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Update on WGIN at the John Innes Centre Simon Griffiths

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Page 1: Update on WGIN at the John Innes Centre€¦ · cv Par cv wPt-7541 1A 0 0 1 1 wPt-3870 1A 1 1 1 1 wPt-8773 1A 0 0 1 1 wPt-0128 1A 1 1 1 1 wPt-3870 1A 1 1 1 1 deletions mutant control

Update on WGIN at the John Innes Centre

Simon Griffiths

Page 2: Update on WGIN at the John Innes Centre€¦ · cv Par cv wPt-7541 1A 0 0 1 1 wPt-3870 1A 1 1 1 1 wPt-8773 1A 0 0 1 1 wPt-0128 1A 1 1 1 1 wPt-3870 1A 1 1 1 1 deletions mutant control

Delivery of genetic improvement in wheat- The role of UK science

PlantBreeders

PublicRoles

WGIN

LINKUnderstanding

TraitsGenes/Alleles

Prioritising Targets-Breeders and Government

New Varieties

ProcessorsUK agriculture

Rural livelihoodsGlobal agriculture

BiodiversityLandscape

GeneticsGenetic analysisPositional CloningMutants

GermplasmCollectionsMapping populationsMutant populations

GenomicsBioinformaticsMarkersTranscriptsTargeted mutagenesis

MechanismsHow do genes change phenotype?

Page 3: Update on WGIN at the John Innes Centre€¦ · cv Par cv wPt-7541 1A 0 0 1 1 wPt-3870 1A 1 1 1 1 wPt-8773 1A 0 0 1 1 wPt-0128 1A 1 1 1 1 wPt-3870 1A 1 1 1 1 deletions mutant control

WGIN resource development at JICAvalon X CadenzaChinese Spring X ParagonJIC synthetic X ParagonEMS and gamma Paragon

GeneticsGenetic analysisPositional CloningMutants

GermplasmCollectionsMapping populationsMutant populations

GenomicsBioinformaticsMarkersTranscriptsTargeted mutagenesis

MechanismsHow do genes change phenotype?

Page 4: Update on WGIN at the John Innes Centre€¦ · cv Par cv wPt-7541 1A 0 0 1 1 wPt-3870 1A 1 1 1 1 wPt-8773 1A 0 0 1 1 wPt-0128 1A 1 1 1 1 wPt-3870 1A 1 1 1 1 deletions mutant control

WGIN resource development at JICDevelopment of Near Isogenic Lines• Height• Flowering time• Yield and yield components

GeneticsGenetic analysisPositional CloningMutants

GermplasmCollectionsMapping populationsMutant populations

GenomicsBioinformaticsMarkersTranscriptsTargeted mutagenesis

MechanismsHow do genes change phenotype?

Page 5: Update on WGIN at the John Innes Centre€¦ · cv Par cv wPt-7541 1A 0 0 1 1 wPt-3870 1A 1 1 1 1 wPt-8773 1A 0 0 1 1 wPt-0128 1A 1 1 1 1 wPt-3870 1A 1 1 1 1 deletions mutant control

WGIN resource development at JIC

• AE Watkins collection • Gediflux• Paragon EMS• Paragon gamma

GeneticsGenetic analysisPositional CloningMutants

GermplasmCollectionsMapping populationsMutant populations

GenomicsBioinformaticsMarkersTranscriptsTargeted mutagenesis

MechanismsHow do genes change phenotype?

Page 6: Update on WGIN at the John Innes Centre€¦ · cv Par cv wPt-7541 1A 0 0 1 1 wPt-3870 1A 1 1 1 1 wPt-8773 1A 0 0 1 1 wPt-0128 1A 1 1 1 1 wPt-3870 1A 1 1 1 1 deletions mutant control

WGIN resource development at JIC

• Brachy and rice for gene content prediction• Development of gene based markers• Tilling and gamma for reverse genetics and custom alleles

GeneticsGenetic analysisPositional CloningMutants

GermplasmCollectionsMapping populationsMutant populations

GenomicsBioinformaticsMarkersTranscriptsTargeted mutagenesis

MechanismsHow do genes change phenotype?

Page 7: Update on WGIN at the John Innes Centre€¦ · cv Par cv wPt-7541 1A 0 0 1 1 wPt-3870 1A 1 1 1 1 wPt-8773 1A 0 0 1 1 wPt-0128 1A 1 1 1 1 wPt-3870 1A 1 1 1 1 deletions mutant control

WGIN resource development at JIC

As we begin to clone key genes andelucidate genetic pathways in wheatcan we predict which genes are most malleable for future manipulation and what type of allele we want to look for.

GeneticsGenetic analysisPositional CloningMutants

GermplasmCollectionsMapping populationsMutant populations

GenomicsBioinformaticsMarkersTranscriptsTargeted mutagenesis

MechanismsHow do genes change phenotype?

Page 8: Update on WGIN at the John Innes Centre€¦ · cv Par cv wPt-7541 1A 0 0 1 1 wPt-3870 1A 1 1 1 1 wPt-8773 1A 0 0 1 1 wPt-0128 1A 1 1 1 1 wPt-3870 1A 1 1 1 1 deletions mutant control

Use of Avalon x Cadenza

• Open resource for research- high quality genotype and phenotype data publicly available.

• Available to various disciplines egphysiologists to ask questions relevant to UK agriculture, using material relevant to UK agriculture.

Page 9: Update on WGIN at the John Innes Centre€¦ · cv Par cv wPt-7541 1A 0 0 1 1 wPt-3870 1A 1 1 1 1 wPt-8773 1A 0 0 1 1 wPt-0128 1A 1 1 1 1 wPt-3870 1A 1 1 1 1 deletions mutant control

Discovering what genetic variation UK wheat breeders are using-

height

• Variation in final plant height is the result of a wide range of developmental effects of direct relevance to yield, yield stability, lodging, biomass, and resource use efficiency.

• What genes do wheat breeders use to manipulate final plant height?

