Triticum monococcum, TILLING, genetics and pathology

www.WGIN.org.uk

Kim Hammond-Kosack

WGIN Management meeting 21st Feb 2007

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

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

Triticum monococcum as a model

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

T. monococcumAmAm

Sexual gene transfer

T. monococcum (Am Am, 2n=2x=14 ) is not the A genome donor for bread wheat

Research highlights - Q3 and Q4 2006• Screen for novel traits

• Resistance to various pathogens• Mycosphaerella graminicola (Septoria tritici blotch)• Take-all• Eyespots• Yellow rust (Lesley Boyd, JIC)

• Other traits• Agronomic and morphological traits• Grain features• Abiotic stresses

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

• TILLING• Generation of mutagenised populations• RAR1 EcoTILLING

Resistance to Septoria tritici leaf blotch,UK No.1 wheat disease

Bread wheat cv. Hereward T. monococcum

Field assessment (3 years)

Resistance to M. graminicola isolate IPO323MDR308 (resistant) MDR002 (susceptible)

Controlled environment: high inoculum levels – attached leaves Responses of 120 accessions to nine differential M. graminicola isolates (gift from James Brown / Gert Kema)

Resistance of Triticum monococcum to Mycosphaerellagraminicola, the wheat Septoria tritici blotch pathogen

Resistance to M. graminicola

Manuscript submitted to New Phytologist Dec 06

Full title

Genetic analysis of pathogen resistance in T. monococcum: Developing Tm mapping populations

** 72 F3 lines were screened for segregation of resistance / susceptibility to Septoria tritici blotch

Accession Septoria Fusarium Eyespot SBCMV Polymyxa

R S

SS

Yellow rust

MDR308 R ND S R

MDR002 S* S R R

Female Male F1 F2 F3

DV92(MDR308) MDR002 18 400 100**

* growth room only

Generation of an SSR marker map for Tm: Results before June 06

128 SSR bread wheat A genome markers using conventional primers / low-melting agarose gel system

46 SSR markers tested using the LICOR systemIn total,130 amplified good products80% give polymorphic bands between accessions Some markers gave up to 6 alleles

Six Tm accessions tested with

Follow-up23 SSR markers used to examine the genetic diversity

of 107 einkorn wheat

Tm SSR markers: Transferability

Chr 1A 2A 3A 4A 5A 6A 7A Total

SSRs 39 43 34 32 39 28 41 256

Wheat A genome SSR markers

Results so far

Total Monomorphicmarkers

No amplification

Not tested yet

Polymorphic Markers*

256 41 85 22 108100 16.0 33.2 8.6 42.2

* Polymorphic between DV92 and MDR002

Tm SSR markers: F3 genotyping

F3 individuals P1 P2

P1: MDR002 P2: MDR308

Develop 1st SSR marker map (Based on 65 SSRs)

1Am 3Am 4Am 5Am 7Am

6Am

2Am

Septoria resistance Screening the F3 MDR002 x MDR 308 population

The 2nd leaf of 21-d-old F3 seedlings inoculated with IPO323 (St8) or IPO89011(St3) and 5 bioassays conducted

• In planta /attached assay (IPO323 & IPO89011)

• Detached assay (IPO323 & IPO89011)

• Attached /detached assay (IPO89011)

•72 F3 lines in each assay

Screening the F3 MDR002 x MDR 308 population

In planta /attached assay

2nd leaves of 21-d seedlings inoculated with IPO323 or IPO89011

Keep with lid-off for another 17 days

Sporulation observation/spore amount counting

Keep the whole plants in a humid box for 3-d under light

Growth with lid-on for another 3-d

Screening the F3 MDR002 x MDR 308 population

In planta /attached assay

MDR002 MDR308

A sporulation-controlling locus on 7Am

Key point- Stops the sporulation of two differential Mg isolates- Stb mediated resistance is located on otherchromosomes in hexaploid wheat

