Development of gene based markers and their application JIC · 33.1wPt-5647 55.3wPt-7285...
Transcript of Development of gene based markers and their application JIC · 33.1wPt-5647 55.3wPt-7285...
Development of gene based markers and their application
JIC• Why gene based markers?• Which platform?• SSCP• Tool and Resources progress• Next steps
Non-genic marker classes
• AFLP• DArT• Retro-border markers• SSR
Define deletions with DArT
WGIN maps
SSR
SSR
SSR
SSR
DArT
DArT
GENE
GENE
GENE
Sequenced genomes
Breeders map
SSRSSR
SSRSSR
SSR
SSR
SSR
SSR
SSR
GENE BASED MARKERS MAXIMISE RESOURCE INTEGRATION
MutantsQTLGermplasm
Gene based marker platforms
• Parallel eg Illumina, padlock, SFP
• One by one eg primer extension, SSCP
XPhyA1
Xpsb67
Xmwg518
psr162
Xcmwg733
Xbcd304
XUbi
Ppd-H2 eam8
1H
Avalon X Cadenza1D
Alignment of wheat and barley flowering time effects; 1D v 1H
Single Strand Conformation Polymorphism is now a high throughput technique
Conventional SSCP ran onnon-denaturing acrylamide
ABI3730 capillary electrophoresis(modified polymer, CAP3)
wPt-3707
wPt-7092
wmc336b0.0wPt-37382.7
wPt-379016.7wPt-896016.8wPt-494216.9wPt-419617.0gwm45830.9barc240a31.7cfd1933.01H(b)35.0Glu-D138.4wPt-374339.2
wPt-468789.6wPt-854589.8wPt-656090.1wPt-769791.1wPt-1263wPt-153191.3wPt-771191.4wPt-498892.2wPt-289793.3
gwm330.0wPt-223010.3wPt-346510.6wPt-156011.0wPt-2988Taglgap11.1gwm26411.7wPt-556218.5wPt-816834.5wPt-345135.2barc240b35.9gwm1836.4gwm1136.51H(a)40.0Glu-B144.5wPt-140355.7
wPt-0944wPt-4532wPt-3475wPt-252672.0wPt-412972.
5wmc4485.4
wPt-4721111.4barc80115.8
wPt-40290.0wPt-97525.6
wmc336a23.1
wPt-390439.7barc11944.1gwm49849.6gwm16450.4wPt-6046INTR10-2W150.7psp302750.9Glu-A154.5wPt-975755.8
gwm9972.9
1A 1B 1D
wmc245b
GWM261
gwm3490.0
gwm32010.1
barc11a0.0wPt-02982.1
2.2wPt-83303.3gwm5398.0wPt-372812.1
barc124a0.0COS2P5.0COS2Q10.0cfd3617.5
20.0
wPt-999740.9
gwm210a0.0wPt-33883.5
wmc243c18.8
gwm25739.1wPt-966847.3wPt-356147.5wPt-499747.6wPt-570747.7INTR4-5W264.0stm17TCAC65.0wPt-033566.7wPt-568068.7wPt-009470.8wPt-856970.9wPt-095071.4wPt-313271.6wmc175a72.1wPt-114072.3wPt-740472.7wPt-935073.1wPt-550273.3wPt-973674.0gwm501wPt-004977.0barc15979.8COS2H100.0wPt-2397102.2gwm526c120.1
gwm614b0.0
barc124b23.3
wPt-564733.1
wPt-728555.3wPt-208756.9gwm35963.2
wPt-705682.2COS2R90.0stm528TCAC91.0gwm12293.2wPt-730694.1wPt-420195.1gwm445102.9
wmc181a130.6
2A 2B 2D
2D
2D
0.00.91.92.7
11.226.528.131.232.035.743.8
63.770.1
89.7
3A
stm02TCACgwm52gwm456gdm72wmc505c
COSEx13(a)3Bgwm389
0.0wPt-80938.1COSEx4(3D)10.0barc7513.0barc13316.0wPt-108120.2wPt-275720.5gwm49324.4cfd79b38.1wPt-823849.
0 wPt-697366.9wPt-510567.4wPt-917067.6wPt-701567.7wmc505a69.2barc16471.8wPt-1625wPt-9310wPt-459772.9wPt-5716wPt-714273.0gwm28576.4wPt-576992.7wPt-3005102.1wmc326115.
