Specific research activities within WGIN –how science can identify new traits and the tools to exploit them

Kim Hammond-Kosack

Wheat Pathogenesis ProgrammePlant-Pathogen InteractionsDivision

23rd June 2004

The Defra WGIN Core ProjectAims:To Underpin Wheat Improvement by Plant

BreedersApproaches:1. Characterisation and provision of genetic resources2. Genetic mapping and marker development3. Trait identification 4. Identification and generation of novel variation in

key traits : using non-GM approaches5. Central storage of grain from field trials6. Liaison and communication

Genetic mapping and marker development

• Establish a reference UK mapping population

Avalon Cadenza

Avalon x Cadenza -

204 double haploid lines

• Switch to ‘within the gene’ molecular markers

> 500,000 wheat ESTs available

Trait identification 1. Nitrogen utilisation and economy (NUE)

2. Plant architecture traits to lower disease pressure

Septoria leaf blotch Fusarium ear blight

3. Grain quality • Field trial at RRes, 0,100, 200, 350 kg nitrogen,31 cultivars, UK(20), Fra (5), Ger (5), Poland (1)

• Desktop scoping study NUE (U of Notts, RRes, ADAS)

WGIN cultivar list for Year 1 field trial

HerewardFlandersEnormELS 02-30EinsteinCappelle-DesprezCaphornCadenzaBeaverBatisAvalonArche

PetrusPBIS 00/77=PrivilegParagon (Spring)Opus MonopolMerciaMaris Widgeon MalaccaLynxIsengrain

ZytaXi19SparkSolsticeSoissonsSokratesScorpion 25RibandRialto

Blue = public molecular data availableGreen = Broadbalk long term exp RRes

Underlined – parent of public DH mapping population

Year 1: Field trial at Rothamsted Research

3 replicates, 4 N treatments, plot size 3 m x 14 m

Split N application

kg N/ha Mar Apr May0100 50 50 0200 50 100 50350* 50 250 50

Tillering GS31/32 GS37

* PGR applications very important to prevent lodging

Year 1: Field trial at Rothamsted Research

Sowing date: Last week November

Emergence: scored December

Residual soil N – deep soil cores taken Feb

Established protocols to measure the variousplant architecture traits

GS 31, 39, 65 and 92 determined for each genotype / treatment

Peter Barraclough, Dimah Habash,Darren Lovell, Caroline Shepherd

Exploitation of diploid wheat

Why ? A rich source of additional gene diversity Ability to test the function of individual alleles

Species: T. monococcum (AA genome)

AA x BB

AABB x DD

AABBDD

T. urartuT. monAA

+ mycotoxins

Novel resistance to problematic pathogens

Septoria leaf blotch

Tapesia eyespot

Fusarium ear blightPolymyxa graminis- Soil borne cerealmosaic virus

Infected roots

T. monococcum collection – three sources

Vavilov Institute, St. Petersburg, Russia

24 accessions all land races, 17 accessions collected prior to 194019 countriesall previously shown to exhibit resistance to multiple pathogens and insect pests in Russia

Sort Number MDR VIR nomber Variety Origin Country Year1 MDR 24 K-105 flavescens, hornemannii Chechen-Ingushetia 19042 MDR 25 K-8365 flavescens, macedonicum Crimea, Ukraine 19233 MDR 26 K-8555 macedonicum, symphaeropolitanum Crimea, Ukraine 19234 MDR 27 K-18105 macedonicum Azerbaijan 19275 MDR 28 K-20399 flavescens Germany 19276 MDR 29 K-20491 flavescens Spain 19277 MDR 30 K-20589 monococcum Spain 19278 MDR 31 K-20994 vulgare, macedonicum Turkey 19279 MDR 32 K-21308 vulgare Italy 1927

10 MDR 33 K-23032 vulgare Yugoslavia 192811 MDR 34 K-23653 hornemannii Armenia 192812 MDR 35 K-25968 vulgare Austria 193013 MDR 36 K-29603 flavescens, monococcum Czechoslovakia 193214 MDR 37 K-30086 macedonicum Armenia 193415 MDR 38 K-30090 monococcum Armenia 193416 MDR 39 K-31683 hornemannii Georgia 193417 MDR 40 K-38079 macedonicum Bulgaria 194018 MDR 41 K-39417 nigricultum, flavescens Albania 195019 MDR 42 K-39471 macedonicum Balkans region 195020 MDR 43 K-39722 vulgare Greece 195021 MDR 44 K-45024 hornemannii Turkey 196522 MDR 45 K-45927 vulgare Denmark 197023 MDR 46 K-46748 macedonicum, vulgare Romania 197024 MDR 47 K-46752 macedonicum Hungary 1970

* also 96 samples from National Small Grains Collection, Aberdeen, USA and 3 from JIC, Norwich will be included in assessment

Description of T. monococcum accessions from VIRNote: Each accession is a land-race NOT a pure line

T. monococcum collection

3 accessions from John Innes Centre, UK

1. EMS mutagenised population of 600 linesavailable (V97031)

2. Can be regenerated in vitro3. Transformable by Agrobacterium - mediated

method ( H. Jones, RRes, 2003)

