INRA Clermont-Ferrand, France Genetics, Diversity & Ecophysiology of

Cereals

The wheat genome sequence: a foundation

for accelerating improvement of bread

wheat

Catherine Feuillet

ACTTGTGCATAGCATGCAATGCCATATATAGCAGTCTGCTAAGTCTATAGCAGACCCTCAACGTGGATCATCCGTAGCTAGCCATGACATTGATCCTGATTTACACCATGTACTATCGAGAGCAGTACTACCATGTTACGATCAAAGCCGTTACGATAGCATGAACTTGTGCATAGCATGCAATGCCATATATAGCAGTCTGCTAAGTCTATAGCAGACCCTCAACGTGGATCATCCGTAGCTAGCCATGACATTGATCCTGATTTACACCATGTACTATCGAGAGCAGTACTACCATGTTACGATCAAAGCCGTTACGATAGCATGAACTTGTGCATAGCATGCAATGCCATATATAGCAGTCTGCTAAGTCTATAGCAGACCCTCAACGTGGATCATCCGTAGCTAGCCATGACATTGATCCTGATTTACACCATGTACTATCGAGAGCAGTACTACCATGTTACGATCAAAGCCGTTACGATAGCATGAACTTGTGCATAGCATGCAATGCCATATATAGCAGTCTGCTAAGTCTATAGCAGACCCTCAACGTGGATCATCCGTAGCTAGCCATGACATTGATCCTGATTTACACCA

BGRI 2012 Technical Workshop September 1-4, Beijing

 

Gene and QTL

mapping

Map-based cloning

Candidate genes

Perfect markers

Allele mining

The future is in an integrated toolbox Tr

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Improved wheat varieties

Genetics and genomics resources (early 2000)

1. Genetic mapping • Molecular markers: low throughput RFLP and SSR

ü  Wheat : 1634 RFLPs/2946 SSRs ü  ESTs : > 1 million of wheat ESTs /

• Mapping populations: A few references with low marker coverage, numerous biparental populations of small sizes (100), a few specific high resolution F2/RILs for map-based cloning projects

Map-based cloning laborious and inefficient

3. Genome sequence: None but…. NGS sequencing revolution opened perspectives

Marker assisted selection not broadly deployed, not cost efficient for most breeders

2. Physical mapping • BAC libraries

ü  Wheat : 1x ABD-genome (CS), 1x AB-genome, 1x A-genome, 1x D genome, Chromosome specific libraries: 3B, 1-4-6D

• Physical maps: None (D genome Ae. tauschii, IWGSC chromosome based roadmap)

Funding or Scientific Contributors

64  members,  22  countries   www.wheatgenome.org  

Launched  in  2005  on  the  ini0a0ve  of  Kansas  Growers    

Sequencing Consortium

23  Sponsors  

~  500  members  

     40  countries  

An integrated and ordered wheat genome sequence

Phenotyping Genetic mapping Physical mapping Sequencing

The Breadwheat genome is……

1.  Big: 17Gb (5 x human genome, 40 x rice…)

2.  Polyploid: 2n= 42 = 6x

T. urartu

Ae. speltoides (?)

Ae. tauschii

3. Full of TEs (>90%)

T. aestivum

T. turgidum

1 MYA

8-10KYA

§ Chromosomes: 605 - 995 Mbp (3.6 – 5.9% of the genome)

Dissection of the genome to single chromosomes (arms) representing

individual (sub)genomes Triticum aestivum (2n = 6x = 42) 1C ~ 17,000 Mbp

AA BB

DD

§ Chromosome arms: 225 - 585 Mbp (1.3 – 3.4% of the genome)

D

B

;

A

Doležel et al., Chromosome Res. 15: 51, 2007

Sheath fluid

Deflection plates

Excitation light

Waste

Laser

Scattered light

Fluorescence emission

Flow chamber

A chromosome-based approach

•  Chromosome specific BAC libraries (End 2012) •  Amplified DNA for chromosome survey (June 2012) IEB

Combined strategies to establish a wheat reference genome sequence

Physical mapping of individual chromosomes

MTP sequencing

A reference sequence anchored to the genetic and phenotypic maps

Gene catalog Virtual order

Markers Short term

Survey sequencing of individual chromosomes

Anchored and ordered sequence Intergenic regions

Markers Long term

Physical mapping of individual chromosomes

An international effort

T.  aes/vum  cv    Chinese  Spring  

2A   3A   4A   5A  1A   6A   7A  

2B  1B   3B   4B   5B   6B   7B  

1D   2D   4D   5D   6D   7D  3D  

Physical map of the 1Gb chromosome 3B chr 3B 1000 Mb

1 283 contigs (average size = 749 kb) with FPC

961 Mb coverage (97% chromosome)

ü 4367 molecular markers (SSRs, ISBPs,unigenes…)

