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
Robert KoebnerJuly 042
• Watkins collection
• EMS mutagenesis
Objective 2 Plant Genetic Resources
Robert KoebnerJuly 043
•Homogenisation of 900 lines(4 plants per line, individually bagged)
•DNA collected on FTA card
Watkins collection
Robert KoebnerJuly 044
Variation in:•Height•Flowering time•Mildew resistance
Robert KoebnerJuly 045
EMS mutagenesis
Current spring variety (Paragon) mutagenised – 3500 plants
Robert KoebnerJuly 046
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
Robert KoebnerJuly 047
Avalon x Cadenza DH population
• mappable (polymorphic) SSRs identified. Mapping to start post summer harvesting
Robert KoebnerJuly 048
SNP development
Loci identified from ESTs, literature and genomic sequence
Robert KoebnerJuly 049
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
Robert KoebnerJuly 0410
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
STMP (sequence tagged microsatellite profiling)
Robert KoebnerJuly 0411
Robert KoebnerJuly 0412
Robert KoebnerJuly 0413
Robert KoebnerJuly 0414
Robert KoebnerJuly 0415
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
Objective 3 Genetic mapping and marker development
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)
Robert KoebnerFebruary 0417
Objective 3 Genetic mapping and marker development
Activities -2
SNP development
SNP development from ESTs (CR). Differential length primer platform optimised. One locus genotyped over Gediflux set, others in progress
Robert KoebnerFebruary 0418
Objective 3 Genetic mapping and marker development
Activities -3
STMP (sequence tagged microsatellite profiling)
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)
Robert KoebnerFebruary 0419
Objective 3 Genetic mapping and marker development
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
Robert KoebnerFebruary 0420
Objective 3 Genetic mapping and marker development
Activities –5
Mapping ESTs to BACs
In negotiation with UC-Riverside to share overgoprobe technology and resources
Robert KoebnerFebruary 0421
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
Robert KoebnerFebruary 0422
Objective 7 Mutagenesis
Activities
de novo mutagenesis based on the spring wheat cvParagon
About to be initiated for spring sowing (LA)
Robert KoebnerFebruary 0423
Objective 17 To establish and maintain international collaborations
Coordination of BAC screening with barley equivalent at UC-Riverside
Robert KoebnerFebruary 0424
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)
Robert KoebnerFebruary 0425
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)