“Omics and drought” by John Bennett, IRRI IRRI - unibo.it · Anthers of field-grown rice cv...
Transcript of “Omics and drought” by John Bennett, IRRI IRRI - unibo.it · Anthers of field-grown rice cv...
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Anthers of field-grown rice cv IR74
WUEMED Drought Course,Bologna, 4-10 July 2006:5 lectures on “Omics and drought”by John Bennett, IRRI IRRI
1. Integration of gene and allele discovery with breeding for drought tolerance
2. Microarrays and proteomics as tools to generate new hypotheses
3. Microarrays: experimental design, statistical analysis and gene clustering
4. Reversible and irreversible responses of gene networks to drought stress
5. Potential and limitations of proteomics and metabolomics in drought research
Omics and Drought: Lecture Outline
Transcriptomics Proteomics Metabolomics
JX Chen GH Salekdeh Cook et al. (2004)
IRRI
John BennettInternational Rice Research Institute
Los Baños, Philippines
1. Integration of gene and allele discoverywith breeding for drought tolerance
WUEMED – Drought Course
IRRI
IRRI’s research is organized into 12 projects
1 2 3 7 8 9
Functional genomics
Germplasm conservation and allele mining
Breeding - irrigated ecosystem
Breeding - rainfed ecosystems
Consortium for UnfavorableRice Environments
Activities relatedto abiotic stresses
IRRI
Natural resource management- for rainfed ecosystems
Abiotic stress is the focus in 5 outputs of Project 7
1
2
3
4
5
6
IRRI
Lowlands – drought and submergence
Lowlands – salinity, deep-water, poor soils
Uplands – drought, poor soils
Aerobic rice – water deficit, water saving
Highly nutritious rice
Novel breeding methods for national programs
Outputs:
Stop press: IRRI’s new Strategic Plan features Drought as a Frontier Project
Early-seasondrought
Drought tolerance: a complex mix of traits• Tolerance – retaining water, protecting tissues, maintaining fertility • Avoidance – deep roots, penetrating roots• Escape – short duration
IRRI
Reproductive stage drought tolerance
Varieties with improved reproductive stage drought tolerance reduce the risk of complete crop failure and increasethe “floor” yield in severe droughts.
Raipur, 2005
G. Atlin IRRI
Focus on understanding how drought stress affects yield components differently
a = panicles m-2
b = spikelets panicle-1
c = filled grain (%)
d = single grain weight (mg)
Yield = 10-5 x abcd g m-2
G T PI F M
G T PI F M
G T PI F M
G T PI F M
= stress period
Boonjung and Fukai (1996) Field Crops Res 48:47-55 IRRI
Biodiversityof rice
Farmers’fields
Traditional breeding
Genebank Tissue
culture
DNAtechnology
Marker-aided
selection(MAS)
GenomicsGenetically-engineered
rice Gene
discovery Exoticgenes
1977 1985
1988
1998
1991
Biotechnology at IRRI:Helping the breeding program
1960
1994
1999
2000
2000 China (Bt, Xa21)2002 Philippines (Xa21)
19961993
2004
IRRI
International Rice Molecular Breeding Program
• A method of identifying genes and alleles important for drought tolerance (applicable to other traits also).
• It combines gene/allele discovery and gene mapping with backcross breeding and gene network discovery
• Many institutions may be involved, backcrossing a common set of ~300 donors to ~3 local elite varieties
• The reproductive-stage drought stress is strong enough to kill bothparents.
• Mapping using ~100 markers per genome allows networks of co-selected unlinked genes to be discovered and novel alleles effective in many genetic backgrounds identified.
Li et al. (2005). Genome-wide introgression lines and their use in genetic and molecular dissection of complex phenotypes in rice (Oryza sativa L.). Plant Mol Biol. 59:33-52. Xu et al. (2005). QTLs for drought escape and tolerance identified in a set of randomintrogression lines of rice. Theor Appl Genet. 111:1642-50. IRRI
A six-step molecular breeding strategy
1. Participatory rural appraisal*2. Mechanism discovery#
3. Gene and allele discovery#
4. Pre-breeding for trait reconstruction5. Molecular breeding for variety development6. Participatory variety selection
*Varieties popular with farmers often contain useful genes- salt-tolerance in Bangladesh- drought tolerance in Bangladesh
#Mechanisms, genes or alleles can be discovered any order
IRRI
1.Simple geneticsSimple screen
2.Simple geneticsDifficult screen
4.Complex geneticsDifficult screen
3.Complex geneticsSimple screen
Enhancing breeding efficiency at IRRI throughmolecular approaches
DNA based selection
Genetic analysis, gene discovery, DNA based selection
Advancedscreens
Package of traitsrequired for
adoption by farmers
Pedigree breeding +trait-based selectionBackcross breeding +
DNA based selection
Four types of traits
IRRI
Backcrossing for enhancing drought tolerance
• Improve drought tolerance of an already popular variety by introgression of major genes or QTLs from different donorsusing DNA marker-assisted backcrossing (MAB)
Donor 1(with major gene1or major QTL1)
Donor i(with major geneior major QTLi)
Recurrent parent(popular variety)
RP + 1
RP + i
Recurrent parent+ 1 + … + i
• There may be a practical upper limit of ~5 genes that can be introgressedsimultaneously into a recurrent parent using MAB.