Page 10: Update on WGIN at the John Innes Centre€¦ · cv Par cv wPt-7541 1A 0 0 1 1 wPt-3870 1A 1 1 1 1 wPt-8773 1A 0 0 1 1 wPt-0128 1A 1 1 1 1 wPt-3870 1A 1 1 1 1 deletions mutant control

Genes controlling plant height in Avalon X Cadenza population

2A 2D 3A

6A4D 6B

Page 11: Update on WGIN at the John Innes Centre€¦ · cv Par cv wPt-7541 1A 0 0 1 1 wPt-3870 1A 1 1 1 1 wPt-8773 1A 0 0 1 1 wPt-0128 1A 1 1 1 1 wPt-3870 1A 1 1 1 1 deletions mutant control

Novel genetic variation from Watkins material

Page 12: Update on WGIN at the John Innes Centre€¦ · cv Par cv wPt-7541 1A 0 0 1 1 wPt-3870 1A 1 1 1 1 wPt-8773 1A 0 0 1 1 wPt-0128 1A 1 1 1 1 wPt-3870 1A 1 1 1 1 deletions mutant control

Investigating genetic pathways in wheat- vernalization as an example

LONG DAYS

VRN3

VRN2

FLOWERING

SHORT DAYS COLD

VRN1

Page 13: Update on WGIN at the John Innes Centre€¦ · cv Par cv wPt-7541 1A 0 0 1 1 wPt-3870 1A 1 1 1 1 wPt-8773 1A 0 0 1 1 wPt-0128 1A 1 1 1 1 wPt-3870 1A 1 1 1 1 deletions mutant control

Vrn1- Spring or Winter alleles?

Winter allele-recessive wild type

Spring allele-Dominant mutation

Page 14: Update on WGIN at the John Innes Centre€¦ · cv Par cv wPt-7541 1A 0 0 1 1 wPt-3870 1A 1 1 1 1 wPt-8773 1A 0 0 1 1 wPt-0128 1A 1 1 1 1 wPt-3870 1A 1 1 1 1 deletions mutant control

Searching for new genetic variation in response to vernalization

• Adaptation of UK crops to climate change will require the fine tuning of growth habit beyond the classical spring/winter division.

• In a screen of of modern UK spring and alternative wheats- Vrn-A1 promoter duplication and Vrn-B1 intron deletion.

• Screen 800 Watkins lines with all available assays- Do we find spring types that do not have the known alleles?

Page 15: Update on WGIN at the John Innes Centre€¦ · cv Par cv wPt-7541 1A 0 0 1 1 wPt-3870 1A 1 1 1 1 wPt-8773 1A 0 0 1 1 wPt-0128 1A 1 1 1 1 wPt-3870 1A 1 1 1 1 deletions mutant control

winter

spr ing

new

inconclusive

Distribution of growth habit alleles in wheat varieties around the world in 1930’s

AfghanistanAustralia

China

USSR

United Kingdom

Spain

Iran

Canary Islands

Page 16: Update on WGIN at the John Innes Centre€¦ · cv Par cv wPt-7541 1A 0 0 1 1 wPt-3870 1A 1 1 1 1 wPt-8773 1A 0 0 1 1 wPt-0128 1A 1 1 1 1 wPt-3870 1A 1 1 1 1 deletions mutant control

What next?

• New alleles of Vrn1? Sequence.• Alleles of Vrn2, Vrn3?• Previously un-described genes?• All of the above could provide new genetic

variation for manipulation of growth habit.

Avalon cross

Page 17: Update on WGIN at the John Innes Centre€¦ · cv Par cv wPt-7541 1A 0 0 1 1 wPt-3870 1A 1 1 1 1 wPt-8773 1A 0 0 1 1 wPt-0128 1A 1 1 1 1 wPt-3870 1A 1 1 1 1 deletions mutant control

Update on gene based marker development at JIC

Page 18: Update on WGIN at the John Innes Centre€¦ · cv Par cv wPt-7541 1A 0 0 1 1 wPt-3870 1A 1 1 1 1 wPt-8773 1A 0 0 1 1 wPt-0128 1A 1 1 1 1 wPt-3870 1A 1 1 1 1 deletions mutant control

Single Strand Conformation Polymorphism is now a high throughput

technique

Conventional SSCP ran onnon-denaturing acrylamide

ABI3730 capillary electrophoresis(modified polymer)

Page 19: Update on WGIN at the John Innes Centre€¦ · cv Par cv wPt-7541 1A 0 0 1 1 wPt-3870 1A 1 1 1 1 wPt-8773 1A 0 0 1 1 wPt-0128 1A 1 1 1 1 wPt-3870 1A 1 1 1 1 deletions mutant control

What’s new with gene based markers at JIC?

• Good progress with development of new markers in theBBSRC Tools and Resources grant- half of the 800 have been designed and are being tested- screened for polymorphism and mapped.

• Rapid adoption by UK wheat community- visits to JIC from RReS,NIAB, university groups, and extensive use in a number of programmes.

Page 20: Update on WGIN at the John Innes Centre€¦ · cv Par cv wPt-7541 1A 0 0 1 1 wPt-3870 1A 1 1 1 1 wPt-8773 1A 0 0 1 1 wPt-0128 1A 1 1 1 1 wPt-3870 1A 1 1 1 1 deletions mutant control

Summary

• WGIN resources are an integrated set of genetic and genomic tools that can deliver outcomes of agricultural significance from UK science. The most important of these outcomes is identification of novel and useful genetic variation.

• The first step in the identification of novel genetic variation is to describe what we already have.

• Novel variation has been identified for sustainability traits including genetic variation that can be exploited in response to climate change.

Page 21: Update on WGIN at the John Innes Centre€¦ · cv Par cv wPt-7541 1A 0 0 1 1 wPt-3870 1A 1 1 1 1 wPt-8773 1A 0 0 1 1 wPt-0128 1A 1 1 1 1 wPt-3870 1A 1 1 1 1 deletions mutant control

Acknowledgements

• AXC phenotyping- Liz Sayers• AXC genotyping, Vrn Watkins work- Leodie Alibert• Paragon mutant analysis- Simon Orford• Gene based marker- Michelle Leverington Waite and

Lorelei Bilham• James Simmonds, Lesley Fish• John Snape

Page 22: Update on WGIN at the John Innes Centre€¦ · cv Par cv wPt-7541 1A 0 0 1 1 wPt-3870 1A 1 1 1 1 wPt-8773 1A 0 0 1 1 wPt-0128 1A 1 1 1 1 wPt-3870 1A 1 1 1 1 deletions mutant control

WGIN UPDATE February 2008

Glasshouse and Field Population Trials at the John Innes Centre

Simon OrfordLeodie AlibertSimon Griffiths

John Snape

Page 23: Update on WGIN at the John Innes Centre€¦ · cv Par cv wPt-7541 1A 0 0 1 1 wPt-3870 1A 1 1 1 1 wPt-8773 1A 0 0 1 1 wPt-0128 1A 1 1 1 1 wPt-3870 1A 1 1 1 1 deletions mutant control

