WGIN JS Feb 04 Woolpit · Robert Koebner February 04 1 ... Mapping popn agreed to - Avalon x Cadenza (JS) Robert Koebner February 04 4 Objective 3 Genetic mapping and marker development

Robert KoebnerFebruary 041

WGIN meeting, Woolpit Feb 26th 2004

Core research activities since start of project

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


Objective 2 Plant Genetic Resources

Activities

Genotyping and general trait characterisation of sample subsets of the varietal collection and mutants generated

Watkins collection (LA) - ca. 3600 plants now growing under glass with FTA DNA preps taken


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)



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

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)



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


Ongoing

Ongoing

Since Aug03, ~50

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

07/04

07/04

By end July each year

02/01

02/02

02/03

Years 1- 5 ongoing (JIC)


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

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

07/04

07/04

01/04

07/04

07/04

07/04

02/04

02/05

02/06

03/01

04/01

07/01

Year-01 (JIC)

WGIN workpackages undertaken at Rothamsted Research

Update 26th February 2004

Kim Hammond-Kosack

Objective 5: Trait identification

Year 1: Field trial at Rothamsted Research

30 winter wheat and 1 spring wheat

UK 20France 5 Germany 5 Poland 1

Old, recent past and current cultivars

NABIM 1 and 2


Nitrogen use efficiency

Plant architecture traits to low disease pressure

- Septoria leaf blotch

- Fusarium ear blight

Grain quality


Who actively contributed to the cultivar list ?

3 UK wheat breeding companies

Dalgety – comprehensive unpublished datasetson NUE

2 German wheat breeding companies

Peter Barraclough (RRes) – Broadbalk exp

Parents of available DH mapping populations

HerewardFlandersEnormELS 02-30EinsteinCappelle-DesprezCaphornCadenzaBeaverBatisAvalonArche

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

ZytaXi19SparkSolsticeSoissonsSokratesScorpion 25RibandRialto(Spring)

WGIN cultivar list for Year 1 field trial

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

Underlined – parent of public DH mapping population


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

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

Split N application

Tillering GS31/32 GS37

* PGR applications very important to prevent lodging


Sowing date: Last week November

Emergence: scored December

Residual soil N – deep soil cores taken Feb

Writing up the SOPs to measure the variousplant architecture traits listed in the CSG7

Peter Barraclough, Dimah Habash,Darren Lovell, Caroline Shepherd

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

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

The UK pathogens selected

Septoria leaf blotch

Tapesia eyespot

Fusarium ear blight

Soil borne cereal mosaic virus and itsvector Polymyxa graminis

T. monococcum collection – complete n = 123

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 JIC

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

T. monococcumPI 119422

T.aestivumGabo

Comparison of ear morphology

Resistance to Septoria leaf blotch

Resistance to Septoria leaf blotchSeptoria leaf blotch is the No1 disease of wheat crops in the UK

12

34 5

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

VIR collection to Septoria tritici

Hexaploids

Claire R lesionsExcept R lesionsSpark R lesionsRiband S lesionsConsort S lesions

All 27 accession No lesions

Septoria disease progress – Feb 04

Good natural winter epidemic

Diploids

Disease measurements based on thermal time

Resistance to Soil-borne Cereal Mosaic Virus and its vector Polymyxa graminis

Phase 3 Soil Borne Cereal Mosaic Virus in leavesdetected by ELISA

Susceptible wheat genotypes

Roots infected withPolymyxa in the autumn

Phase 1

Cereal plant

Virus particles move in early springinto the stem

Phase 2

Polymyxa graminis - SBCMV

Infected roots

Cereal plant

Polymyxa graminis - SBCMV

Infected roots

Best resistant hexaploid wheat genotypes

Roots still become infectedwith Polymyxabut SBCMV particles do notspread into aerial tissues

stop

No virus detected by ELISA in leaf tissue

Glasshouse screening of T. monococcum accessions for resistance to SBCMV and Polymyxa graminis

+ - + -K-39722 12 7 5 12 -MDR-1 7 - 7 7 -

K-38079 10 - 10 10 -MDR-2 7 7 - 7 -

MDR-50 7 - 7 7 -

sample number of plantsELISA test

leaves roots

ELISA test to detect SBWMV

Results:

• Individual plant tests confirm the resistance and susceptible phenotypes for lines K-38079 and K-3972, respectively.

• accession K- 39722 segregates for susceptibility and resistance to SBWMV.

• new data about reaction of three accessions to SBWMV – two of them are susceptible and one of them is resistant to this pathogen;

• all tested samples are susceptible to Polymyxa graminis.