2007: Further characterisation of M. graminicola resistance in T. monococcum

Fine mapping using further markers• SSR markers• EST markers

A consensus molecular marker map (2007)• Test more SSR markers• DArT markers• COS markers

T. monococcum (AmAm)T. turgidum (AABB) X

Triploid (AmAB) /Amphiploid (AmAmAABB)

(male)

GA spray

Embryo rescueMature seeds

Embryo rescueMature seeds

GA spray

Hybrids

BC1F1 seeds

T. aestivum (AABBDD)(female)

XT. monococcum (AmAm)(male)

X

T. aestivum (AABBDD)(female)

X

Utilisation of useful traits: Introgression

1 2

Bread wheat Hybrids T. monococcum

Seed-set

Embryo-rescue

Tm Tm F1 TaF1 Ta

Trait introgression

F1 hybrid responses to Mg infection

M308 F1 M308 x Riband Riband

A big issue for trait introgression

Necrosis is restricted to reproduction organs in F1 hybrids

T. monococcum (AmAm)(PI355520) containing

two genes confer female fertility

XXT. aestivum

(AABBDD)

X Hybrids

T. monococcum (AmAm)

An unique T. monococcum accession

BC1F1

Cox et al., (1991) Plant Breeding 107: 105-118.

T. aestivum(AABBDD)

Hairy Black awn

Genetics:Associations between traits and SSR markers

Genetics: Trait and marker associationsGrain texture associated with Chr region containing hardness locus

Peter Shewry/Paola Tosai/Mark Wilkinson

‘Hard’

‘Soft’

*

** Mapping population

Genetics : Trait and marker associations

23 SSR markers and 30 T. monococcum accessions

TILLING: Mutagenised populations

Natural accessions: 263

EMS populations: 1,500 M3 lines of MDR050 (Hungary for M4lines 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)

Ion-beam treated diploid and hexaploid wheat

Dosage Total sown Ungerminated Germinated Germination percentage

2x1016 N+/cm2 1268 34 1234 97.35x1016 N+/cm2 1164 45 1119 96.18x1016 N+/cm2 1172 100 1072 91.5Total 3604 3425

Wu and Yu, (2001) Radiobiological effects of a low-energy ion beam on wheat. Radiation and Environmental Biophysics 40, 53-57

Dosage Total sown Ungerminated Germinated Germination percentage

2x1016 N+/cm2 961 417 544 56.65x1016 N+/cm2 1206 510 696 57.78x1016 N+/cm2 1259 761 498 39.6Total 3426 1733

DV92 (T. monococcum)

Cadenza (T. aestivum)

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

RAR1/SGT1 mediated resistance

Shirasu et al., 1999) Cell, 99:355-366

Austin et al., (2002) Science, 295:2077-2080

Ler-0 rar1-10 sgt1b

Powdery mildew (Blumeria graminis f.sp. hordei)

Arabidopsis downy mildew (Peronospora parasitica)

R gene

Barley Mla6

ArabidopsisRPP5

•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 Posadadposada@uvigo.es

Bread wheat complexity

AABBDDHexaploid (2n=42, ~17,000Mb)

TaRAR1A

Homoeologous genes

TaRAR1B TaRAR1D

Resistance phenotypes

60% 40%0%

A B D

2007 TILLING work plan

• Field phenotyping of EMS populations(Hungary 2007)

• Identify knock-out and variant alleles of RAR1 and functional tests

• Population mutation frequency (AFLP)

• Select and screen new gene targets

AcknowledgementsRRes (PPI)Hai-Chun JingKim Hammond-KosackKostya KanyukaJason RuddRichard GutteridgeDarren LovellKim OldhamAlison FergusonGrégoire GerinHelen ChapmanJean Devonshire(Bioimaging Centre)

www.WGIN.org.uk

JIC/Sainsbury LabSimon OrfordRobert KoebnerLesley BoydJohn SnapeKen Shirasu (RIKEN)