2 wPt-9368wPt-8021132.1
wPt-8845145.1wPt-4412157.3gwm547159.0
cfd79a0.0wPt-24789.5gwm369wmc505bbarc19wPt-9215
wmc264wPt-1562
gwm155wPt-4725
wPt-5133
3B
3D
gwm1940.0
gwm62416.1
Rht-D10.0
wPt-580910.3wPt-043110.5wmc89a12.6
wPt-12720.0wPt-86500.7wPt-02461.9
wPt-53340.0wPt-39910.8wmc89b1.4barc202.1
gwm5137.6
gwm53818.9
gwm3500.0wPt-86571.9wPt-46203.1wPt-51724.0wPt-53545.0
barc7813.4
wPt-3795wPt-208417.5wPt-136217.7wPt-780718.0
wPt-709632.0barc70a33.3wPt-990134.9
4A
4B
4B 4D
4D
gwm2120.0
barc11036.4
wPt-059653.1
wPt-040065.3
gwm1900.0
GWM17419.0
gwm15935.0gwm358a40.4
gwm2340.0wPt-13020.7
wPt-126121.3wPt-393121.6wPt-5914wPt-5346wPt-6348wPt-517527.0wPt-145734.9wPt-195135.1barc7436.7gwm21339.7wPt-125049.4wPt-424650.8COS5R60.0COS5Q65.0gwm40870.7barc14284.2
wPt-1179105.4stm660acag115.0COS5V2120.0
wPt-3922135.0wPt-0484138.2wPt-7036144.0wPt-8553145.8
wPt-50960.0gwm1269.2
gwm291b33.2
COS5BCOS5FwPt-37940.0gwm293b1.2wPt-36201.9gwm156a6.3wPt-413110.2
gwm617a28.7
5A
5A
5B 5D
5D
barc173a0.0
barc17553.0
wPt-76420.0wPt-7662wPt-18520.4wPt-77770.5wPt-6994wPt-3304wPt-48670.7wPt-8239wPt-1089wPt-9990wPt-1922wPt-3130wPt-47206AwPt-7150
0.8
COSEX171.0wPt-69881.9wPt-493018.2wPt-278631.9wPt-881433.5wPt-525633.6wPt-124134.1wPt-485839.2wPt-757639.3wPt-559639.4wPt-841240.4wPt-330940.5wPt-540840.6wPt-373354.7wmc10556.3gwm21968.5
barc134a85.2wPt-993087.2
wPt-132595.9
wPt-71270.0wPt-25730.8wPt-96901.6wPt-88333.5wPt-30913.7barc23a7.4barc17116.2gwm57018.0gwm617b20.0wPt-164221.0COS7T22.0wPt-720422.3
gwm3340.0
wPt-9382wPt-166418.2
6A
6A 6B6D
wPt-11000.0
barc70b23.2
wPt-576538.7 wPt-256538.8 wPt-126939.8
wPt-81060.0 wPt-88901.0 wPt-94672.9 barc1763.8
wmc51721.1
wPt-534337.4wPt-481441.6
gwm57764.9wPt-060071.5wPt-120979.9 wPt-119681.0 wPt-267781.9 wPt-859882.3 wPt-974683.6
wPt-60190.0 wPt-1259wPt-1023
0.1 gwm3441.0 COS4E2.0 wPt-64953.4 wPt-55333.6 wPt-79475.0
cfa2040a20.8
stm216acag0.0
gwm635b23.0
wPt-515349.0
COS7Cbarc1270.0 wPt-77341.8 wPt-9796wPt-38831.9 COS2B5.0 stm549TCAC7.0COS7K15.0wPt-197420.8wPt-463727.2 wPt-723628.4 barc2930.6 barc49b32.7
7A
7A
7A
7B
7B
wPt-45550.0
cfd21a9.8gwm43715.4stm535acag20.0psp311325.3stm517TCAC30.0
gwm4280.0 COS7S5.0 cfa2040b6.2 wPt-392310.9 wPt-567411.0 wPt-205413.9 wPt-982214.5 wPt-162814.9 barc9717.8
barc76c28.8
7D
7D
Gene based markers on Avalon X Cadenza map
Tools and resources work
• Marker design• Phase 1: Species panel• Phase 2: Wheat mapping• Phase 3: Selected SNP discovery
COS marker design1 2 3 4 5 6 7
A
B
D
abcdabcdabcd
Rice 1
Homologysearch
100-300bp intron?
YESNO Conserved primer sites? YES!NO
Phase 1 testing primers on species panel
Phase 2- wheat mapping
• Thicker blanket coverage- Rice is the predictor
• Targeting- Telomeres so far- groups 4, 6, and 1 to come- Focus on AxC
• Markers screned on AvalonXCadenza and OpataX Synthetic
Phase 3- SNP or indel description
• 100 selected polymorphisms from Avalon Cadenza
• Conversion to breeders markers?
Follow on work
• Mapping in the other grasses
• Widen wheat mapping panel for unmapped markers and homeologues
• Reduce data point cost- multiplex, reagents….
Mapping candidate genes
• In many programmes it is necessary to map a set of genes.
• HFN, stature/stress CSI, grain shape CSI
• Many more- phenology, yield, primary (starch) and secondary (anthocyanins) metabolomics.