- -

Activities planned for Q1-Q2, 2004

•Continue glasshouse trial aimed at testing the reaction of T. monococcum accessions to SBCMV – ELISA test will be used for the assessment

•Try to identify T. monococcum plants resistant to Polymyxa graminis – light microscopy and quantitative real-timePCRtechnique (with Elaine Ward)

•Isolate, clone and sequence SBCMV isolate used in the glasshouse trials (with Kostya Kanyuka)

•Re-sow T. monococcum accessions from VIR collection and continue field experiment aimed to identify the reaction of T. monococcum samples to natural UK populations of Septoria tritici in the spring (possibly inoculate ears with Fusarium)

•Start screening VIR accessions for the resistance and susceptibility to Tapesia yallundae / T. acuformis

•Cross the best R and S genotypes with each other to create mapping population

Objective 9 PCR TILLING

Progress on other WGIN objectives

12. Website – www.WGIN.org.UK

Aim to pre-test site in MarchPopulating the site in Feb

17. International activities - CIMMYT

WGIN grain store at RRes – under construction

Avalon = Maris Ploughman1 / Bilbo21 Cappelle *4 / Hybrid 48 // 2* Maris Widgeon /3/ Viking 2 Vilmorin 29 / VG 8058 // Cappelle Desprez

/4/ CI 12633 / Cappelle Desprez/3/ Heines 110 / Cappelle Desprez // Nord Desprez

Cadenza = Axona1 / Tonic21HPG-522-66/Maris Dove2RPB-87-73/RPB-94-73

Pedigree of Avalon and Cadenza

PHENOTYPEGENE

1. IDENTIFICATION OF NOVEL ALLELES OF TARGET GENES IN EXISTING POPULATIONS

2. ISOLATION OF NEW ALLELES OF TARGET GENES FROM MUTAGENIZED POPULATIONS

3. FUNCTIONAL GENOMICS IN WHEAT – MATCHING PHENOTYPE TO GENE

THE WHEAT TILLING PROGRAMME

IDENTIFICATION AND GENERATION OF NOVEL VARIATION IN KEY TRAITS USING NON-GM APPROACHES

SEED

TILLING(Targeting Induced Local Lesions in Genomes)

MUTATE

McCallum C.M., Comai L., Greene E.A. and Henikoff S. (2000) Targeting induced local lesions in genomes (TILLING)

for plant functional genomics. Plant Physiol., 123, 439-442.

CHEMICAL MUTAGENESIS

HNN

N

N

O

OP-O O

CH2OPO

-O O

H2N

O

NO

OP O-O

H2COPOO-O

N

NH2

O

CYTOSINE

GUANINE

DNA (CG PAIR)

NN

N

N

O

OP-O O

CH2OPO

-O O

H2NNO

OP O-O

H2COPOO-O

N

NH2

O

CYTOSINE

O6-ETHYL-GUANINE

OC2H5

X

NN

N

N

O

OP-O O

CH2OPO

-O O

H2NNO

OP O-O

H2COPOO-O

NH

O

O

THYMINE

O6-ETHYL-GUANINE

O

C2H5

H3C

NN

N

N

O

OP-O O

CH2OPO

-O O

NO

OP O-O

H2COPOO-O

NH

O

O

THYMINE

ADENINE

H2NH3C

DNA (TA PAIR)

EMS

H3C S O

O

O

C2H5

SEED M2POPULATION(segregating)

SELF

M2 SEED


MUTATE

M1POPULATION(heterozygous)

DNA ISOLATION

POOL

PCR WITHGENE-SPECIFIC

PRIMERS

PCR PRODUCTS


GENE-SPECIFICPCR PRODUCTS

MELT &RE-ANNEAL CEL1

DENATURE

GEL ANALYSIS&

SEQUENCE

REPEAT WITHINPOOL TO IDENTIFY

INDIVIDUALS

WGIN TILLING TARGETS: GA BIOSYNTHESIS- STATURE- PHS

GA20

GA1

GA8

GGPP GA53

GA 20-OXIDASE eg SD-1

GA 3-OXIDASE

GA 2-OXIDASE

INITIAL WGIN TILLING TARGETS: PLANT STATURE

rht Rht1 Rht2 Rht1Rht2

Rht3 Rht2Rht3

KEY ISSUES

• IDENTIFY OPTIMAL MUTAGEN DOSAGE- HIGH MUTATION FREQUENCY- LOW MORTALITY

• IDENTIFY HOMOEOLOGUE-SPECIFIC PRIMERS

• MAXIMIZE POOL SIZES FOR PCR

• WHICH DETECTION PLATFORM?- LABELLED PRIMER + GEL SEPARATION- WAVE HPLC

• CAN MUTATIONS IN ONE HOMOEOLOGUE HAVE A PHENOTYPE?

