuclease
Whiskers Maize IPK1
CRISPRCas Protoplasts Rice PDS
Controlling gene
expression
TALE repressor
(SRDX)
Stable
integration
Arabidopsis RD29A transgene
Zinc-finger
activator (VP16)
Stable
integration
Brassica
napus
KasII
Continue
62
Application of genome editing1 Introduction of precise and predictable modifications directly in an elite
background
2 multiple traits can be modified simultaneously
3 NHEJ enables gene knockout and targeted modifications
4 Introduction of transgenes at defined loci that promote high-level
transcription and do not interfere with the activity of endogenous genes
5 Site-specific nucleases also allow targeted molecular trait stacking -low
risk of segregation
6 CRISPR Cas is a transgene free approach ndash No regulatory burdens
7 frequency of off-target mutations is well below that caused by chemical
and physical mutagenesis techniques
8 In future can be utilized for metabolic engineering and molecular farming
63
Six-way Venn diagram showing the distribution of shared genefamilies (sequence clusters) among M acuminata P dactyliferaArabidopsis thaliana Oryza sativa Sorghum bicolor and Brachypodiumdistachyon genomes
A DrsquoHont et al Nature 2012 doi101038nature11241
64
Achieving super-DomesticationINTROGRESS-RECREATE-CREATE-DOMESTICATEREDOMESTICATE
bull Mobilizing left out genetic
variation still available in
land races and wild and
weedy species
bull Replaying the evolutionary
tape( resynthesis in
polyploids)
bull Domesticateredomesticate
65
We can admire and emulate how indigenous people still domesticateplants create biodiversity and manage it to sustain their future Thereis no equivalently dynamic or flexible crop breeding in modernagriculture to promote biodiversity we still have much to learn from
traditional knowledge Jan Salick 2012
62
Application of genome editing1 Introduction of precise and predictable modifications directly in an elite
background
2 multiple traits can be modified simultaneously
3 NHEJ enables gene knockout and targeted modifications
4 Introduction of transgenes at defined loci that promote high-level
transcription and do not interfere with the activity of endogenous genes
5 Site-specific nucleases also allow targeted molecular trait stacking -low
risk of segregation
6 CRISPR Cas is a transgene free approach ndash No regulatory burdens
7 frequency of off-target mutations is well below that caused by chemical
and physical mutagenesis techniques
8 In future can be utilized for metabolic engineering and molecular farming
63
Six-way Venn diagram showing the distribution of shared genefamilies (sequence clusters) among M acuminata P dactyliferaArabidopsis thaliana Oryza sativa Sorghum bicolor and Brachypodiumdistachyon genomes
A DrsquoHont et al Nature 2012 doi101038nature11241
64
Achieving super-DomesticationINTROGRESS-RECREATE-CREATE-DOMESTICATEREDOMESTICATE
bull Mobilizing left out genetic
variation still available in
land races and wild and
weedy species
bull Replaying the evolutionary
tape( resynthesis in
polyploids)
bull Domesticateredomesticate
65
We can admire and emulate how indigenous people still domesticateplants create biodiversity and manage it to sustain their future Thereis no equivalently dynamic or flexible crop breeding in modernagriculture to promote biodiversity we still have much to learn from
traditional knowledge Jan Salick 2012
63
Six-way Venn diagram showing the distribution of shared genefamilies (sequence clusters) among M acuminata P dactyliferaArabidopsis thaliana Oryza sativa Sorghum bicolor and Brachypodiumdistachyon genomes
A DrsquoHont et al Nature 2012 doi101038nature11241
64
Achieving super-DomesticationINTROGRESS-RECREATE-CREATE-DOMESTICATEREDOMESTICATE
bull Mobilizing left out genetic
variation still available in
land races and wild and
weedy species
bull Replaying the evolutionary
tape( resynthesis in
polyploids)
bull Domesticateredomesticate
65
We can admire and emulate how indigenous people still domesticateplants create biodiversity and manage it to sustain their future Thereis no equivalently dynamic or flexible crop breeding in modernagriculture to promote biodiversity we still have much to learn from
traditional knowledge Jan Salick 2012
64
Achieving super-DomesticationINTROGRESS-RECREATE-CREATE-DOMESTICATEREDOMESTICATE
bull Mobilizing left out genetic
variation still available in
land races and wild and
weedy species
bull Replaying the evolutionary
tape( resynthesis in
polyploids)
bull Domesticateredomesticate
65
We can admire and emulate how indigenous people still domesticateplants create biodiversity and manage it to sustain their future Thereis no equivalently dynamic or flexible crop breeding in modernagriculture to promote biodiversity we still have much to learn from
traditional knowledge Jan Salick 2012
65
We can admire and emulate how indigenous people still domesticateplants create biodiversity and manage it to sustain their future Thereis no equivalently dynamic or flexible crop breeding in modernagriculture to promote biodiversity we still have much to learn from
traditional knowledge Jan Salick 2012