WGIN marker (and related) work at JIC
• Michelle Leverington• Lorelei Bilham• Leodie Alibert• John Snape• Simon Orford• Liz Sayers
• Peter Isaac• Lesley Fish• James Simmonds• Yingkun Wang• Neil McKenzie
Take – all disease
Kim Hammond-KosackRothamsted Research
WGIN management meeting, Nov 07
The root infecting Take-all fungus
Affects 2nd and 3rd wheat crops
Unused nutrients left behindleach into water courses
infected roots
Lowers yield and grain quality
Emerging disease complex with Fusarium ear blight
The problem
No known resistancein wheat
Screening for genetic variation in Take-allsusceptibility
Richard Gutteridge
To adapt the laboratory and field protocolsdeveloped at Rothamsted over the past 20 years to screen novel fungicides and formulation
Richard GutteridgeGeoff BatemanJohn Jenkins David Hornby
Approach
Take-all Pot test – 5 reps (10 seeds/pot)artificial inoculum (12 Ggt isolates)
Take-all Pot test
CompletedPot Test = 2 (70 genotypes) Field Tests = 2 years
30 Tm genotypes + 5 hexaploid wheatsas a 3rd crop
In progress (2007)
Two-site field test 3rd year30 Tm genotypes + 3 hexaploid wheat, triticale and rye
Autumn pot test
Take-all tests - T. monococcum2005 - 2007
Genetic variation in Take-all susceptibility%
Roo
t with
take
-all
0
10
20
30
40
50
60
T. monococcum accessions Bread wheat
s.e. =3.78Pot test – 5 reps (10 seeds/pot)artificial inoculum (12 Ggt isolates)
Two mapping populations generated and F2 seed available
Triticum monococcum accessionsHereward A B C D E F G H I J K
Roo
t with
Tak
e-al
l (%
)
0
10
20
30
40
50
60
SED=3.718
* *
*
Take-all resistance in T. monococcum
2007 – 2008 field season – Take-all trials
Exp 130 lines - T. monococcum 6 lines - hexaploid wheat
5 reps per genotype
Great Harpenden III(3rd wheat)
Aim: Evaluating the best genotypes identified in the previous field and pot test
2007 – 2008 field season – Take-all trials
Exp 24 lines - T. monococcum 4 lines - hexaploid wheat 1 line – winter triticale1 line – winter rye
5 reps per genotype
Great Field IV(2nd wheat)
To benchmark the disease levels between speciesand the best diploid and hexaploid lines
2007 – 2008 field season – Take-all trials
Exp 3
The Watkins Collection ~ 750 lines(1 rep)
Controls1 oat, 1 triticale, 1 rye, 5 hexaploid lines (5 block)randomised single Hereward plots (20)
Aims- Eliminate the most susceptible linesBenchmark against other species
Long Hoos 1 and 2 ( next to Broadbalk)(3rd wheat) – tested for inoculum build-up
Watkins collection on Long Hoos
Richard hand sowing the last 16 plots
Jing was taking the photo!
Acknowledgements
RRes (PPM)
Take-allRichard GutteridgeGeoff BatemanRRes Farm Staff
Hai-Chun JingSanja TreskiElke AnzingerDan JenkNeil BrownAndrew Beacham
www.WGIN.org.uk
JIC/ Watkins collectionSimon Orford
The Vavilov InstituteDmitry KornyukhinOlga Mitrofanova
Ukraine ScientistAnastasiya Zlatska
Septoria resistance in Triticum monococcum
www.WGIN.org.uk
Hai-Chun Jing
WGIN Management meeting 05 November 2007
Objective 6 Exploiting T. monococcum as a model for detection of traits, genes and variant alleles and for identifying phenotype: genotype relationships
Objective 9 Identification of gene sequence variants with biological relevance by the PCR TILLING technique
Triticum monococcum as a model
Resistance to Septoria tritici leaf blotch,UK No.1 wheat disease
Bread wheat cv. Hereward T. monococcum
Field assessment (4 years)
no lesions !