• The best genes to use may be (a) well-characterized transgenes, (b) major genes, (c) QTLs of large effect, (d) novel alleles identified bymining techniques, and (e) mutant alleles; all must have large effects in diverse backgrounds. IRRI
Biomass production is greatly reduced in dry fields
G. Atlin
Enhancing biomass in dry fields at flowering may help increase yield
IRRI
Screening in paddies drained after transplanting can identify cultivars with improved tolerance to
long periods of dry soil conditions
Raipur, India, 2005G. Atlin
IRRI
410116370.40PSBRC 82421013080.31IR 36485416710.34PSBRC 14519715550.30PSBRC 80733618740.26IR 79670-125-1-1-3757621080.28IR 70213-10-CPA 4-2-2-2
Biomass (kg/ha)
Grain yield (kg/ha)
Harvest indexDESIGNATION
Cultivar differences in biomass production in intermittently-dried field: IRRI WS 2005
G. Atlin
IRRI
• Some varieties have much greater tolerance to flowering–stage stress than IR 36, IR 64, and MTU 1010.
• Tolerance to extreme stress at flowering may be affected by major QTLs.
Tolerance to reproductive-stage stress
R. Venuprasad, J. Bernier, A. Kumar, G. Atlin IRRI
Genes for drought tolerance at IRRI
IRRI*IRMBP = International Rice Molecular Breeding Program
Rice germplasm Genes fromother sources
Gene discovery
Allele pyramids
Allele mining
New drought-tolerant varieties
Promoterdiscovery
Phenotyping, mutants,expression analysis, map-based cloning
TILLING, DArT
Marker-aided selection
Marker-aided backcrossing
Novel alleles
Promoterswitching
Transformation
IRMBP*
Phenotypingof advancedbackcrosslines Rice orthologues
Finding mutants and alleles throughhigh-throughput TILLING and eco-TILLING
Colbert et al. (2001). High-throughput screening for induced point mutations. Plant Physiol. 126: 480-484.
TILLING: Targeting Induced Local Lesions IN Genomes
IRRI
Colbert et al. (2001). High-throughput screening for induced point mutations. Plant Physiol. 126: 480-484.
TILLING using acrylamide gels
IRRI
Slade et al. (2004). A reverse genetic, nontransgenic approach to wheat cropimprovement by TILLING. Nature Biotechnology 23: 75-81.
TILLING of WAXY gene of wheat
IRRI
1. Waxy genes encode granule-bound starch synthase1 (GBSS1) 2. Wheat with only one or two functional GBSSI genes produces starch with
intermediate levels of amylose and is referred to as partial waxy wheat.3. Waxy starches, including those of maize and rice, are composed almost entirely of
amylopectin with little or no amylose; they have unique physiochemical propertiesand economically valuable functional qualities.
4. Commercial uses of full waxy wheat exist in the food, chemical and paper industries.
Slade et al. (2004). A reverse genetic, nontransgenic approach to wheat cropimprovement by TILLING. Nature Biotechnology 23: 75-81.
Screen for mutations in the wheat waxy genes
IRRI
TILLING of WAXY gene of wheat
Slade et al. (2004). A reverse genetic, non-transgenic approach to wheat cropimprovement by TILLING. Nature Biotechnology 23: 75-81. IRRI
Model for induction of rd29A Gene expression under abiotic stress conditions
Liu et al. (1998) Plant Cell 10:1391-1406 IRRI
RNA gel blot analysis of DREB1A and DREB2Atranscripts
Liu et al. (1998) Plant Cell 10:1391-1406
IRRI
Freezing and drought tolerance of 35S:DREB1Aband 35S:DREB1Ac transgenic plants
Liu et al. (1998) Plant Cell10:1391-1406 IRRI
Phenotypes of 35S:DREB1A and rd29A:DREB1Atobacco plants in relation to control plants
Kasuga et al. (2004) Plant Cell Physiol. 45:346-350 IRRI
OsLip9 Promoter T-NosHPT
LBRB
pAg7
H H : H : Hind IIIHind IIIE: E: Eco R IEco R IB: B: Bam H IBam H I
AtDREB1AP-Nos
EB
Lip9::AtDREB1A
Lip9::OsDREB1B
DREB Constructs for Rice Transformation
OsLip9 Promoter T-NosHPT
LBRB
pAg7
H
OsDREB1BP-Nos
EB
Variety Genotype Drought tolerance
Palmar Indica, Lowland +Cica 8 Indica, Lowland ++CT6241 Japonica, Upland +++
Rice Genotypes used for this work
Z. Lentini, M. Ishitani, CIAT IRRI
10 days 30 days
80 days Maturity
Z. Lentini, M. Ishitani, CIAT
Transgenic rice with OsLIP9::AtDREB1A construct
IRRI
0.00.20.40.60.81.00.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
NTR
JL 24RD 2
FTSWK. Sharma, V. Vadez, ICRISAT
Applying advanced ideas in drought physiology to transgenic rd29A::AtDREB1A groundnut
NTR:Normalized transpiration rate
FTSW: fraction of transpirable soil waterJL 24:Non-transgenic control plantsRD 2:rd29A::DREB1A transgenic line
IRRI
Asian Rice Biotechnology Network: Working together on biosafety
1. Adopt science-based biosafety regulations2. Harmonize regulations to facilitate bidirectional trade3. Encourage post-graduate training of regulators4. Optimize costs of regulation5. Focus of transparency and public information
IRRI’s greenhouse for GM rice has operated since 1994
IRRI
Genes for drought tolerance at IRRI
IRRI*IRMBP = International Rice Molecular Breeding Program
Rice germplasm Genes fromother sources
Gene discovery
Allele pyramids
Allele mining
New drought-tolerant varieties
Promoterdiscovery
Phenotyping, mutants,expression analysis, map-based cloning
TILLING, DArT
Marker-aided selection
Marker-aided backcrossing
Novel alleles
Promoterswitching
Transformation
IRMBP*
Phenotypingof advancedbackcrosslines Rice orthologues