WGIN workpackages undertaken at Rothamsted Research · TILLING OBJECTIVES: YEAR 2 1. Grow M 2 generation of EMS-mutagenized Mercia Rht3. 2. Collect leaf material for genomic DNA

WGIN workpackages undertaken at Rothamsted Research

Update 6th July 2004

Kim Hammond-KosackHai-Chun Jing

Objective 6 : Using diploid wheat as a modelto explore novel sources of disease resistance to important UK pathogens

Species: T. monococcum (AA genome)

T. urata is the source of the A genome in hexaploid bread wheat

Dmitry Kornyukhin – Rothamsted International FellowDarren Lovell – PPI DivisionKostya Kanyuka –PPI Division

Other researchers

T. monococcum genotype collection at RRes

26 accessions (land-races) Vavilov collection (St Petersburg, Russia) (1904 – 1970)

94 samples from National Small Grains Collection, USA

3 lines from JIC1. EMS mutagenised population of 600 lines available (V97031) (MDR50)2. Can be regenerated in vitro (MDR1) 3. Transformable by Agrobacterium – mediated method

(H. Jones, RRes, 2003) (MDR2)

Line DV 92 - BAC library available (Dubrovsky)

Total size of collection = 124 accessions

Screening T. monococcum accessions / lines for resistance and susceptibility to important fungal and viral pathogens

PATHOGEN Susceptible Resistant Mixed reaction

TOTAL number of accessions screened

Polymyxa graminis 90 0 0 124 (34 retesting)

SBCMV 872 (retesting 34

putative R) 1 124Mycosphaerella graminicola 4 24 (Immune) 0 28 (VIR+JIC)

Tapesia yallundae 21 829

(VIR+JIC+DV92)

Tapesia acuformis in progress in progress in progress29

(VIR+JIC+DV92)

Fusarium culmorum, F. graminearum in progress in progress in progress 28 (VIR+JIC)

SUMMARY OF PROGRESS

T. monococcumK-20589

T. aestivumcv. Consort

Septoria fieldtrial

photographedmid-Feb 2004

Diagnostic PCR for Septoria triticiβ-Tubulin Cytochrome B

f Tm Ta f Tm Ta

T. monococcum

T. aestivumcv. Consort

Photographed 2nd May 2004

All T. monococcum accessionsdisease free

All T. aestivum cultivars are heavily diseased

Pathogen Trial type MDR 1 MDR 2 MDR 50/ V97031 DV 92

Polymyxa graminis Pot S S S work in progress

SBCMV Pot R S R work in progress

Mycosphaerella graminicola Field then pot R S (lesions in Feb) S (lesions in Feb no data available

Tapesia yallundae Pot S S S S

Tapesia acuformis Pot work in progress work in progress work in progress work in progress

Fusarium culmorum, F. graminearum Field work in progress work in progress no data available no data available

Plant status

single plants 3 (+1) 5 (+1) 4 (+1) pure line pure line

how many seeds harvested 20 seeds/ plant 20 seeds / plant 20 seeds/plant 100 seeds

Attributes in vitro

regeneration Transformable EMS population BAC library

Source JIC JIC JIC Dubrovsky

SUMMARY OF PATHOGEN TESTS ON T.monococcum: TWO ACCESSIONS AND TWO PURE LINES

SPECIFIC ACCESSIONS tested as single plants to SBCMV plus thetwo accessions with M. graminicola (Septoria) lesions in February

Pathogen Trial type MDR-90/ K-39722 MDR-87 / K-38079 MDR-73 / K-8555MDR-77 / K-

20589

Polymyxa graminis Pot S S S S

SBWMV Pot Mixed R and S R S S

Mycosphaerella graminicola Field then pot R R S (Feb lesions) S (Feb lesions)

Tapesia yallundae Pot Mixed R and S S S S

Tapesia acuformis Pot in progress in progress in progress in progress

Fusarium culmorum, F. graminearum Field in progress in progress in progress in progress

Plant status

single plants 6 8 5 5

how many seeds harvested 20 seeds/ plant 20 seeds/ plant 20-30 ears / plant 20-30 ears / plant

Attributes - - - -

Source VIR VIR VIR VIR

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

Testing T. monococcum with a different set of Septoriaisolates from bread wheat and Durum wheat

Isolates provided by Gert Kema (Wageningen) and James Brown (JIC)

Aim

To identify which Stb resistance genes are presentin each accession / line

MethodPot experiment, artifically inoculation of seedlings under environmentally controlled growth room Conditions (16 C)

Arabidopsis (2000)Rice, Maize, Zebrafish - (conference abstracts / web)Diploid and Hexaploid Wheat - (pers comm)