ü  Minimal Tiling Path (8448 clones)

h8p://urgi.versailles.inra.fr/projects/Tri0cum/index.php  

Paux  et  al,  Science  2008;  Rustenholz  et  al,  Plant  Physiol  2011  

http://urgi.versailles.inra.fr/cgi-bin/gbrowse/wheat_FPC_pub/

Combined strategies to establish a wheat reference genome sequence

Physical mapping of individual chromosomes

MTP sequencing

A reference sequence anchored to the genetic and phenotypic maps

Gene catalog Virtual order

Markers Short term

Survey sequencing of individual chromosomes

Anchored and ordered sequence Intergenic regions

Markers Long term

Physical mapping of individual chromosomes

Amplified  Sorted  DNA  

(IEB)  

~50X  Survey  sequence  of  all  

individual    chromosomes  

Assembly  of  gene  catalog  for  each  

chromosome/arm  (TGAC)    

ComparaPve  –  “Genome  Zipper”  (MIPS)    

Virtual  Gene  Order  of  the  21  Bread  Wheat  

Chromosomes  

Sequencing  Survey  IniPaPve  

Illumina reads (2*108 bp/) PE 0.5 kb Min 50 x

Chromosome Survey Sequencing

Assembly (ABySS) K-mer 71

Contigs > 200bp N50 = 2.4 kb

• 1,526 genes average per short arm • 2,460 genes average per long arm • Total: 83,977

Amplified DNA/sorted chromosomes

IEB

Map your favorite gene in silico

Ø  Anyone  can  register  to  get  a  login  and  password  through  signing  the  data  release  policy  agreement  

Ø  Click  on  a  chromosome  to  have  access  to  the  survey  sequence  with  blast  search  and  viewers  

Ø  BLAST  against  all  or  selected  surveys  Ø  Download  your  best  hit  sequences    

http://urgi.versailles.inra.fr/Species/Wheat/Sequence-Repository

An unlimited source of markers

Low  copy  fracPon:  10%  

Gene  density:  1  /  104  kb  

Candidate  genes  

RepePPve  fracPon:  90%  

ISBP  density:  1  /  5  kb  

Anonymous  markers  

Resequencing  4  European  wheat  elite  lines  

(Premio,  Renan,  Robigus  and  Xi19)  

IWGSC  chromosome  arm  survey  sequences  

X  

Paux  et  al  Plant  J  2006;  Plant    Biotech  J  2010  

ISBP-derived SNPs: 3 millions Average polymorphic ISBP density: 1 / 20 kb

Average SNP density: 1.8 SNPs / ISBP

è High density isolated anonymous SNPs

An unlimited source of markers

Intergenic region-derived SNPs: 2,1 Millions variable density

Average SNP density: 2.2 SNPs / kb

è Low density blocks of anonymous SNPs

Gene-derived SNPs: 670,000 Average gene density: 1 / 104 kb

Average SNP density: 2.9 SNPs / gene

è Low density blocks of "candidate" SNPs

And integration of 12’175 ESTs, 1181 DArTs, 38’905 GBS and 7000 gene SNPs from the 9K infinium array

Combined strategies to establish a wheat reference genome sequence

Physical mapping of individual chromosomes

MTP sequencing

A reference sequence anchored to the genetic and phenotypic maps

Gene catalog Virtual order

Markers Short term

Survey sequencing of individual chromosomes

Anchored and ordered sequence Intergenic regions

Markers Long term

Chr 3B physical map 1282 BAC-contigs 8448 BACs

922 pools

Pool of 10 BACs (Roche 454 GSFLX Titanium, 8 Kb MP)

Sorted chr. 3B

(2*108 bp) PE 0.5 kb

Illumina (82X)

Sanger

(2*600 bp)

ü Annotation (TriAnnot) ü Anchoring/orientation (ISBP SNPs) ü Resequencing and polymorphisms analyses ü Transcription map (15 RNASeq)

3B SEQuencing Project (1Gb)

454 scaffolds

Illumina contigs

Super-scaffolds

BAC pool

BAC-ends

3B sequence automated annotation

Leroy et al, Frontiers in Plant Science 2012)

7975  non  redundant    genes  with  expression  profiles  

Assembly v2

•   5109  scaffolds  •   995  Mb • N50  =  463  Kb  (Max  1,6  Mb)

RNASeq data from 15 samples

An integrated and ordered wheat genome sequence

Phenotyping Genetic mapping Physical mapping Sequencing

An integrated and ordered wheat genome sequence

SSR RFLP ISBP SNP SFP DAr

T GBS STS AFLP

Others Tot.