Overview of Resources• Paragon EMS Population

• Paragon Gamma Population

• Watkins Collection

• Avalon x Cadenza mapping population

• EU GEDIFLUX Collection

Page 24: Update on WGIN at the John Innes Centre€¦ · cv Par cv wPt-7541 1A 0 0 1 1 wPt-3870 1A 1 1 1 1 wPt-8773 1A 0 0 1 1 wPt-0128 1A 1 1 1 1 wPt-3870 1A 1 1 1 1 deletions mutant control

Studies of ‘Gamma’ Material

• Developed to M3 generation • Around 500 of the interesting 250gy types

plus other doses total 2000 lines• Potential for more material to be irradiated

following promising DArT findings and further generation development of current lines

Page 25: Update on WGIN at the John Innes Centre€¦ · cv Par cv wPt-7541 1A 0 0 1 1 wPt-3870 1A 1 1 1 1 wPt-8773 1A 0 0 1 1 wPt-0128 1A 1 1 1 1 wPt-3870 1A 1 1 1 1 deletions mutant control

DArT Analysis• 90 gamma lines sent to DArT (duplicated DNA

samples) screened with a new set of wPtmarkers

• Majority of lines have evidence of deletions of at least one marker, or a block of several markers on each chromosome

• Publication being prepared, awaiting completion of data from Triticarte

Page 26: Update on WGIN at the John Innes Centre€¦ · cv Par cv wPt-7541 1A 0 0 1 1 wPt-3870 1A 1 1 1 1 wPt-8773 1A 0 0 1 1 wPt-0128 1A 1 1 1 1 wPt-3870 1A 1 1 1 1 deletions mutant control

Principle of Diversity Array Technology (DArT)

‘A flourescent ‘reverse’ RFLP cocktail’

Page 27: Update on WGIN at the John Innes Centre€¦ · cv Par cv wPt-7541 1A 0 0 1 1 wPt-3870 1A 1 1 1 1 wPt-8773 1A 0 0 1 1 wPt-0128 1A 1 1 1 1 wPt-3870 1A 1 1 1 1 deletions mutant control

DArT CHRM

GAM132A-1

GAM132A-1

GAM133A-1

GAM133A-1

GAM134A-1

GAM134A-1

wPt-3991 4B 1 1 1 1 0 0

wPt-3991 4B 1 1 1 1 0 0

wPt-1046 4B 0 0 1 1 1 1

wPt-8756 4B 0 0 1 1 1 1

Results from DArT

Using this approach from a screen of approx 500 markers we will get a good representation of deletions created

mutant a mutant b mutant c

Page 28: Update on WGIN at the John Innes Centre€¦ · cv Par cv wPt-7541 1A 0 0 1 1 wPt-3870 1A 1 1 1 1 wPt-8773 1A 0 0 1 1 wPt-0128 1A 1 1 1 1 wPt-3870 1A 1 1 1 1 deletions mutant control

Results from DArT

DArT CHRM

GAM130A-1

GAM130A-1

Par cv

Par cv

wPt-7541 1A 0 0 1 1

wPt-3870 1A 1 1 1 1

wPt-8773 1A 0 0 1 1

wPt-0128 1A 1 1 1 1

wPt-3870 1A 1 1 1 1

deletions

mutant control

80cM

Chromosome 1A worked on to check the concept.Using Triticatre DH maps deletions are physically lined up

Page 29: Update on WGIN at the John Innes Centre€¦ · cv Par cv wPt-7541 1A 0 0 1 1 wPt-3870 1A 1 1 1 1 wPt-8773 1A 0 0 1 1 wPt-0128 1A 1 1 1 1 wPt-3870 1A 1 1 1 1 deletions mutant control

Chromosome 1A

Mutant 130a

1 2 3 4 5 6A

Mutant deletion Mutant deletion

7

B

D

Genome

Developing a Knockout Deletion Panel

Mutant aMutant b

Selected

Mutant cMutant d

Mutant eMutant f

lines

Page 30: Update on WGIN at the John Innes Centre€¦ · cv Par cv wPt-7541 1A 0 0 1 1 wPt-3870 1A 1 1 1 1 wPt-8773 1A 0 0 1 1 wPt-0128 1A 1 1 1 1 wPt-3870 1A 1 1 1 1 deletions mutant control

Vrn Markers Screened on Gamma Population

A genome knockouts

D genome knockouts

B genome knockouts(paragon appears to not amplify naturally)

Page 31: Update on WGIN at the John Innes Centre€¦ · cv Par cv wPt-7541 1A 0 0 1 1 wPt-3870 1A 1 1 1 1 wPt-8773 1A 0 0 1 1 wPt-0128 1A 1 1 1 1 wPt-3870 1A 1 1 1 1 deletions mutant control

Paragon EMS

• Crossing of interesting mutants - stature, stay greens, heading and others carried out on Avalon and Cadenza

• More recently to a collection of 32 diverse spring cultivars to discover more polymorphisms

Page 32: Update on WGIN at the John Innes Centre€¦ · cv Par cv wPt-7541 1A 0 0 1 1 wPt-3870 1A 1 1 1 1 wPt-8773 1A 0 0 1 1 wPt-0128 1A 1 1 1 1 wPt-3870 1A 1 1 1 1 deletions mutant control

A E Watkins Collection

• A collection of great diversity originating from the 1930s from 32 countries

Origins of Watkins Collection

0

20

40

60

80

100

120

140

Alg

eria

Can

ary

Isla

nds

Egy

pt

Eth

iopi

a

Mor

occo

Tuni

sia

Afg

hani

stan

Bur

ma

Chi

na

Indi

a

Iran

Iraq

Pal

estin

e

Syr

ia

Turk

ey

Bul

garia

Cre

te

Cyp

rus

Finl

and

Fran

ce

Gre

ece

Hun

gary

Italy

Pol

and

Por

tuga

l

Rom

ania

Spa

in

Uni

ted

Kin

gdom

US

SR

Yug

osla

via

Aus

tralia

Bra

zil

Countries

Num

ber o

f Acc

essi

ons

Page 33: Update on WGIN at the John Innes Centre€¦ · cv Par cv wPt-7541 1A 0 0 1 1 wPt-3870 1A 1 1 1 1 wPt-8773 1A 0 0 1 1 wPt-0128 1A 1 1 1 1 wPt-3870 1A 1 1 1 1 deletions mutant control

Targets for Watkins

• Assess the homogenous nature of the accessions

• Collect data on heading time, height and vernalisation requirement for all

• Other potential points of interest eg mildew res/sus

• Obtain stocks for future experiments

Page 34: Update on WGIN at the John Innes Centre€¦ · cv Par cv wPt-7541 1A 0 0 1 1 wPt-3870 1A 1 1 1 1 wPt-8773 1A 0 0 1 1 wPt-0128 1A 1 1 1 1 wPt-3870 1A 1 1 1 1 deletions mutant control