Steven Henikoff and Luca Comai (Seattle, USA)

Two types - Mutagenesis TILLING or EcoTILLING

PCR TILLING – to explore allele variation

Targeting Induced Local Lesions IN Genomics

Henikoff S and Comai L. 2003. Single nucleotide mutations forfunctional genomics. Ann Review Plant Biology 54: 375-401

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

Demonstration PCR TILLING project No 2

PATHOGEN RECOGNITION

Plant cell signalling

A MULTI-COMPONENTRESISTANCE RESPONSE

3 COMPONENTS TO INDUCIBLE PLANT DEFENCE

TILLING wheat defence signalling regulators NPR1

NPR1ArabidopsisTobacco Rice Barley Wheat

Function Late signalling component

Gene copy 5 8 1 (?) 2 (?)

Gene size Genomic 2401 4239 4270cDNAs 1781 1767 1745 417* 1788AA 593 588 635 137* 609

Variant alleles 10 (1 gene)

**TILLING region 88 – 1386 nt 1 – 321 aa

* Partial sequence only for barley** CODDLE software

Chromosome 1

RAR1ArabidopsisTobacco Rice Barley Wheat

Function Early signalling regulator

Gene copy 1 1 1 1 1

Gene size Genomic 1770 2338 3561 ~3600*cDNAs 676 666 629 695 690AA 226 221 210 232 230

Variant alleles 8 1 2

TILLING region 2-1300nt, 1 - 69 aa

TILLING wheat defence signalling regulators RAR1

Chromosome 6 ? * Determined by PCR

SGT1ArabidopsisTobacco Rice Barley Wheat

Function Interactor with RAR1, HSP90 and SCF

Gene copy 2 2 1 1 1

Gene size Genomic 2204 4826 ~4500*cDNAs 1077 1110 1104 1119 1128AA 358 370 367 373 377

Variant alleles 9 2 1 1

TILLING region 899 - 2197nt, 120 - 357aa

TILLING wheat defence signalling regulators SGT1

Chromosome 1* Determined by PCR

NPR1, RAR1 and SGT1 structure in T. monococcum

Tm-RAR1 (~ 3.6 kb)

TILLING region

?

TILLING region

Tm-SGT1 (~ 4.5kb)

1 2 3 4 5

1 2 3 4 5 6 7 8 9 10

Tm-NPR1, 4270 bp

TILLING region

1 2 3

http://www.wgin.org.uk

Launch date: 7th June 2004

3 new selection boxes (see next slide)

Three new selection boxes on WGIN website

WGIN field trial 2004 – 2006

Focus:

Nitrogen use efficiency

Canopy architecture traits to lower disease pressureof Septoria leaf blotch and Fusarium ear blight

Key decision:30 genotypes Main nitrogen regime and the 2nd nitrogen regime

Experimental design:30 wheat genotypes at one N rate, and 12 selected varieties at a 2nd N rate 126 plots

100 Kg N / ha (environmentally sensitive areas)200 Kg N / ha to give 8 tonnes / ha yield

Objective 6 Diploid wheat (Year 2 )

Select two T. monococcum accessions, multiple seed from single plant, create mutagenised M2

population (each > 5K) and archive seed

Create up to 2 F2 mapping populations and phenotype for their disease response

Use PCR TILLING to identify variant alleles of SGT1, RAR1 and NPR1 in diploid accession collection. Identify specific allele: trait associations

1

2

3

Objective 9 PCR TILLING Year 2

Identification of novel alleles of SGT1, RAR1 and NPR1 genes in hexaploid wheat.

Compare results with diploid dataset

Manuscripts to prepare in Year 2

1. Responses of T. monococcum accessions to different fungal and viral pathogens

2. Sequence variation in the NPR1, RAR1 and SGT1genes in different T. monococcum accessions

3. Successful PCR Tilling in T. monococcum

WGIN Objective 9:TILLING in hexaploid wheat

Year 1

WGIN TILLING PROGRAMME

Aims:

1. To assess the utility of PCR TILLING for identification of novel variation in key performance genes in wheat.

2. To generate an EMS-mutagenized population of an elite hexaploid wheat and establish a TILLING resource.

3. To generate new alleles of Rht affecting plant stature as proof-of-concept

M2POPULATION(segregating)

SELF

STORE FAMILIALLINES AS SEED

TILLING(Targeting Induced Local Lesions in Genomes)

SEED

MUTATE

M1POPULATION(heterozygous)

DNA ISOLATION

POOL

PCR WITHGENE-SPECIFIC

PRIMERS

PCR PRODUCTS

TILLING(Targeting Induced Local Lesions in Genomes)