WGIN TILLING MATERIALS

EMS-MUTAGENIZED MERCIA Rht3 POPULATION FOR VARIANTS OF Rht (M1 GENERATION IN FIELD NOV 2003)

EMS-MUTAGENIZED CADENZA FOR VARIANTS OF GA20OX, GA3OX (M1 GENERATION IN FIELD AND/OR GLASSHOUSE FEB/MAR 2004)

HISTORIC POPULATIONS OF WHEAT (GEDIFLUX SET): ECO-TILLING FOR HEIGHT/DIEASE RESISTANCE

DIPLOID ACCESSIONS: ECO-TILLING FOR HEIGHT/DIEASE RESISTANCE

EMS-MUTAGENIZED DIPLOID LINES FOR VARIANTS OF DISEASE RESISTANCE GENES

MUTAGENESIS STRATEGY

EMS-TREAT SEED (M0)

GROW M1 POPULATION

COLLECT M2 SEEDS

GROW M2 POPULATION – ONE SEED PER M1

COLLECT TISSUE FOR DNACOLLECT SEEDS -> STORE

PROGRESS TO DATE

• KATIE TEARALL APPOINTED – STARTED 01-01-2004

• 20,000 SEEDS WHEAT CV. MERCIA RHT3 TREATED WITH EMS AT THREE DOSES AND PLANTED IN FIELD 09-10-2003

• DUE TO OBTAIN FULL GENOMIC SEQUENCES FOR RHT ALLELES 02-03-2004

• PRELIMINARY RHT PRIMER PAIRS TESTED

•VISITED SCRI TO DISCUSS TILLING STRATEGIES 25-02-2004

A

B C

D

MERCIA RHT3 M1 POPULATION

Minutes of WGIN Breeders meeting, held at Nickerson, Woolpit, 9 January 2004 Attending: Bill Angus Nickerson Chris. Chapman (Chair) Nickerson David Feuerhelm Elsoms Mike Field Advanta Phil Howell Syngenta Richard Jennaway Saaten Union Tom Jolliffe Advanta Steve Smith Cebeco Richard Summers PBI Monsanto Peter Werner CPB Twyford While this is not a BSPB group, it opinions represent a consensus of the key wheat breeders in the UK. Breeders stress that their opinions stem partly from their understanding of the technologies which is not comprehensive. They would welcome further discussion on any point with researchers. It was agreed that minutes of this meeting would go to those attending, Peter Shewrey and John Snape as heads of departments at Rothamsted and the John Innes Centre respectively; and Donal Murphy-Bokern of the Science Directorate at DEFRA 1. The group are aware that, compared with typical breeding budgets, significant public funding is going into wheat research applicable to wheat genetics and breeding, but we have no clear overview of this work. This hampers useful interactions between the research and breeding communities. We propose that a catalogue of UK publicly funded research programmes, present and recent, with applications to wheat genetics and breeding should be established by WGIN on its web site, with links to project reports as appropriate. This should be extended in time to include overseas research programmes. Initially this group will assemble a listing of all the projects it is aware of as a starting point. Action: Chris to circulate a list of all the projects he can trace. Other breeders to add to this or amend. 2. There is a general concern that research projects should focus on deliverables to the breeder as end points, and bear in mind that the various programmes have differing capabilities. In this respect industry needs to be more active in leading LINK projects and, working with the funding bodies, emphasize practical rather than academic output. The rigor which now accompanies the initiation and management of LINK projects should be extended to all WGIN research. In particular genes/traits need to be tested and delivered in elite backgrounds (e.g. recent Recommended List varieties) rather than model varieties or alien species. The lack of a ‘germplasm bridge’ is seen as a major constraint to practical application of research results. Markers need to be robust, and of a type most breeders can deploy. The more recently developed types may require technology that some breeders cannot presently justify