Watkins Collection Field Results

Data for 814 lines from 32 countriesAssessment made on accession uniformity

Page 35: Update on WGIN at the John Innes Centre€¦ · cv Par cv wPt-7541 1A 0 0 1 1 wPt-3870 1A 1 1 1 1 wPt-8773 1A 0 0 1 1 wPt-0128 1A 1 1 1 1 wPt-3870 1A 1 1 1 1 deletions mutant control

Summary of Watkins Data

Days to Heading Watkins Trial Church Farm 2006

60

65

70

75

80

85

90

95

100

105

110

115

120

125

130

135

Height (cms) of Watkins Collection Church Farm 2006

404550556065707580859095

100105110115120125130135140145

Winter / Spring Habits of Watkins Collection Church Farm 2006

Winter Type - 100

Spring Type - 688

Heterogenous Type - 35

77 Days

128 Days

44 cm

140 cm

Spring

Winter

Paragon90 Days

Paragon85 cm

Page 36: Update on WGIN at the John Innes Centre€¦ · cv Par cv wPt-7541 1A 0 0 1 1 wPt-3870 1A 1 1 1 1 wPt-8773 1A 0 0 1 1 wPt-0128 1A 1 1 1 1 wPt-3870 1A 1 1 1 1 deletions mutant control

2007/08 Field Trials• Over 50 candidates from 2006/07 low N trial EMS lines

replicated in 1m2 plots in both 240kg N and 20kg N. Senescence to be recorded

• 60 GEDIFLUX cvs also under the same conditions –smaller ‘snapshot’ representation of the 500 line collection derived from SSR data

• Further studies into N efficiency of mutants through hydroponic approach

• Avalon x Cadenza field trials

Page 37: Update on WGIN at the John Innes Centre€¦ · cv Par cv wPt-7541 1A 0 0 1 1 wPt-3870 1A 1 1 1 1 wPt-8773 1A 0 0 1 1 wPt-0128 1A 1 1 1 1 wPt-3870 1A 1 1 1 1 deletions mutant control

More Interest in the MutantCollections

Previously Reported• At the JIC (BBSRC / INRA): nitrogen use efficiency• CIMMYT (Turkey and Mexico): flowering time and stature• Two areas of research at NIAB: phytate pathway and root development• RRes (WGIN and CSI): stay green and grain shape• INRA Clermont-Ferrand: monoculm and tillering• ADAS: leaf size • RAGT• CPB Twyford

Recently Underway• Two JIC pathology groups have shown interest in mildew and senescence variation

(EMS)• Tilling in operation for starch mutants at the JIC Genome Centre (EMS)• A Nottingham University Phd screening for chromosome breakage (Gamma)• Ph1 locus pairing publication from Graham Moore at JIC (Gamma)

Page 38: Update on WGIN at the John Innes Centre€¦ · cv Par cv wPt-7541 1A 0 0 1 1 wPt-3870 1A 1 1 1 1 wPt-8773 1A 0 0 1 1 wPt-0128 1A 1 1 1 1 wPt-3870 1A 1 1 1 1 deletions mutant control

Acknowledgements

• Gamma –Andrzej Kilian (Triticarte) and Nicola Hart

• EMS – RAGT Seeds

• Watkins – Mike Ambrose

• Also Leodie Alibert, Simon Griffiths and John Snape

Page 39: Update on WGIN at the John Innes Centre€¦ · cv Par cv wPt-7541 1A 0 0 1 1 wPt-3870 1A 1 1 1 1 wPt-8773 1A 0 0 1 1 wPt-0128 1A 1 1 1 1 wPt-3870 1A 1 1 1 1 deletions mutant control
Page 40: Update on WGIN at the John Innes Centre€¦ · cv Par cv wPt-7541 1A 0 0 1 1 wPt-3870 1A 1 1 1 1 wPt-8773 1A 0 0 1 1 wPt-0128 1A 1 1 1 1 wPt-3870 1A 1 1 1 1 deletions mutant control

The Development of Mutagenised Populationsand Screening Platforms

for Functional Genomics and Wheat Improvement

Andy PhillipsRothamsted Research, UK

Page 41: Update on WGIN at the John Innes Centre€¦ · cv Par cv wPt-7541 1A 0 0 1 1 wPt-3870 1A 1 1 1 1 wPt-8773 1A 0 0 1 1 wPt-0128 1A 1 1 1 1 wPt-3870 1A 1 1 1 1 deletions mutant control

A Multidisciplinary Approach to Crop Improvement

Candidategenes

Novel variationAllele mining

Fieldexperiments

Understandingthe biology

QTLmapping

Smartscreens

Validation of QTLs&

candidate genes

Breedingprogrammes

Novel alleles forcrop improvement

Page 42: Update on WGIN at the John Innes Centre€¦ · cv Par cv wPt-7541 1A 0 0 1 1 wPt-3870 1A 1 1 1 1 wPt-8773 1A 0 0 1 1 wPt-0128 1A 1 1 1 1 wPt-3870 1A 1 1 1 1 deletions mutant control

CHEMICAL MUTAGENESIS

HNN

N

N

O

OP-O O

CH2OPO

-O O

H2N

O

NO

OP O-O

H2COPO

O-ON

NH2

O

CYTOSINE

GUANINE

DNA (CG PAIR)

NN

N

N

O

OP-O O

CH2OPO

-O O

H2NNO

OP O-O

H2COPO

O-ON

NH2

O

CYTOSINE

O6-ETHYL-GUANINE

OC2H5

X

NN

N

N

O

OP-O O

CH2OPO

-O O

H2NNO

OP O-O

H2COPO

O-ONH

O

O

THYMINE

O6-ETHYL-GUANINE

O

C2H5

H3C

NN

N

N

O

OP-O O

CH2OPO

-O O

NO

OP O-O

H2COPO

O-ONH

O

O

THYMINE

ADENINE

H2NH3C

DNA (TA PAIR)

EMS

H3C S O

O

O

C2H5

Page 43: Update on WGIN at the John Innes Centre€¦ · cv Par cv wPt-7541 1A 0 0 1 1 wPt-3870 1A 1 1 1 1 wPt-8773 1A 0 0 1 1 wPt-0128 1A 1 1 1 1 wPt-3870 1A 1 1 1 1 deletions mutant control

CONSTRUCTION OF MUTAGENISEDWHEAT POPULATIONS

12x8 array ofCadenza M2 lines

• Assess lethal dose of EMS – aim for 30-60% survival.