GENE-SPECIFIC,END-LABELLEDPCR PRODUCTS

MELT &RE-ANNEAL CEL1

DENATURE

GEL ANALYSIS&

SEQUENCE

REPEAT WITHINPOOL TO IDENTIFY

INDIVIDUALS

WHEAT GENETIC IMPROVEMENT NETWORK

1. ESTABLISH TILLING TECHNIQUE IN WHEAT

2. GENERATE EMS-MUTAGENIZED LINES OF RHT3 BREAD WHEAT

WHEAT TILLING FOR NOVEL ALLELESIN PERFORMANCE-RELATED GENES:

GREEN REVOLUTION II:DWARF WHEAT INSENSITIVE TO GA

rht Rht1 Rht2 Rht1Rht2

Rht3 Rht2Rht3

ESTABLISHING TILLING TECHNIQUEIN WHEAT

1. Obtained RHT sequence information from N. Harberd, JIC

2. Designed and tested homeologue-specific primers for Rht-B1 and Rht-D1.

3. PCR-amplified Rht sequences from tall (Rht-B1a) and dwarf (Rht-B1c) and identified point mutations.

4. Digested pooled, annealed PCR products with CEL1 to generate cleavage products.

5. Consulted existing TILLING programmes at SCRI Dundee (barley) and U. Washington, Seattle (Maize, Arabidopsis) for advice on detection platform (WAVE dHPLC, slab gel sequencer, capillary sequencer).

WHEAT GENETIC IMPROVEMENT NETWORK

1. ESTABLISH TILLING TECHNIQUE IN WHEAT

2. GENERATE EMS-M2 LINES OF RHT3 BREAD WHEAT

WHEAT TILLING FOR NOVEL ALLELESIN PERFORMANCE-RELATED GENES:

Rht-D1b(Rht2)

Rht-D1a

Rht-B1b(Rht1)

Rht-B1c(Rht3)

Rht-B1a

*

*

*

Homoeologue-specific primers for wheat Rht genes

EMS-MUTAGENESIS OF RHT3 (RHT-B1c)

1. 20,000 Mercia Rht3 seeds treated overnight with EMS at 0.3% (24mM), 0.6% (48mM) or 1.2% (97mM) in water. M1 seed field-sown Nov 2003.

2. Seeds with lower EMS treatment germinated and established. No germination from highest rate.

3. Anthesis mid-June 2004 – no evidence of widespread sterility (cf. barley). Problem with tall haplotypecontamination.

4. Evidence of lethal mutants in higher EMS rate (up to 50% undeveloped grain).

5. Seed from individuals to be harvested ~Aug 2004.

WGIN Objective 9:TILLING in hexaploid wheat

Year 2 Objectives

TILLING OBJECTIVES: YEAR 2

1. Grow M2 generation of EMS-mutagenized MerciaRht3.

2. Collect leaf material for genomic DNA.

3. Identify tall individuals.

4. Establish high-throughput TILLING (8-fold pooling, 96-well format).

5. Screen tall individuals for mutations in Rht3 by TILLING.

ADDITIONAL TARGETS:GIBBERELLIN BIOSYNTHESIS & SIGNALLING

1. Long-established association between GAs and germination/α-amylase suggest a link with pre-harvest sprouting and PMAA.

2. Recent work from Australia identifies a GA 20-oxidase on barley 5HL linked to major (70%) QTL for sprouting.

3. Orthologous gene in wheat is GA20ox1, isolated at LARS in 1998. Expressed in germinating seed (embryo & scutellum), elongating leaves, stem nodes, developing embryos. Three homoeologues with 10x difference in expression levels.

REDUCING GA LEVELS BYSUPPRESSING GA 20-OXIDASE

GGPP CPPCPS

ent-KAURENE

KS

ent-KAURENOIC ACID KO

GA53

KAO

GA20

GA20OX

GA1

GA3OX

GA8

GA2OX

THE GREEN REVOLUTION:SEMI-DWARF RICE DEFICIENT IN GA

sd1 (OsGA20ox2) MUTATION IN RICE

Robert KoebnerJuly 041

WGIN meeting, JIC July 2004

Progress on Research Goals since start of project

Leodie Alibert – Robert Koebner – Christian Rogers –John Snape – Pauline Stephenson


• Watkins collection

• EMS mutagenesis

Objective 2 Plant Genetic Resources


•Homogenisation of 900 lines(4 plants per line, individually bagged)

•DNA collected on FTA card

Watkins collection


Variation in:•Height•Flowering time•Mildew resistance


EMS mutagenesis

Current spring variety (Paragon) mutagenised – 3500 plants


Objective 3 Genetic mapping and marker development

• Avalon x Cadenza mapping population

• SNP development

• STMP (sequence tagged microsatellite profiling)