Resistance to M. graminicola isolate IPO323MDR308 (resistant) MDR002 (susceptible)
Controlled environmentResponses of 120 accessions to nine differential M. graminicola isolates (gift from James Brown / Gert Kema)
Days post inoculation
4 6 8 10 12 14 16 18 20 22
0
500
1000
1500
2000
2500
3000
3500
Resistance to M. graminicola isolate IPO323
MDR002
DN
A (p
g/le
af)
Fungal biomass qPCR
MDR002
Resistance is achieved by preventing hostProgrammed Cell Death (PCD): DNA laddering
M308
6 8 10 13 15 17 20Days post inoculation
No sporulationResistance : No PCD
6 8 10 13 15 17 20Days post inoculation
M002
SporulationSusceptibility: PCD
Keon et al. (2007) MPMI
Resistance to M. graminicola isolate IPO323
*72 F3 lines were screened for segregation of resistance / susceptibility to Septoria tritici blotch
**all the 68 F3 lines were screened for segregation of resistance / susceptibility to Septoria triticiblotch
Female Male F1 F2 F3
DV92(MDR308)MDR002
MDR002MDR043
1822
30002500
100*68**
M002F1(M308xM002)
Resistance is dominant and prevents host cell death induced by M. graminicola
M308 F1(M002xM043) M043
11 Days post inoculation
Fungal biomass (DNA ug/leaf)0 200 400 600 800 1000 1200
Cou
nt
0
2
4
6
8
10
12
14
Resistance to M. graminicola isolate IPO323
Fungal biomass (ug DNA/0.5ul)0 2000 4000 6000 8000 10000
Cou
nt
0
5
10
15
20
25
30
M308xM002 F3 M002xM043 F2
M002 M002
M043M308
Histogram of fungal biomass in F2/F3 progeny
Xcfd390.0
Xwmc11010.6
Xcfa214120.0
Xcfa2234 Xgwm415Xwmc805 Xgdm68Xwmc705
0.0
Xbarc1800.7
Xwmc79523.1
Xgwm44327.3
Xgwm63936.2
Xwmc84549.7
5Am
Xwmc8610.0
Xwmc46613.1
Xwmc84322.1
Xgwm229.8
Xcfa213447.7
Xwmc110.0
Xbarc3218.0Xbarc5710.0
19.8 Xbarc69
12.0 Xcfa2193
0.0 Xcfa2183
3Am
17.6
Xgdm330.0Xgwm1361.6Xcfa21532.2
Xwmc336
Xgwm7520.0Xbarc1482.1Xwmc716 Xwmc469Xwmc611 Xwmc2786.9
Xbarc830.0Xgwm1351.1
1Am
Xwmc4070.0Xgwm3122.3
Xgwm2960.0
Xwmc17716.8
Xwmc3228.4
Xgwm12235.8Xwmc29639.3
Xgwm27545.1Xgwm24947.6Xgwm44848.6Xwmc42052.4Xwmc77953.7Xwmc64456.3
Xgwm3110.0
Xgdm935.9
Xgwm52615.1
Xgwm60123.3
2Am
Xdupw40.0
Xwmc16129.9
Xgwm74858.4
Xgwm11819.6
4Am
Xbarc1710.0
Xwmc7537.4
Xwmc78614.5
Xgdm336.6
Xbarc1468.5
6Am
A microsatellite linkage map ofTriticum monococcum
7Am
Xgwm3440.0
Xcfa204029.2Xwmc52533.0
Xdupw25446.9
Xgwm74852.5
Xwmc79059.3
Xcfa212364.9
Xbarc17272.8Xwmc48875.7
Xwmc28380.0
Xwmc60385.7Xwmc596 Xbarc10886.6Xcfa204989.0
Xwmc1799.1
Xbarc174107.6
Xwmc405124.4
A sporulation-controlling locus on 7Am
Wheat Stb genes for resistance to Septoria tritici blotch
IPO323
IPO90012
IPO94269
IPO89011
IPO88004
IPO94269
IPO88004
IPO87019
IPO87019IPO88004
Stb11
A
B
D
1 2 3 4 5 6 7
Stb10
Stb9
Stb6
Stb2
Stb1
Stb15
Stb3
Stb8
Stb5Stb4
Stb12
Stb7
Stb11Stb11
A
B
D
1 2 3 4 5 6 7
Stb10Stb10
Stb9Stb9
Stb6Stb6
Stb2Stb2
Stb1Stb1
Stb15Stb15
Stb3Stb3
Stb8Stb8
Stb5Stb4Stb5Stb4
Stb12Stb12
Stb7Stb7
Genetics of M. graminicola resistancein T. monococcum
Fine mapping and for introgression• SSR markers• EST markers• DArT markers – Australia collaboration• COS markers – Ian King IGER (CSI)
Conserved resistance/susceptibility mechanisms in diploid and hexaploid wheat
T. monococcum (AmAm)T. turgidum (AABB) X
Triploid (AmAB) /Amphiploid (AmAmAABB)
(male)
GA spray
Embryo rescueMature seeds
Embryo rescueMature seeds
GA spray
Hybrids
BC1F1 seeds
T. aestivum (AABBDD)(female)
XT. monococcum (AmAm)(male)
X
T. aestivum (AABBDD)(female)
X
Utilisation of useful traits: Introgression
1 2
-Resides at location different to known STB loci ?