• Plant EMS-imbibed seeds and grow to maturity. Collect 1 M1 ear per plant.

• Sow 1 M2 seed from each ear in 12x8 format.

• Harvest young leaf tissue into 12x8 deep-well microplates for genomic DNA isolation.

• Note any obvious phenotypes and harvest M3 seed -> archive.

• M2 ears sent to Martonvasar, Hungary, for fixing to M6 and field phenotyping

Page 44: Update on WGIN at the John Innes Centre€¦ · cv Par cv wPt-7541 1A 0 0 1 1 wPt-3870 1A 1 1 1 1 wPt-8773 1A 0 0 1 1 wPt-0128 1A 1 1 1 1 wPt-3870 1A 1 1 1 1 deletions mutant control

CURRENT STATE OF MUTAGENISED POPULATIONS

Species

T. monococcum (2n: A)

T. durum (4n: A,B)**

T. aestivum (6n: A,B,D)

M2

3,000*

4,500

4,300

* 1,500 donated by Kay Denyer, JIC** Under EU programme “Optiwheat”

Page 45: Update on WGIN at the John Innes Centre€¦ · cv Par cv wPt-7541 1A 0 0 1 1 wPt-3870 1A 1 1 1 1 wPt-8773 1A 0 0 1 1 wPt-0128 1A 1 1 1 1 wPt-3870 1A 1 1 1 1 deletions mutant control

THE HEXAPLOID WHEAT EMS POPULATIONCONTAINS A VARIETY OF MUTANT PHENOTYPES

Awn suppressor Disease lesionmimic Extra florets Extra spikelets

Page 46: Update on WGIN at the John Innes Centre€¦ · cv Par cv wPt-7541 1A 0 0 1 1 wPt-3870 1A 1 1 1 1 wPt-8773 1A 0 0 1 1 wPt-0128 1A 1 1 1 1 wPt-3870 1A 1 1 1 1 deletions mutant control

FIELD CHARACTERISATION OF MUTAGENISED WHEAT

Character % phenotype

Time of ear emergence 8.0

Ear glaucosity 2.1

Plant height 2.4

Ear structure 6.0

Ear shape 0.8

Ear length 3.4

Virus susceptible 1.2

Sterile ears 1.6

2,200 lines evaluated by HEALTHGRAIN at MartonvasarAssessed under the UPOV/DUS system

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White wheats – putative R gene mutantsScreened 4,300 M4 lines by NaOH treatment

Identified five putative mutants, four confirmed in M5

Mariann RakszegiZoltan Bedo

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Starch biosynthesis: mutants of SGP1 (SSII)

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

Francesco Sestilli & Domenico Lafiandra

Univ. Tuscia

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WildtypeMutant

PCR

Melt, anneal

CEL1 digest

Tilling in the bread wheat EMS population

700nm 800nm

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MUTATION FREQUENCIES IN PLANT SPECIES

Species PloidyMutation freq.

(muts./1000 bp/1000 lines)

Lines for 95% probability of

truncation in each homoeologue*

Reference

Arabidopsis Diploid 3.3 ~18,000 Greene et al. 2003

Barley Diploid 1.0 ~60,000 Caldwell et al. 2004

Durum wheat Tetraploid 26 ~2,800 Slade et al. 2005

40 ~1,800 Slade et al. 2005

50 ~1,500 This workBread wheat Hexaploid

*Assuming 5% of mutations in coding region result in a truncation.

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GIBBERELLIN SIGNALLING IN WHEAT

GGPP CPPCPS

ent-KAURENE

KS

ent-KAURENOIC ACIDKO

GA53

KAO

GA20GA20oxGA1GA3ox

GA8GA2ox

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A B D TOTALSequenced 13 5 4 22Identified 10 8 10 28

Total 23 13 14 50

eg. Ta20ox1A

TILLING in wheat GA20ox1 genes

Splice site Invariant Gly

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Progress

Target Trait Primer design TILLING Alleles

GA20ox1 Stature/PHS Done Done A&B 53

GSP1 Starch quality Done Done A&D 17

PMS1 DNA repair Done Done 6 (short region)

GAMyb PHS/PMA Done In progress

GID1 Stature Done OptimisingSSIII Starch quality Done Optimising

Isoamylase Starch quality Done Optimising

TILLING – Progress and current targets

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****** ****** 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

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HIGH-RES MELT ANALYSIS FOR MUTATION DETECTION

Labelled primers

PCR from genomic

Cel Digest

Desalt

Licor gel analysis

Identify mutations(manual)

Total: 8 hours

Unlabelled primers

PCR from genomic

Melt analysis

Identify mutations(automatic)

Total: 2 hours

No labelled primersNo CelINo Gels

Direct sequencing

TILLING MELTing

Lightscanner

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HIGH-RES MELT ANALYSIS FOR MUTATION DETECTION

PCR from genomic DNA

Melt analysis

Sign

al

Raw data Normalized Subtracted from WT

Positive samples of human DNA)

Temp

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HIGH-RES MELT ANALYSIS FOR MUTATION DETECTION

Wheat 20ox1DHRM Internal primer pair LSD4

(219 bp) 8 samples, 2 replicates per sample.

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Triticum monococcum as an emerging genetic and molecular resource for wheat genetic improvement

www.WGIN.org.uk

Hai-Chun Jing

WGIN management meeting 25 February 2008

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Objective 6 Exploiting T. monococcum as a model for detection of traits, genes and variant alleles and for identifying phenotype: genotyperelationships

Objective 9 Identification of gene sequence variants with biological relevance by the PCR TILLING technique

Triticum monococcum as a model

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T. monococcum (AmAm, 2n=2x=14 )an cultivated diploid einkorn wheat

Barley Bread wheat(cv. “Kavkaz”)

T. monococcum

5mm140cm

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T. monococcum, an ancient grain

17000BC 10000BC 7800BC 7000BC 6000-7000BC 4700BC

Collecting and eating wild Einkorn and Emmer

Bread Wheat

Domestication of Einkorn and Emmer

Stone Age (1.5m -5000 yr ago) Bronze Age

Durum WheatPasta wheat

Neolithic period

Golden Age of T. monococcum

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Gill et al. (2004) Genetics 168: 1087–1096

Origin of bread wheatT. monococcum AmAm

Sexual gene transfer

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T. monococcum collection at RRes