The Vavilov InstituteDmitry KornyukhinOlga Mitrofanova

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

RRes (CPI)Katie TearallCarlos BayonAndy PhillipsAngela DohertyHuw JonesRowan MitchellPaola TosaiPeter Shewry

Ukraine ScientistAnastasiya Zlatska

STB loci in bread wheat

St3(IPO89011)

St8(IPO323)

Isolate RR No. OriginIPO87019 ST01 Uruguay

IPO88004 ST02 Ethiopia

IPO89011 ST03 Netherlands

IPO94269 ST04 Netherlands

IPO92006 ST05 Portugal

IPO001 ST06 Netherlands

IPO90012 ST07 Mexico

IPO323*** ST08 Netherlands

IPO95052 ST09 Triticum durum

Differential M. graminicola strains

Isolates vesus STBs

RRes Code Isolate Mating

type Origin Resistant bread cultivars Effective

STB gene(s) References

St1 IPO87019 MAT1-1 Uruguay TE 911, Catbird, Kavkaz-K4500 L.6.A.4

STB7, STB12

(McCartney et al., 2003; Chartrain et al., 2005c)

St2 IPO88004 MAT1-1 Ethiopia Senat, Israel 493, Veranopolis, Chaucer, Olaf, Riband, Longbow, Baldus

STB2, STB3 STB15

(Adhikari et al., 2004a) (Arraiano et al., 2007)

St3 IPO89011 MAT1-2 Netherlands Tonic, Courtot STB9 (Chartrain, 2004) St4 IPO94269 MAT1-2 Netherlands Gene, Mentana, Frontana STB5,

STB10 (Arraiano et al., 2001a; Chartrain et al., 2005a)

St5 IPO92006 MAT1-2 Portugal Chaucer ? (Chartrain et al., 2004) St6 IPO001 MAT1-1 Netherlands Equinox, Reaper ? (Chartrain et al., 2004) St7 IPO90012 MAT1-1 Mexico Olaf, TE 911, Veranopolis,

Israel 493 STB11 (Chartrain et al., 2005c)

St8 IPO323 MAT1-1 Netherlands Numerous cultivars STB6 (Brading et al., 2002; Chartrain et al., 2005b)

St9 IPO95052 MAT1-2 Durum wheat

hexaploid wheat ? ?

Screening the F3 MDR002 x MDR 308 population

Detached assay

M308 M002 Riband M308 M002 RibandControl (No spray) IPO323

Detach the 2nd leaves of 21-d seedlings

Keep growth under light and high humidity for another 17 days

Sporulation observation/ spore amount counting

Screening the F3 MDR002 x MDR 308 population

Spray inoculated with IPO323 or IPO89011

Detached assay

MDR002 MDR308

Screening the F3 MDR002 x MDR 308 population

Keep attached with lid off until 14 dpi

Detachment and incubate on damp paper

Sporulation observation/ spore amount counting

2nd leaves of 21-d seedlings inoculated with IPO323 or IPO89011

Keep the whole plants in a humid box for 3-d under light

Screening the F3 MDR002 x MDR 308 population

Attached/detached assay

The use of ph1 mutant?

•Recombination between 1Am and 1A, 3Am and 3A, and 5Am and 5A increased in ph1 background

•Cross T. monococcum to ph1b mutant in Chinese Spring background and then to elite wheat variety

• Cross T. monococcum with amphiploid between Aegilops speltoides and durum wheat and bread wheat

Update on germplasm and marker development at JIC

WGIN management group meeting 21st February 2007Simon Griffiths

WGIN at JIC• Avalon X Cadenza mapping population

• Watkins germplasm collection

• Paragon mutants EMS/gamma

• Markers -COS development- Map with existing (SSRs) and emerging (DaRT)

INTEGRATION

Avalon x Cadenza Mapping Population (202 DH)

• Avalon• Parents: Maris Ploughman x

Bilbo • -Winter wheat• Pinb-D1b allele present• 5B-7B translocation• 1/6+8/2+12 HMW proteins• Rht-D1 dwarf

• Cadenza• Parents: Axona x Tonic• Spring type wheat• Pinb-D1c allele present• Normal 5B, 7B• N/14+15/5+10 HMW

proteins• no dwarfing genes

Routine phenotyping• Yield• TGW• Specimen ears• Ear emergence• Height• Lodging• NIR

Data now on www.wgin.org.uk

Mutated Paragon Population

• EMS chemically induced mutations 1% for 16hrs – point mutations.