• Functional multilocus DNA fingerprinting

• Mapping ESTs to BACs


Avalon x Cadenza DH population

• mappable (polymorphic) SSRs identified. Mapping to start post summer harvesting


SNP development

Loci identified from ESTs, literature and genomic sequence


STMP (sequence tagged microsatellite profiling)

A B D1 8 21 7 362 13 27 17 573 14 38 15 674 12 15 8 355 8 26 10 446 14 22 7 437 11 22 20 53

80 171 84 335

new loci definedand mapped


STM primer PCR conditions STM primer sequenceAnchor

primer name Location

stm506tgag 25x @ 55'C CTACTAGGAATAGGAATGAGGGAAG (tg)7(ag)4 4A

stm507acag 25x @ 55'C AGCTCAGCTTGCTTGGTACC (ac)7(ag)4 5B

stm507tgag 25x @ 55'C CAACTGGCATTGCATTACATCCTTG (tg)7(ag)4 7A

stm508tctg 30x @ 50'C GAAAACTTTGGGAACAAGAGCA (tc)7(tg)4 2B







Objective 4 Hexaploid diversity screen

• GAIT (BBSRC funded) database now available via the internet

• Gediflux (EU funded)

One SSR per chromosome arm for all 500+ entries

NBS profiling … 3 primers x 3 RE’s completed:~50 loci

Transposon-based fingerprinting (SSAP):~70 loci

Robert KoebnerFebruary 0416


Activities -1

Using sequence variants within ESTs (SNPs and SSRs) to add genic markers to the key mapping populations

Mapping popn agreed to - Avalon x Cadenza (JS)



Activities -2

SNP development

SNP development from ESTs (CR). Differential length primer platform optimised. One locus genotyped over Gediflux set, others in progress



Activities -3


Writing up and imminent deposit to Graingenes of data wrt ~230 primer pairs (~350 mapped loci)

(extension of collaborative project with Value Adeed Wheat CRC, University of Sydney)



Activities -4

Functional multilocus DNA fingerprinting

NBS profiling now working very well. Three independent motifs, in association with three RE’s. Tested over Gediflux materials



Activities –5

Mapping ESTs to BACs

In negotiation with UC-Riverside to share overgoprobe technology and resources


Objective 4 Hexaploid diversity screen

ActivitiesGAIT (BBSRC funded)

Database now available to the community via the internet

Gediflux (EU funded)One SSR per chromosome arm for all 500+ entries …. CompletedNBS profiling … 3 primers x 3 RE’s completed. Has yielded ~50 lociTransposon-based fingerprinting (SSAP) …. Completed , has yielded ~70 loci from 3 primer combinations


Objective 7 Mutagenesis

Activities

de novo mutagenesis based on the spring wheat cvParagon

About to be initiated for spring sowing (LA)


Objective 17 To establish and maintain international collaborations

Coordination of BAC screening with barley equivalent at UC-Riverside


02/01

02/02

02/03

07/04

07/04

By end July each year

Objective 2 Plant genetic resources (recurrent)Regeneration and cytogenetic/marker genotype verification of aneuploid and precise genetic stocks

Collect and record morphological data on new accessions entered into the main collection and update associated databases

Service requests for information and seed for 70 requests each year

Ongoing

Ongoing

Since Aug03, ~50

Years 1- 5 ongoing (JIC)


02/04

02/05

02/06

03/01

04/01

07/01

07/04

07/04

01/04

07/04

07/04

07/04

Objective 2 Plant genetic resourcesObserve and fill gaps in morphological records for the 500 lines from the main wheat collection, selected on the basis of maximised ecogeographic and available phenotypic data

Regenerate and bulk the CSxSQ1 doubled haploid lines as bagged seed and make available. Confirm spike phenotype to archival reference set

Make available through the JIC web site, listings of Wheat Precise Genetic Stocks holdings.

Objective 3 Genetic mapping & marker developmentin silico screening performed to define EST-SNPs, appropriate primers designed and validated

Objective 4 Hexaploid diversityGenotyping of Gediflux set (ca. 500 entries) at a minimum of 25 SSR loci

Objective 7 Hexaploid mutagenesisSeed multiplied and prepared for mutagenesis

Plants in field at Church Farm

Now using Av x Cad

Ongoing activity

Poor return on effort. Change of strategy. Some SNP assays generated & validated

SSR completed (42 loci). >120 other loci assigned. Now switching to SNP

Preparing for Spring 04 sowing

Year-01 (JIC)

Documents

WGIN workpackages undertaken at Rothamsted Research · TILLING OBJECTIVES: YEAR 2 1. Grow M 2 generation of EMS-mutagenized Mercia Rht3. 2. Collect leaf material for genomic DNA