Now increased to = 263

Origin Country Numbers Origin Country Numbers Variety Numbers Other features NumbersAlgeria 1 Ukraine 2 MDR050 1 SeasonalityChechen 1 Armenia 3 DV 92 1 Spring 207Czechoslovakia 1 Austria 3 kaploutras 1 Winter 35Denmark 1 Georgia 3 kelcyras 1 Facultative 1French 1 United States 3 mansfeldii 1 Intermidiate 1Iran 1 Germany 4 viridivulgare 1Israel 1 Romania 4 laetissimum 2 Earlist collection time Year 1904Kenya 1 unkown 7 sofianum 3Russian 1 Yugoslavia 7 atriaristatum 5South Africa 1 Balkans region 8 hohensteinii 6Syria 1 Greece 9 nigricultum 6Azerbaijan 2 Italy 9 monococcum 9 Transformable accessions 2Ethiopia 2 Spain 9 flavescens 13Hungary 2 Bulgaria 11 hornemannii 21Iraq 2 Europe 39 macedonicum 28Morocco 2 Albania 45 vulgare 66Sweden 2 Turkey 55 unknown 79Switzerland 2 Total 246 Total 244

Accession with ion beam irradation populations

1

Accession with BAC library

1

Accessions with EMS populations

2

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Research highlights

•Screen for novel traits•Resistance to various pathogens

•Mycosphaerella graminicola (Septoria tritici blotch)•Take-all (See Kim’s talk)•Eyespots, Fusarium ear blight, ergot, yellow rust, leaf rust, aphid, powdery mildew

•Other traits•Agronomic and morphological traits•Grain features•Salt tolerance•Drought tolerance

•Generation of novel tools and resources•A SSR map•Mapping populations •Trait / Gene introgression

•TILLING and VIGS•Generation of mutagensied populations•RAR1 EcoTILLING

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MDR308 F1 MDR002

MDR002 MDR308

6 8 10 13 15 17 20Days post inoculation (d)

Novel locus (7Am)

6 8 10 13 15 17 20

DNA laddering

Dominant resistance

Days post inoculation (d)

Resistance to M. graminicola

Triticiummonococcum

Hexaploid wheatField assessment over 4 years

no lesions !

Glasshouse assay (120 Tm accessions x 9 Mg isolates)

TmStb10.0

Xbarc17423.5

Xbarc10836.8Xwmc596Xwmc603

39.3

Xwmc48854.3

C

39.5

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Molecular resourcesA microsatellite linkage map of Triticum monococcum

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 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

92 SSR markers

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Molecular resourcesA DArT linkage map of Triticum monococcum

3047300.011649412.711647013.6470024 37631914.147024414.446983415.146957121.547047922.612054223.646989525.837659325.946947031.446978032.8408446 11652112087939.3

34490240.746958842.034528142.137344843.540838343.947002946.946927557.8312865 46977359.7120689 12027060.037626660.1119890 11989162.038193964.837856665.011609681.0115722 34841383.8119753100.4116612103.2119652106.4469368106.9469358109.5116703121.6469337126.9378465131.5305752 469462131.9120517135.5470583135.7469139135.8116359136.1469974136.4345110136.7469559137.1375933138.8115316143.3470486149.1120598161.7348684165.8379024166.0377950166.1470004166.2469623176.1469665177.8469403184.3469447184.8116616185.5119900185.7470051185.8470275186.2470387189.9115260193.4469291193.7470661193.8120049197.2119672204.3469997205.2120840206.6120674206.8470194207.6120262209.3470239210.4120194211.6120760228.5116166231.4

1

3057930.0470049 3778840.14698166.11163987.63451227.846977410.312118610.846916211.611602312.146985012.546955229.446965830.312028232.646959135.847040736.546963436.712002745.447010150.512082653.747035557.937507858.846970859.138076259.334359261.846968667.247043468.246962568.646952568.846997273.412002975.834644376.947040477.546940480.037630681.947023586.9469720 34621487.947064288.038107288.137696788.246953588.734734390.346996290.4116321102.2469631104.4116733104.8345853105.5470097114.1470344123.2469509128.2470334134.3469283138.7469456 470219469811140.4310884151.1469825 469383153.2470457159.8120585174.0120579174.1408336174.3

2

4699750.03112007.13769747.43489877.947050910.646956911.637719312.337660813.137726115.834549520.734364924.0469198 46996825.847024830.431213533.811634035.346943335.646982735.846941836.247021036.411994336.937642437.0344391 376425469602 37644937.1376448 37636337.234628537.537860737.8116930 11708037.912066138.947043239.347049039.411653939.847028340.146960945.246952245.431129647.446984547.737668548.511970150.430506751.5115379 34739530542351.712001258.037691871.947001676.046920577.847024578.711640684.5

3

4694160.01170055.746991618.546933020.446915527.137663238.037685640.246938741.847018841.937655144.511547846.134798950.611966750.746951152.834768156.934653357.137456359.437902859.937673160.034567175.1

4

1160460.037589417.737693819.212068419.3373941 46970122.637597025.347011528.546961041.746984745.3310698 31215247.947025866.037647066.347061768.146947568.2

5

4698650.047035014.711951915.746981018.547065118.937717319.047030122.034627722.147036222.437904522.737606423.511984024.537712527.847051436.946968441.5

6

3743390.03485922.63761442.73765486.41160076.5469296 4699896.71169566.834377712.1345060 34635418.3

7

4693600.04694272.7312516 4697797.34698269.447038113.312111619.712020822.7

8

4697020.0

37673026.0469650 11558232.546967534.4

9

•16 accessions, 846 polymorphic DArT markers•300 polymorphic in a mapping population

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Genetic resources

In total 18 different mapping populations in T. monococcum

cross female male F1 F2 F3 Comments1 MDR 037 MDR046 6 sptoria tritici blotch2 MDR 037 MDR229 19 septoria tritici blotch, eyespots3 MDR308 MDR002 18 1200 104 septoria tritici blotch, cereal viruses4 MDR002 MDR043 22 6 68 septoria tritici blotch, cereal viruses5 MDR043 MDR040 38 6 100 septoria resistance allelism, cereal viruses 6 MDR308 MDR044 1 400 100 septoria resistance allelism, cereal viruses 7 MDR308 MDR043 43 1200 - septoria resistance allelism, cereal viruses 8 MDR037 MDR002 8 septoria susceptibility allelism

Population for disease resistance

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T. monococcum (AmAm)(PI355520) containing

two genes confer female fertility

X XT. aestivum(AABBDD)

X Hybrids(Female)

T. monococcum (AmAm)

Novel trait introgression

BC1F1

T. Aestivum(AABBDD)

Hairy Black awn

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Bread wheat Hybrids T. monococcum

Seed-set

Embryo-rescue

Tm Tm F1 TaF1 Ta

Trait introgression

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TILLING: Natural and mutagenised populations