- 2 -

Movement of technologies, materials or germplasm into breeding programmes must not be constrained by IP restrictions or licenses, including MTAs. 3. The main activity areas in the original research application were reviewed with the following comments:- The breeders will be happy to assist in germplasm evaluation (aspect of activities 1, 2 and 3) in respect of traits in which the breeders have an interest. [A particular question was asked concerning the recent work on the Watkins collection: is the data compiled and available?] With regards to mapping populations work (activity 2), that it should be aligned on the one hand with international mapping initiatives, on the other with the specific mapping populations which will be required to mark specific traits. Markers and data should be fully available. In principle breeders would be willing to assist in population development, or contribute mapping populations they have already developed. The value of a diversity screen per se (activity 3) is unclear to the breeders. However, identifying useful alleles and traits in northern European wheats is seen as extremely useful as this variation is much more easily accessible when compared to more exotic materials. In general keeping the germplasm base underlying UK breeding broad is seen as important. The traits and trials being considered under activity 4 need to be driven by the breeding and industry rather than research institutes. Otherwise it is unlikely that the output will be taken up by breeders. [See specific comments on nitrogen below]. The recourse to diploid species as a source of novel traits (activity 5) is seen as a last resort only used when more closely related sources have been exhausted, and breeders do not support this activity. In this respect they were surprised to learn of work on SBWMV resistance originating from Triticum monococcum had been placed under WGIN, when sources in existing cultivars are only just being investigated. The work on mutagenised hexapolid populations (activity 6) to generate novel variation is unlikely to be of practical utility. Breeders do recognize its value as a research tool to investigate gene activity, and as a test bed for PCR TILLING (activity 7) which may uncover useful alleles. 4. The wheat breeders were especially concerned about the conduct of the nitrogen use efficiency trials. The origins of the experiment now underway at Rothamsted were unclear; its initiation precedes the desk study which should underpin it; the structure and materials being used run contrary to advice sought from and given by breeders. It is expected to give a poor return for the public funds expended. Breeders draw attention to broadly similar BSPB quality strips underway, and suggest that these could made available to supplement Rothamsted work, with the researchers taking additional information they require on disease ratings and harvest index. [Since the meeting, the matter has been raised with BSPB. They point out there are significant differences and restrictions in their trials that would require discussion.] 5. There was a general consideration of general research priorities as the breeders see them which is outlined in table 1. Blank cells indicate either this aspect is adequately covered in present breeding work, or that it is unlikely to be relevant up to the medium term.

- 3 -

The major interests lie in pests, diseases and quality. The upstream research on genomics and transformation appears remote from practical application. The importance of moving genes from exotic into elite backgrounds, and the technology to do so, was emphasised. In several cases there is a need for better understanding of the biology of a trait, and better means of evaluation. The possible loss of these skills in relevant old technologies from public institutes in competition with new technologies was a particular concern. 6. A more detailed review for diseases is shown in table 2. In general where there was a research priority, the disease needed covering in depth. The important role of the service function of institutes, e.g. in maintaining disease races or advising on screening protocols, is emphasized. We draw attention to the special case of take-all, a major disease of second wheats, where no resistance is known. Here high-risk research strategies would be rational. For pests, the emphasis is on orange blossom midge and wheat bulb fly (table 3). 7. Whilst quality parameters tend to emphasize bread (table 4) breeders are well aware that feed is the largest end use and should not be overlooked. Breeders are unclear what it is end users are seeking on grain size and shape. Action: Chris to approach CCFRA for clarification on this point. 8. For agronomic traits, there is great interest in the under researched area of roots. In particular more needs to be known about ‘second wheat syndrome’ where rooting and soil borne pathogens must both be implicated. There is also value in researching lodging, but phenology, drought tolerance, winter hardiness and plant architecture are not priorities. 9. Breeders note that several of the priority areas are already the subject of LINK projects, but that generally only a few breeders are in any one. Whilst these priorities reflect breeders interests, any project on these proposed under WGIN must be judged on its own merits. Action: Chris to circulate minutes to breeders for comment by Friday January 16 Action: Breeders to return comments by Friday January 23

Table 1. General wheat breeding research priorities

TraitsDiseases & pests Quality

Agronomy1

intensive extensive

Biology/ evaluation +++ ++ +

elitepoorly adapted ++exotic and alien +++

Mapping and QTLs + +++ +++ +

Fine mapping and gene based markers

++ ++ +

Gene structure and function

Transformation1 see item 8

Yield

Germplasm sources2

2 This relates to ease of transfer of traits from various sources. Mapping and other research on elite stocks may be required to clarify the genetics and biology of a trait.

Minutes WGIN breeders meeting 09/01/04

WGIN JS Feb 04 Woolpit · Robert Koebner February 04 1 ... Mapping popn agreed to - Avalon x Cadenza (JS) Robert Koebner February 04 4 Objective 3 Genetic mapping and marker development

Documents