-Stops the sporulation of two different Mg isolates
A sporulation-controlling locus on 7Am
Next steps
•Develop more closely-linked markers for introgression
•Establish gene-for-gene relationship
•Map-based cloning
AcknowledgementsRRes (PPI)Kim Hammond-KosackJason RuddKostya KanyukaRichard GutteridgeDarren LovellKim OldhamAlison FergusonGrégoire GerinDaniel JenkJean Devonshire(Bioimaging Centre)
www.WGIN.org.uk
JIC/Sainsbury LabSimon OrfordRobert KoebnerLesley BoydJohn SnapeKen Shirasu (RIKEN)
SCRIJane ShawChristophe Lacomme
The Vavilov InstituteDmitry KornyukhinOlga Mitrofanova
RResSteve Hanley (PIE)Salvador Gezan (BAB)Alan Todd (BAB)Lesley Smart (BCH)
RRes (CPI)Carlos BayonKatie TearallAndy PhillipsAngela DohertyHuw JonesRowan MitchellPaola TosaiPeter Shewry
Ukraine ScientistAnastasiya Zlatska
WGIN Programme:Mutagenesis and TILLING
in wheat
WildtypeMutant
PCR
Melt, anneal
CEL1 digest
TILLING IN THE BREAD WHEAT EMS POPULATION
eg. Ta20ox1A
TILLING in wheat GA20ox1 genes
503012Potential KOs
2810810Located
224513Sequenced
TOTALDBA
Splice site Conserved Gly->Asp
Candidate for effects on height and PHS
White wheats – putative R gene mutantsCadenza has only one functional R gene (on 3D)
Screened 4,300 M4 lines by NaOH treatment (Healthgrain)
Identified five putative mutants, four confirmed in M5
Mariann Rakszegi, Martonvasar
Starch biosynthesis: mutants of SGP1
Screened using SDS-PAGE of starch proteins11 lines identified from 500 M4 lines of Cad-EMS
Cadenza SGP1-A1- SGP1-B1- SGP1-D1-
SGP-A1SGP-D1SGP-B1
Sestilli & Lafiandra,Univ. Tuscia,Viterbo
****** ****** Localised melting and drop in fluorescence
Add intercalating dye
HIGH-RES MELT ANALYSIS FOR MUTATION DETECTION
Heat
LCGreen binds dsDNA– UV fluorescence********* ********
Heteroduplex fromAnnealed PCR products
HIGH-RES MELT ANALYSIS FOR MUTATION DETECTION
Exon1 Exon2 Exon3
F3 R3 969 bp
F1 R1 279 bp
F4 R4 110 bp
Test using known mutations in Arabidopsis GA20ox3 gene
Melting curve Subtraction plot
Use nested PCR: F3-R3 1st roundF1-R1 or F4-R4 2nd round
F3-R3 -> F4-R4 F3-R3 -> F4-R4
ACKNOWLEDGEMENTSRothamsted: Carlos Bayon
Antonio Hernandez-Lopez
Hai-Chun Jing
Kim Hammond-Kosack
Martin Parry
Pippa Madgwick
Thanks to:
Martonvásár: Mariann Rakszegi
Zoltan Bedö
Viterbo: Francesco Sestilli
Domenico Lafiandra
WGIN Update WGIN Update November 2007November 2007
Phenotyping Mutant Phenotyping Mutant PopulationsPopulations
Simon Orford, Leodie Simon Orford, Leodie Alibert, Simon Griffiths Alibert, Simon Griffiths
and John Snapeand John Snape
Starting Material Starting Material Paragon Spring WheatParagon Spring Wheat
RAGT’s NABIM Group 1 varietyRAGT’s NABIM Group 1 variety
Lower yield than other spring Lower yield than other spring varieties but higher protein varieties but higher protein contentcontent
Relatively tall at 93cm approx Relatively tall at 93cm approx 10% higher than other current 10% higher than other current spring varietiesspring varieties
Generally good disease resistance, Generally good disease resistance, with good resistance to mildew, with good resistance to mildew, rusts and Septoriarusts and Septoria
* Source HGCA * Source HGCA
Ethyl Methane Sulphonate Ethyl Methane Sulphonate (EMS) Population(EMS) Population
7000 seed treated with a 1% EMS solution for 16hrs 7000 seed treated with a 1% EMS solution for 16hrs
Fifty per cent germination rate achievedFifty per cent germination rate achieved
Resulting in small / single base pair changesResulting in small / single base pair changes
Two seeds taken per plant at MTwo seeds taken per plant at M2 2 harvest resulting in 7000 harvest resulting in 7000 lineslines
Single Seed Descent (SSD) to MSingle Seed Descent (SSD) to M5 5 generation.generation.
6500 M6500 M55 lines entered to field trial in 2006 lines entered to field trial in 2006
Paragon Mutant Paragon Mutant DevelopmentDevelopment
Single Seed Descent under glass at the JIC
Specimen Ear ArchivingSpecimen Ear Archiving
club type sterility awn suppressorspelt type
EMS Paragon Field EMS Paragon Field TrialsTrials
MM55 seed drilled as 1m seed drilled as 1m rows in field 2006rows in field 2006
Regular records made on Regular records made on developmentdevelopment
Specimen ear maintained Specimen ear maintained for future referencefor future reference
MM66 seed available and Mseed available and M33
DNA. Check via database DNA. Check via database www.wgin.org.ukwww.wgin.org.uk
Ear EmergenceEar Emergence
clump dwarf early ear emergence
Ear Emergence 85 –120 days (clump dwarf not included in trial)
Paragon 93 days following March16th sowing
Height VariationHeight Variation
Heights 30 Heights 30 --104cm in field trial104cm in field trialaverage average –– Paragon 84cmParagon 84cm
Many more shorter and later than early and tall. Approx Many more shorter and later than early and tall. Approx 1000 lines differing by 10% or more1000 lines differing by 10% or more
Paragon
Height Variation of Paragon EMS Mutants
0
20
40
60
80
100
120
Hei
ght (
cms)
Majority of lines 80 to 90cm
Segregation of Dwarf TypesSegregation of Dwarf Types
F2 Distribution of Plant Heights of Paragon Mutant 2566b x Cadenza
0
1
2
3
4
5
6
29cm
32
cm35
cm38
cm41
cm44
cm
47cm
50cm
53cm
56cm
59cm
62cm
65cm
68cm
71cm
74cm
77cm
Heights
No. o
f Ind
ivid
uals
Acc 2566b Cadenza
Strategy for Tapping the Strategy for Tapping the ResourceResource
Crossing lines of interest to Paragon parent to ‘clean’ Crossing lines of interest to Paragon parent to ‘clean’ backgroundbackground
Crossing to other adapted varieties and Crossing to other adapted varieties and phenotypicallyphenotypicallyselecting for mutation selecting for mutation
We are investigating the early proof of concept We are investigating the early proof of concept –– BSA to BSA to genotypicallygenotypically rough map (DArT) rough map (DArT) -- dealing with known or dealing with known or novel alleles?novel alleles?