Natural accessions: 263

EMS populations: 1,500 M3 lines of MDR050 (Hungary for M4 lines for 2007 phenotying spread rows)1,800 M2 lines of MDR308 (1000 M3 lines for field phenotyping in Hungary)

Low-energy ion beam irradiation(~5,000 seeds MDR308 treated with three dosages,1733 M2 lines obtained)

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RAR1

R proteinType 2 CC-NBS-LRR

EDS1

OB

SGT1

HR

NDR1

NO

R proteinType 1kinase

R proteinType 4RPW8

R proteinType 3 TIR-NBS-LRR

TILLING:Global regulators as candidate genes

A greater number of interacting partners can be integratedEarly defence responses

HSP90

Key signalling complex

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•MDR001 ( MDR002, MDR024, MDR030, MDR032, MDR033, MDR034, MDR035, MDR038, MDR041, MDR047, MDR036, MDR039, MDR046, MDR308, MDR042)

•MDR027 (MDR037, MDR040, MDR043, MDR044, MDR049, MDR050)

•MDR031

•MDR026

•MDR028

•MDR025

•MDR045

•MDR048

•MDR029

RAR1 EcoTILLING: ESTs are very conserved

No null alleles

TCS1.21David [email protected]

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Virus induced gene silencing (VIGS)

Category 3 building for VIGS at RRES(Not part of WGIN, but candidates genes from WGIN will be tested using this technique)

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Long term goals: Reference species for gene function discovery and validation

Gene function validation (Genotype : phenotype)

candidate gene selection

Tm homologues

VIGS TILLING Transformation

Hexaploid wheat genetic improvement

Bioinformatics and Information from reference species

Mutational analysisMap-based cloningTrait-marker association

omics tools

Introgression/Transgenes

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Conclusions

•WGIN has successfully exploited T. monococcum for many useful traits

•WGIN developed many molecular genetic resources and tools for T. monococcum

•WGIN has built a solid basis for future research using T. monococcum as a real model for wheat genetic improvement

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AcknowledgementsRRes (PPM)Hai-Chun JingKim Hammond-KosackJason RuddKostya KanyukaRichard GutteridgeDarren LovellKim OldhamAlison FergusonGrégoire GerinDaniel JenkJean Devonshire(Bioimaging Centre)

www.WGIN.org.uk

JIC/Sainsbury LabSimon OrfordRobert KoebnerLesley BoydSimon GriffithJohn SnapeKen Shirasu (RIKEN)

SCRIJane ShawChristophe Lacomme

RRes (PSC)Carlos BayonKatie TearallAndy PhillipsAngela DohertyHuw JonesPaola TosaiPeter Shewry

Ukraine ScientistAnastasiya Zlatska

The Vavilov InstituteDmitry KornyukhinOlga Mitrofanova

RResSteve Hanley (PIE)Salvador Gezan (BAB)Alan Todd (BAB)Lesley Smart (BCH)

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WGIN Diversity and Double Haploid Trials:

What variation in NUE?Malcolm Hawkesford, Feb 2008

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Rothamsted WGIN field trials and nitrogen

Outline• WGIN Rothamsted Diversity Trial and A x C mapping

population– Summary of acquired data on Diversity Trial (2004-08)– The Avalon x Cadenza trial (2007 and 2008)

• Gene based approaches

2004

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Why Nitrogen?

major determinant of yield, cost implications, environmental concerns

2007 Yields WGIN Diversity Trial

0

2

4

6

8

10

12

0 100 200 350

N level (kg/ha)

yiel

d (t

/ha

85%

DM

)

•Surprisingly little information in the public domain on UK cultivars•Is there any variation in N-use efficiency in wheat cultivars?•Need for good basic data on the complex trait of N-efficiency•Need to identify good performers and the traits responsible to aid breeding programmes

•Current varieties have been selected under high-inputs

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WGIN Diversity Trial summary (2004-2008)

Diversity trial

• 2004: 0, 50, 200 & 350N, 32 varieties*• 2005: 0 & 200N, 20 varieties*• 2006: 0, 100 & 200 N, 24 varieties*• 2007: 0, 100, 200 & 350N, 24

varieties*• 2008: 0, 100, 200 & 350N, 24

varieties*

*Varieties varied with core set identical. 2007 and 2008 will be identical.

N usually 0, 100, 200 and 350 kg/ha.

2007: randomised block design, 3 replicates, 18 x 3 m plot size

2007

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Avalon

BAtis

Beaver

CadenZa

CLaire

COrdiale

MErcia

MOnopol

NApier

PAragon

RIband

RObigus

SAvannah

SHamrock

SoiSsons

SoKrates

SoLstice

XI19

Green = Broadbalk @ RRes

Underlined = parent of public DH mapping population

HEreward

HUrley

IStabraq

LYnx

Malacca

Maris Widgeon*

Purple = spring variety

*Tall variety

WGIN 2006-08 varieties

Soissons N100

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Soil N measurements: WGIN Diversity Trial, 2007

01020304050

kg ha-1

Soil N0

30

60

90

cm

Depth 0-30 cm

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Varieties do not perform in same rank order at different N inputs

Yields, WGIN 2004

0.00

2.00

4.00

6.00

8.00

10.00

12.00C

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Variation in yield between varieties at different N inputs

LSD (5%) = 1.313

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NUE, NUpE and NUtE• NUE has two independent

components: uptake efficiency and utilisation efficiency

• N-uptake efficiency (NUpE) is total crop uptake divided by N supply from soil and fertilizer (uptake/supply) – root trait?

• N-utilisation efficiency (NUtE) is grain yield (100%DM) divided by total N uptake (yield/uptake)

• Overall N-use efficiency, NUE = NUpE x NUtE (=yield/supply)

• Variation observed in all traits amongst WGIN varieties

• For all component traits, multiple pathways, enzymes, genes and control sites/forms of regulation involved

NutE vs NupE (200 kg/ha)

20

30

40

50

60

0.4 0.5 0.6 0.7 0.8

NupE (kg/kg)

Nut

E (k

g/kg

)

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Evidence for genetic diversity: variation in NUE and component traits(WGIN 2004 data)

LSD (5%) = 0.197 LSD (5%) = 5.6 (10.7)LSD (5%) = 6.33

uptake use efficiency overall

Note – data for varieties at the different N inputs are ranked independently

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 31Variety

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 31Variety

Nut

E(k

g-D

M/k

g-N

)N0N1N2N3

Ranked NUE

0

10

20

30

40

50

60

1 4 7 10 13 16 19 22 25 28 31Variety

NU

E (k

g-D

M/k

g-N

)

