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 simon.orford@bbsrc.ac.uk simon.orford@bbsrc.ac.uk

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