Being used in the current BBSRC / INRA Nitrogen Use Being used in the current BBSRC / INRA Nitrogen Use Efficiency projectEfficiency project
Stay Green CandidatesStay Green CandidatesNitrogen Use Efficiency?Nitrogen Use Efficiency?
Tested under Low Nitrogen treatment 2007Tested under Low Nitrogen treatment 2007
60 candidate mutants replicated and more 60 candidate mutants replicated and more intensively investigated 2008 (intensively investigated 2008 (--N and +N)N and +N)
Hydroponic testing planned on promising linesHydroponic testing planned on promising lines
Early Senescence Early Senescence VariationVariation
Early senescingMutant ratio16/6500
Leaf Colouration Leaf Colouration VariationVariation
zebra leaf expressionto cold 11/6500
Yellowinglines
Leaf Leaf DiscolourationsDiscolourations
variegation1/6500
Anthocyanin expression
Grain ShapeGrain Shape
grain shape6/6500?
ParagonAcc 732a
Stem and LeafStem and LeafVariationVariation
Waxless variationratio – 11/6500
Monoculmappearance ratio - 15/6500
Awn Suppressor and Awn Suppressor and WaxlessWaxless
Awn return ratio– 8/6500
Gamma Paragon MutantsGamma Paragon Mutants
Nicola Hart Phd Nicola Hart Phd -- Larger deletions but smaller Larger deletions but smaller population sizepopulation size
Initial tests with gamma at Norfolk and Norwich Initial tests with gamma at Norfolk and Norwich Hospital Hospital
Irradiated at IAEA Austria 25Irradiated at IAEA Austria 25--250 Grays. Higher 250 Grays. Higher dose showing more interestdose showing more interest
Developed to M3 generation Developed to M3 generation –– further further development planneddevelopment planned
PublicityPublicity
Cereals Day January 2007 (Norwich)Cereals Day January 2007 (Norwich)
EWAC Conference May 2007 EWAC Conference May 2007 (Istanbul)(Istanbul)
Cereals 07 June (JIC NIAB Alliance)Cereals 07 June (JIC NIAB Alliance)
Early Public DemonstrationsEarly Public Demonstrations
Research described as ‘chemically induced Research described as ‘chemically induced genetic changes’genetic changes’
Directed away from the use of the word Directed away from the use of the word ‘mutation’ ‘mutation’
One Year Later....One Year Later....
Cereals 2007 Event
Branch off projectsBranch off projects
At the JIC (BBSRC / INRA)At the JIC (BBSRC / INRA)CYMMIT (Turkey and Mexico), CYMMIT (Turkey and Mexico), RAGT RAGT CPB TwyfordCPB TwyfordTwo areas of research at NIABTwo areas of research at NIABRRes (WGIN and CSI) RRes (WGIN and CSI) INRA ClermontINRA Clermont--FerrandFerrandADAS ADAS Interest in nitrogen use efficiency, root development, Interest in nitrogen use efficiency, root development, phytatephytate pathways, stay green, pathways, stay green, monoculmmonoculm, plant size, grain , plant size, grain shape and breeder interest shape and breeder interest
Mutant Collection StorageMutant Collection Storage
Extensive Extensive specialist specialist storage storage facilities at facilities at the JICthe JIC
Low temp Low temp and and humidity humidity giving giving approx 20 approx 20 year shelf year shelf life life
Requests?Requests?
Substantial list of mutations recorded Substantial list of mutations recorded
Visit Visit www.wgin.org.ukwww.wgin.org.uk to view database to view database
Seeds requests to Seeds requests to [email protected] [email protected]
AcknowledgementsAcknowledgements
DEFRA funding and all involved in the DEFRA funding and all involved in the WGIN groupWGIN groupLeodie Alibert and for early EMS set up. Leodie Alibert and for early EMS set up. Richard Summers and Peter Jack: Initial Richard Summers and Peter Jack: Initial Paragon donation Paragon donation Steve Reader for field trial managementSteve Reader for field trial management
MUTATION!MUTATION!