96 samples from National Small Grains Collection, Aberdeen, USA

Complete collection n = 123

Comparison of ear morphology

T. monococcumPI 119422

T.aestivumGabo

Resistance to Septoria leaf blotchScreening for resistance to Septoria leaf blotch –the No1 disease of wheat

crops in the UK

12

34 5

Field experiment at RRes (2003-2004) to explore reaction of 24 Triticum monococcum samples to

natural infection by Septoria tritici

Septoria disease progress – Jan 04

Good natural winter epidemic

Hexaploids

Claire R lesionsExcept R lesionsSpark R lesionsRiband S lesionsConsort S lesions

All 24 accession No lesionsDiploids

Disease measurements based on thermal time

T. monococcumK-20589

T. aestivumcv. Consort

Verification of Septoria diseaseon Triticum monococcum plants

Diagnostic PCR for Septoria triticiβ-Tubulin Cytochrome B

f Tm Ta f Tm Ta

Feb 2004

Pycnidia(Trypan blue

staining)

infected leaves

Septoria leaf blotch symptoms on hexaploid bread wheatcv. “Consort”, 5th May 2004

All Triticum monococcum plants (n = 3000+ plants) – no Septoria leaf blotch disease symptoms !

Glasshouse screening of T. monococcum

Host Plants

3 x T. mon; 2 x T. ave

Pathogen

5 x Isolates derived from T. monococcum3 x Isolates derived from Cv. Consort

3 x Isolates derived from Cv. Claire

Both hexaploid cultivars were heavily infected by all Septoria

isolates

Introgression of gene diversity from diploid wheat

Interspecies sexual crosses

T. aestivumAABBDD

T. monococcumAA

X

fertile F1 hybridAABBDD

deploy cytogenetics to select for 42 chromosomes

Overview of diploid wheat plant inoculation results

Septoria leaf blotch – most accessionsextremely resistant

Tapesia eyespot

Fusarium ear blight

Soil borne cereal mosaic virus – both resistantand susceptible accessions

vector Polymyxa graminis – only susceptible

Expected variation among accessions

SBWMV/Polymyxa

Tapesiaeyespot

Fusariumear blight

Ideal accessions for mutagenesis

Identification and generation of novel variationin key traits

Two demonstration PCR TILLINGprojects involving hexaploid bread wheat and diploid T. monococcum

Steven Henikoff and Luca Comai (Seattle, USA)

Targeting Induced Local Lesions IN Genomics

Originally developed for the model plant - Arabidopsis

PCR TILLING Technique to identify gene variants

P1 P2Gene of interest

PCR primers 1000bp apart

PCR product lines 1- 7

PCR product line 8

Pool of PCRproducts(1000 bp)

Single bp mis-matches cut in heteroduplex DNA by Cel1 enzyme

denaturingelectrophoresis

Wild-type allele

Variant allele

Assess lines carrying variant allele for novel phenotypes

Application of PCR TILLING technique

- chemically mutagenised populations

ethyl methane sulphonate (EMS) causes GC to AT base pair changes

- Diverse germplasm collections (EcoTilling)

Compare the extent of gene diversitybetween diploid and hexaploid wheat

Demonstration PCR TILLING project No 1

Rht3 gene variants - wild-type phenotype extreme dwarf

rht Rht1 Rht2 Rht1Rht2

Rht3 Rht2Rht3

EMS mutagenised - Mercia Rht3 line- Cadenza

Demonstration PCR TILLING project No 2

Global plant defence signalling regulatorsin both cereal and non-cereal species

PATHOGEN RECOGNITION

Plant cell signalling

A MULTI-COMPONENTRESISTANCE RESPONSE

3 COMPONENTS TO INDUCIBLE PLANT DEFENCE

Overall PCR Tilling Objective for wheat

Correlate using the diploid accessionsspecific gene sequences with specific phenotypes (trait types)

Example: the rar1 gene – required for disease resistanceto multiple pathogens

R1 R2 R3

rar1 (null)

Fully Susceptible

stop

R1 R2 R3

rar1 (weak)

Moderately R

stop

R1 R2 R3

rar1 (elite)

Fully Resistant

The Defra WGIN Core ProjectAims:To Underpin Wheat Improvement by Plant

BreedersApproaches:1. Characterisation and provision of genetic resources2. Genetic mapping and ‘within gene’ marker

development3. Trait identification 4. Identification and generation of novel variation in

key traits : using non-GM approaches- chemical mutagenesis- diverse diploid and hexaploidaccessions

- PCR Tilling technique5. Central storage of grain from field trials6. Liaison and communication

http://www.wgin.org.uk/

Wheat Genetic Improvement NetworkMany thanks to ….

Rothamsted ResearchPPI Division CPI DivisionHai-Chun JingDmitry Kornyukhin*Kostya KanyukaDarren Lovell

Peter ShewryAndy PhillipsKatie Tearall

Funded byJohn Innes CentreJohn SnapeRobert KoebnerLeodie AlbertChristian Rogers

Defra *Rothamsted InternationalFellows Programme