• Single Seed Descent (SSD) to M5 generation. Each generation bagged.

• 7000 M3 plants – 6500 M6.

Paragon Mutant Development

Single Seed Descent (SSD) under glass

EMS Paragon Field Trials

• M5 seed drilled as 1m rows in field 2006• Phenotypic notes and photographs taken• Seed harvested to give stocks for future work• Specimen ear maintained• M6 seed available and M3 DNA. Check via

database www.wgin.org.uk

Paragon EMS examples

Stature

Seedshape

Maturity

Data now on ww.wgin.org.uk

Gamma Paragon Mutants

• Deletions

• Irradiated at IAEA Austria 25-250 Grays

• Developed to M3 generation

• Populations used for Ppd and Ph1

A. E. Watkins Collection

• Collection in the 1930s through the London Board of Trade

• Currently 814 accessions from around the world

• 32 Countries

Origins of Watkins Collection

• Africa: Algeria, Canary Islands, Egypt, Ethiopia, Morocco, Tunisia, • Asia and Middle East: Afghanistan, Burma, China, India, Iran, Iraq, Palestine, Syria, Turkey,• Europe: Bulgaria, Crete, Cyprus, Finland, France, Greece, Hungary, Italy, Poland, Portugal, Romania,

Spain, UK, USSR, Yugoslavia, • Australia and Brazil

Data now on www.wgin.org.uk

Single Strand Conformation Polymorphism is now a high throughput technique

Conventional SSCP ran onnon-denaturing acrylamide

ABI3730 capillary electrophoresis(modified polymer)

Data now on www.wgin.org.uk

AXCReference map

SSR

SSR

SSR

SSR

DART

DART

GENE

GENE

GENE

Sequenced genomesBreeders map

SSRSSR

SSRSSR

SSR

SSR

SSR

SSR

SSR

DART

DARTDARTDART

DART

DART

DART

BSA/DART Mapped mutants

INTEGRATION

Update on germplasm and marker development at JIC

WGIN management group meeting 21st February 2007Simon Griffiths

WGIN Programme:Mutagenesis and TILLING

in wheat

AIMS of WGIN TILLING PROGRAMME

• Establish the TILLING technique in different wheat species, including bread wheat and T. monococcum.

• Develop high-density mutagenised population of bread and diploid wheat, including isolation of genomic DNA from all lines.

• Identify novel mutations in all homoeologues of a candidate gene (GA20ox1) as proof of concept.

• Demonstrate feasibility of isolating and crossing mutations in each homoeologue to yield a phenotype.

• Identify and develop further target genes.

• Establish a UK TILLING service in wheat.

ANTICIPATED ISSUES WITH TILLING IN WHEAT

• Bread wheat, Triticum aestivum is allohexaploid: A, B & D genomes from T. urartu, Aegilops speltoides (?) and A. tauschii, leading to high level of genetic redundancy –single mutations unlikely to yield phenotypes.

• Homoeologous sequences are closely related, especially in coding regions (99-99.5% identity).

• Incomplete sequence data, mainly from ESTs – genes expressed at low levels not represented.

• Large genome (~12 x 109 bp), 100x that of Arabidopsis and very GC-rich (60-80%) – difficult PCR conditions.

