triticum monococcum, tilling, genetics and pathologyion-beam treated diploid and hexaploid wheat...
TRANSCRIPT
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
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
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
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
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: Trait and marker associationsGrain texture associated with Chr region containing hardness locus
Peter Shewry/Paola Tosai/Mark Wilkinson
‘Hard’
‘Soft’
*
** Mapping population
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 [email protected]
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
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
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
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.
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
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
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
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)