‘‘the changing of the structure of a gene, the changing of the structure of a gene, resulting in a variant form which may be resulting in a variant form which may be transmitted to subsequent generations, transmitted to subsequent generations, caused by the alteration of single base caused by the alteration of single base units of DNA, or the deletion, insertion, or units of DNA, or the deletion, insertion, or rearrangement of larger sections of genes rearrangement of larger sections of genes or chromosomes’or chromosomes’
-- Oxford English Dictionary Oxford English Dictionary
Nitrogen use efficiency
Malcolm Hawkesford, Nov 2007
outline• WGIN Rothamsted trials summary• De-convoluting NUE?• Evidence for genetic diversity• Usefulness of Avalon x Cadenza lines• Post-anthesis N-remobilisation - gene
discovery approaches• What next?
WGIN trials summaryDiversity trial
• 2004: 4N, 32 varieties*• 2005: 2N, 20 varieties*• 2006: 3N, 24 varieties*• 2007: 4N, 24 varieties*• 2008: 4N, 24 varieties*
*Varieties varied with core set identical. 2007 and 2008 will be identical.
N usually 0, 100, 200 and 350 kg/ha.
DH (Avalon x Cadenza)
• 2007:2 sites (3 + 2 reps)• 2008: 2 sites (3 + 3 reps) + seed
204 lines + parents.N was 200 kg/ha in 2007 and will be 100
kg/ha in 2008.
Classical measures of Nitrogen Use Efficiency
•Nitrogen Uptake Efficiency (NUpE) = N taken up into plant (above ground)/N available in soil
•Nitrogen Utilization Efficiency (NUtE) = grain yield/N taken up into the plant (above ground)
•Nitrogen Use Efficiency (NUE) = NUpE x NUtE = yield/available N
•Nitrogen Harvest Index (NHI) = N in grain/N in whole plant at harvest
Analysis on standing trials
•Remobilization = N transfer from component of canopy to grain in defined developmental window
•Genes and pathways being expressed in this window
De-convoluting the NUE trait
Time post anthesis
Model for resource remobilisation
0
stem
leaf
2
leaf
3 leaf
1
Grain fillingPr
oces
s
De-convoluting the traits
NUpE
NHI NUpE
Yield (carbohydrate)N
PhotosynthesisSenescenceN-export
For all component traits:Multiple pathways, enzymes, genes and control sites/forms of regulation involved
ArchitectureProliferationActivity
SenescenceN-export
Ranked grain yields
0
2
4
6
8
10
12
1 4 7 10 13 16 19 22 25 28 31
Variety
Gra
in y
ield
(t/h
a, 8
5%)
N0N1N2N3
Yield: WGIN studies indicate genetic diversity
Data collected from WGIN trials 2004-2007 and being analysedConsiderable variation in yield and N-input specific effects
Ranked Grain %N
1.0
1.5
2.0
2.5
3.0
1 4 7 10 13 16 19 22 25 28 31
Variety
Gra
in %
N N0N1N2N3
Bread target 2.3% N (13% protein)
Grain %N: genetic diversity
Ranked NUE
0
10
20
30
40
50
60
1 4 7 10 13 16 19 22 25 28 31
Variety
NU
E (k
g-D
M/k
g-N
)
N0N1N2N3
Ranked NupE
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
1 4 7 10 13 16 19 22 25 28 31
Variety
Nup
E (k
g-N
/kg-
N)
N0N1N2N3
Ranked NutE
20
30
40
50
60
70
80
1 4 7 10 13 16 19 22 25 28 31
Variety
Nut
E (k
g-D
M/k
g-N
)N0N1N2N3
Evidence for genetic diversity - Variation in NUE and components(year to year stability being evaluated)
WGIN DH trial at Rothamsted, 2007
Grain %N (2-3%)
Leaf 2 %N (3.4-5%)
Yield (4.6-11.1 t/ha)
3
3.5
4
4.5
5
5.5
1 7 13 19 25 31 37 43 49 55 61 67 73 79 85 91 97 103
109
115
121
127
133
139
145
151
157
163
169
175
181
187
193
199
205
line
%N
/gD
W
1.501.701.902.102.302.502.702.903.103.303.50
line
0
2
4
6
8
10
12
Lines
Time post anthesis
Model for resource remobilisation
0
stem
leaf
2
leaf
3 leaf
1
Grain fillingPr
oces
s
De-convoluting the traits
NUpE
NHI NUpE
Yield (carbohydrate)N
PhotosynthesisSenescenceN-export
For all component traits:Multiple pathways, enzymes, genes and control sites/forms of regulation involved
ArchitectureProliferationActivity
SenescenceN-export
Post anthesis N remobilisation: Varietal differences
0
2
4
6
8
10
0 7 14 21 28 35 42 49 0 7 14 21 28 35 42 49
dpa dpa
N c
onte
nt (m
g)
N1 N4Leaf 1Leaf 2Leaf 3Stem
Current gene discovery approaches - Tools and resources
Aim: link pathways/genes to traits
Small scale variety N trials, varieties:HerewardIstabraqMaris WigeonRibandSoissonsWelford
NMR
H I M R S W
H I M R S W
All genes
subsets
How similar are the profiles?