N0N1N2N3

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Ranked on performance at 0 kg/ha

Rothamsted WGIN-04Combine Grain-NutE

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20

40

60

80Be

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Lynx

Sols

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in-N

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0 kg/ha50 kg/ha200 kg/ha350 kg/ha

Nitrogen utilization efficiency (NUtE) varies between varieties and at different N levels

2004

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N input x genotype interactions and NUtE

NUtE as a function of N rate, WGIN 2006 data

30.0

35.0

40.0

45.0

50.0

55.0

60.0

65.0

70.0

75.0

0 100 200N rate (kg/ha)

NU

tE(k

g/kg

)

Hereward Istabraq Maris W Riband Soissons

Istabraq & Riband – high yield for low N uptake, feed/biscuit type

Hereward, Soissons, MarisWidgeon – require high N, bread type

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WGIN Mapping Population Trial summary

DH (Avalon x Cadenza)

• 2007:2 sites (3 + 2 reps)• 2008: 2 sites (3 + 3 reps) +

seed

204 lines + parents.Sites: Rothamsted and WoburnRandomised block, 3 reps, plot size

was 8 x 2 mN was 200 kg/ha in 2007 and will

be 100 kg/ha in 2008.

Measuring yield, flowering time, N parameters, candidate gene expression

Working on DH population, 19th June 2007

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Challenge to identify processes and respective genes underpinning components of NUE

WGIN 2004

WGIN 2007

WGIN 2008+

subset of varieties

gene discovery

validation

NUtE as a function of N rate, WGIN 2006 data

30.0

35.0

40.0

45.0

50.0

55.0

60.0

65.0

70.0

75.0

0 100 200N rate (kg/ha)

NU

tE(k

g/kg

)

Hereward Istabraq Maris W Riband Soissons

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Example trait: post anthesis N remobilisation as a key component of NUtE

Changes in N content of leaf2/3 post anthesis (Hereward)

Timing and degree of N re-mobilisation depend upon:

• N-input• Genotype

Use this variation to identifying genes involved

0

2

4

6

8

10

0 7 14 21 28 35 42 49

dpa

N c

onte

nt (m

g)

196 kg/ha N

Leaf 1Leaf 2Leaf 3Stem

Sample leaf for N, metabolites and RNA

Final harvest data

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Transcriptomics to assess gene expression

Hereward

N1 N2 N1 N2 N1 N2 N1 N2 N1 N2 N1 N2

Istabraq MarisWidgeon Riband Soissons Welford

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Summary

• Surprising variation in NUE amongst elite wheats

• Variation in component traits

• Performance at different N inputs not always linked

• Segregation in Avalon x Cadenza population

• Candidate genes correlating with processes and indicative of sub-trait performance identified

• Validation of candidate genes required, via wider screening of germplasm (WGIN) and via transgenic proof of function

Diversity trial, 6th July, 2007

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Contributors

• Peter Barraclough• Jonathan Howarth • RRes Farm staff• WGIN team at RRes• Group and field team: Peter

Buchner, Mark Durenkamp, SarojParmar, Janina Jones, Dan Godfrey, Emmanuelle Cabannes, Guillaume N’guyen, Claire Marescal

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WGIN Field experiments 2007-2008 @ Rothamsted

Disease resistance evaluation trials

Kim Hammond-Kosack

2 x Take-all disease

1 x eyespot

25th February 2008

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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

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2007 – 2008 field season – Take-all trials

Exp: 2008/R/WW/802 Field: Long Hoos 1&2 Sown: 23rd Oct

The Watkins Collection 740 lines(1 rep – plot size: 50cm x 50cm, 45 seed over 3 rows)

Controls5 hexaploid lines, 1 triticale, 1 rye, 1 oat (5 block)

randomised single Hereward plots (20)

Aims- Eliminate the most susceptible linesBenchmark against other species

Long Hoos 1 and 2 (nearby to Broadbalk)(3rd wheat) – tested for disease severity in July 2007

Take-all index: 72 % slight – 24.9%, moderate – 31.3, severe 43.8%

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14th February 2008

Long Hoos Take-all trials 2007-2008

Wheat plants in the surrounds will be monitored for take-all each month

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Long Hoos Take-all trials 2007-2008

14th February 2008

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2007 – 2008 field season – Take-all trials

Exp: 2008/R/WW/810 Field: Long Hoos 1&2 Sown: 19th Oct

Diploid T. monococcum wheats - 30 linesHexaploid wheats - 6 lines5 replica plots per genotype

plot size: 50cm x 50cm, 45 seed over 3 rowsAim: Evaluating the best genotypes identified in the previous field and pot test

Original plan

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Two mapping populations generated and F1 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

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2007 – 2008 field season – Take-all trials

Exp: 2008/R/WW/810 Field: Long Hoos 1&2 Sown: 19th Octplot size: 50cm x 50cm, 45 seed over 3 rows

Aim: Evaluating the best genotypes identified in the previous field and pot test

The actual experiment sown

10 reps each of the two resistant parental lines 10 reps of the susceptible parental line5 reps each of 12 other ‘promising’ lines5 reps of the bridging line PI355520

T. monococcum – 16 lines

Other species

5 reps each of 1 triticale, 1 rye, 1 oat5 reps each of cv Hereward

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The stem base infecting eyespot fungi

RRes White Horse 2000

Eye shaped lesionson stem base

Affects all cereal crops including 1st wheats

Crop lodging, ~10% yield reduction

Fusarium can infect aftereyespot causing a stem base complex

The problem

Resistance in wheatPch1, Pch2

2 speciesOculimacula yallundaeO. acuformis

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2007 – 2008 field season – Eyespot trial

Exp: 2008/R/WW/811 Field: Little Knott 1 (1st wheat after oats)plot size: 50cm x 50cm, 45 seed over 3 rows Sown: 19th Oct

10 reps each of the 7 resistant lines10 reps each of the 3 susceptible line5 reps each of the lines PI355520 and L118 + 8 R lines

from pot testT. aestivum

T. monococcum – 20 lines

5 reps - Hereward (4)5 reps – Hyperion 10 reps each of Humber (8), Consort (6) and Lynx

Natural inoculum + artificial inoculum added to the soil surface at 2nd leaf stage (Nov/ Dec) for both species

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Acknowledgements

Rothamsted ResearchSeed counters - Watkins seed collection from JIC Elke AnzingerHelen JenkinsSanja TreskicRichard Gutteridge

Hand Sowers Richard GutteridgeHai-Chun JingAndrew BeachamNeil Brown+ farm staff

John Innes CentreSimon Orford – for the Watkins seed


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