CONSTRUCTION OF MUTAGENISEDBREAD WHEAT POPULATIONS

12x8 array ofCadenza M2 lines

1. Assessed lethal dose of EMS (test 0.3%, 0.6%, 1.2%, 2.4%).

2. Used 0.6% + 0.9% (~30% non-germinating seeds) on ~8000 dry seeds 16h.

3. Sowed seeds and grew to maturity. Collected 1 M1 ear per plant.

4. Sowed 1 M2 seed from each ear

5. Harvested young leaf tissue for genomic DNA

6. Estimated DNA concentrations & archived.

7. Harvested M3 seed & archived.

Species

T. monococcum (2n: A)a

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

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

M1

6,000

4,500

-

M2

3,000*

4,600

4,500

a also >300 natural accessions for EcoTILLING* Generated from two accessions (One pop’n donated by Kay Denyer, JIC)** Under EU programme “Optiwheat”

CURRENT STATE OF MUTAGENISED POPULATIONS

Totals

9,000

9,100

4,500

• Field phenotyping under HEALTHGRAIN (Peter Shewry & Zoltan Bedö)

• Single M2 ear of each line sent to Agricultural Research Institute of the Hungarian Academy of Sciences, Martonvasar, Hungary

• Sown as headrows in March 2006

• Plants (M3) field-phenotyped and seed bulked

• All lines also to be fixed by SSD in Hungary

• T. monococcum population to be field-grown this year

CHARACTERISATION OF MUTAGENISED POPULATIONS

FIELD CHARACTERISATION OF MUTAGENISED CADENZA

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

ION-BEAM MUTAGENESIS OF CADENZA

Dosage Total sown Ungerminated Germinated % Germination

2x1016 N+/cm2

5x1016 N+/cm2

8x1016 N+/cm2

Total

1268116411723604

3445100

1234111910723425

97.396.191.5

Dosage Total sown Ungerminated Germinated % Germination

2x1016 N+/cm2

5x1016 N+/cm2

8x1016 N+/cm2

Total

961120612593426

417 5446964981733

56.657.739.6

510761

T. monococcum (DV92)

Bread wheat (Cadenza)

Seeds treated with low-energy ion-beam in China, March 2006

Beckman robot arraying DNA samples for TILLING.

TILLING IN THE BREAD WHEAT EMS POPULATION

WildtypeMutant

PCR

Melt, anneal

CEL1 digest

TILLING IN THE BREAD WHEAT EMS POPULATION

TILLING IN THE BREAD WHEAT EMS POPULATION

• This example is 96 M2 lines, pooled 2-fold

• Target is 1.3kbp of GA20ox1A gene

• Average hit frequency 1:20 per kbp(two targets, 500 lines each)

• Corresponds to ~200 mutations per kbp across the 4500 individuals in the M2 population.

• Implies that every fifth base-pair of coding region is mutated somewhere in our population!

MUTATIONS IN TARGET GENES

G→A

Mutations identified:

GA20ox1A 30GA20ox1D 24 } Growing up for crossing

(candidate gene for HFN)

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 ~18,000 Greene et al. 2003

Barley Diploid 1 ~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 that 5% of mutations in coding region result in a truncation.

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

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

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

HIGH-RES MELT ANALYSIS FOR MUTATION DETECTION

Tested using plasmid-borne mutations in

GA20ox1A gene

1.3 kbp PCR target

Mutation A

Mutation B

Wild type

HIGH-RES MELT ANALYSIS FOR MUTATION DETECTION

Detection of mutations in RAR1 in T. monococcum (Jing H-C)

NEXT SIX MONTHS

• Identify new alleles of GA20ox1B

• Sequence all alleles to identify KO/KD mutations

• Cross AxD (most highly-expressed homoeologues)

• Develop further targets

• Develop Lightscanner as platform for high-resmelting for mutation discovery (BBSRC proposal submitted Dec 06)

ACKNOWLEDGEMENTS

Rothamsted: Katie Tearall

Carlos Bayon

Hai-Chun Jing

Kim Hammond-Kosack

Martin Parry

Pippa Madgwick

Peter Shewry

Thanks to:

Martonvasar: Zoltan Bedö

Mariann Rakszegi