- Good replication7 and 21dpa are quite distinct- high and low N are distinct for all varieties except Maris Widgeon and Riband at 7 dpa
=21dpa=7dpa
=N1=N2
Variety
22K
gen
es
N2
N1
N2
N1
Principal component analysis of Variety N-trial leaf arrays (coloured by N-level)
7 dpa 21 dpa
-0.10
-0.08
-0.06
-0.04
-0.02
0.00
0.02
0.04
0.06
0.08
0.10
-0.3 -0.2 -0.1 -0.0 0.1 0.2 0.3
t[2]
t[1]
total-std..M11 (PCA-Class(5)), Leaf ctr 28 dayst[Comp. 1]/t[Comp. 2]Colored according to values in variable total-std.(Treatment)
R2X[1] = 0.850244 R2X[2] = 0.11883 Ellipse: Hotelling T2 (0.95)
Series (Settings for Treatment)
MissingFYMN1N4No S
SIMCA-P+ 11 - 28/01/2006 21:00:00
-12
-10
-8
-6
-4
-2
0
2
4
6
9.99
59.
805
9.60
59.
405
9.20
59.
005
8.80
58.
605
8.40
58.
205
8.00
57.
805
7.60
57.
405
7.20
57.
005
6.80
56.
605
6.40
56.
205
6.00
55.
805
5.60
55.
405
5.20
55.
005
4.72
54.
525
4.32
54.
125
3.92
53.
725
3.52
53.
275
3.07
52.
875
2.67
52.
475
2.27
52.
075
1.87
51.
675
1.47
51.
275
1.07
50.
875
0.67
5
Scor
e C
ontri
b(O
bs 8
8-fy
ml-2
8d-z
1-3
- Ave
rage
), W
eigh
t=p1
p2
Var ID (Primary)
total-std..M11 (PCA-Class(5)), Leaf ctr 28 daysScore Contrib(Obs 88-fyml-28d-z1-3 - Average), Weight=p[1]p[2]
5.405
5.2155.2054.615
4.325
4.185
3.975
3.805
3.705
3.665
3.265
2.485
1.1051.035
SIMCA-P+ 11 - 28/01/2006 21:04:08
-16
-14
-12
-10
-8
-6
-4
-2
0
2
9.99
59.
805
9.60
59.
405
9.20
59.
005
8.80
58.
605
8.40
58.
205
8.00
57.
805
7.60
57.
405
7.20
57.
005
6.80
56.
605
6.40
56.
205
6.00
55.
805
5.60
55.
405
5.20
55.
005
4.72
54.
525
4.32
54.
125
3.92
53.
725
3.52
53.
275
3.07
52.
875
2.67
52.
475
2.27
52.
075
1.87
51.
675
1.47
51.
275
1.07
50.
875
0.67
5
Scor
e C
ontri
b(O
bs 6
7-n1
-l-28
d-z1
-3 -
Aver
age)
, Wei
ght=
p1p2
Var ID (Primary)
total-std..M11 (PCA-Class(5)), Leaf ctr 28 daysScore Contrib(Obs 67-n1-l-28d-z1-3 - Average), Weight=p[1]p[2]
5.405
5.205 4.625
4.205
4.035
3.805
3.665
3.265
2.685
2.465
SIMCA-P+ 11 - 28/01/2006 21:06:33
N4N1
Increased SucroseIncreased AromaticsDecreased Glucose Decreased Glycine betaine
Decreased SucroseDecreased AspartateDecreased Glucose Decreased Glycine betaine
Typical metabolite data obtained from NMR, GC & LC MS etc
Parallel metabolite profiling studies
GLU
CYS
GLY
SER
TRP
PHE TYR
ALA
LEU
VAL
HIS
ARGPROGLN
ILE
MET
ASP
ASN
THR
Ornithine
Citrulline
chorismate
pyruvate
OAA
NH3
P-glycerate
2-OG
2-OG
2-OG
NH3*
2-OG
pyruvate
shikimateCO2
GlnGlu
NH3
ketoacid
ketoacidamino acid
5’-ProFAR
urea
GSA
NH3
Amino acid pool and gene expressionchanges between 7 and 21dpa (N2)
= increasing
= decreasing
= stays same
= below detection
Glu
Gly
LYS
Photorespiration
or Asp
What next?
• Validating genes using diversity trial + further steps pre-MAB
• Usefulness of Avalon x Cadenza for N studies• Nitrogen uptake?
Contributors
• Group: Jonathan Howarth, Peter Buchner, Mark Durenkamp, Saroj Parmar, Janina Jones, Dan Godfrey, Emmanuelle Cabannes, Guillaume N’guyen, Claire Marescal
• Field expertise: Peter Barraclough• Metabolomics: Mike Beale, Jane
Ward