Nb 348 99 88 373 114 790 108 30 4 19 1973

Seq 293 40 88 373 114 96 108 0 0 0 1112

Ø Integration of all known markers into the ordered sequence

Ø  3B  consensus  map  (coll  with  wheat  community)  •  Cs  x  Re  as  reference  map  (335  markers)  •  10  addiDonal  maps  (>200  populaDons)  •  1973  markers  (1112  with  sequence  info)  •  metaQTL  analysis  underway  

•  40 genes and QTL mapped on 3B....

Ø  13 map-based cloning projects underway using 3B resources ü  Disease resistance genes (Sr, Lr, Yr, Stb…)

ü  Solid stem (saw fly)

ü  Yield

ü  Drought tolerance

ü  Boron transporter

ü  Flowering time

ü  NUE

ü  Chromosome pairing…

3B physical map and sequence utilization

-> 343 scaffolds accounting for 29 Mb targeting 74 BAC-contigs sequences provided to collaborators

Map-based cloning

E

C

C

D

D

E D

E

YFG

A   B  

1-2 CM

E

C

C

D

D

E YFG

7-­‐8  years  

1-­‐3  years  

A   B  

R locus: a multiple disease resistance region

Sn2

Sr2

Stb2

Yr

Stagonospora nodorum

Puccinia graminis

Septoria tritici

Puccinia striiformis

Fhb1 Fusarium graminearum

R locus 20 Mb

3B

Sv2 Puccinia triticina

Leaf  rust,  incited  by  the  biotrophic  fungus  

Puccinia  tri/cina,  is  one  of  the  most  important  

diseases  of  wheat  worldwide,  causing  annual  

yield  losses  of  about  5-­‐10%  in  ArgenDna  

Some  South  American  varieDes  as  La  prevision    13,  Pergamino  Gaboto,  Sinvalocho  MA,  Buck  

MananDal,  Buck  Poncho  and  El  Gaucho  FA,  among  others,  showed  durable  resistance  

In   Sinvalocho,   the   seedling   resistance   Lr3   in   6BL   and   two   adult   plant   resistance   genes,  

LrSV1  in  2DS  and  LrSV2  in  3BS,  were  idenDfied  

LrSV2:  dominant    

 race-­‐specific  

   Adult  Plant  Resistance  (APR)  

   subtelomeric  3BS  

María  José  Diéguez    

Map-based cloning of LrSv2

•  2 physical contigs (ctg 11 and 344 of >1 Mb ) identified with markers flanking SV2

•  48 new markers developped and tested on parents and populations

•  15 new markers at the SV2 locus

•  a high resolution genetic map Sinvalocho x G6 (1308 F2s = 2616 gametes)

•  Crossover detection Sinvalocho x G6 (3403 F2s = 6806 gametes)

swm13

wmm1104cfp1410

cfb5008cfb5021cfb5018FMOcfb5006cfb5009cfb5023nw1821gpw7080SCAR 40/42cfb5000cfb5007cfb5010cfb5025cfb5026cfb5015cfb5011cfb5013cfp5222cfb5019cfb5061ger9Sr2-CAPSD10F-C5csSr2RKCoAcfb5014cfb5060

cfp41cfp37

cfp5231cfp5243

gwm533

3BS physical (Kb)cfb3417

swm13

cfb5010SV2wmm1104-cfp41cfp37

gwm533stm559stm560

3BS genetic (cM)swm13

wmm1104*cfp1410*

cfb5008*cfb5021*cfb5018*FMO*cfb5006*cfb5009*cfb5023*nw1821*gpw7080*SCAR 40/42*cfb5000*cfb5007*cfb5010*cfb5025*cfb5026*cfb5015*cfb5011*cfb5013*cfp5222*cfb5019*cfb5061*ger9*Sr2-CAPS*D10F-C5*csSr2RK*CoA*cfb5014*cfb5060*

cfp41*cfp37

cfp5231cfp5243

gwm533

crossovers0

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0.26 cM

0.88 cM

0.22 cM

0.04 cM

www.wheatgenome.org  

IWGSC MTP sequencing

Genotyping

Sequencing

Phenotyping

Polygenic traits

Monogenic traits

Some challenges remain…..

Catherine Feuillet

Patrick Wincker

Etienne Paux

Lise Pingault

Acknowledgments

Josquin Daron

Adriana Alberti

Julie Poulain Hadi Quenesville

Michael Alaux

Nicolas Guilhot

Pierre Sourdille

Frédéric Choulet

Philippe Leroy Arnaud Couloux

Delphine Boyer

Sébastien Theil

Institute of Experimental Botany Jaroslav Dolezel Hana Simkova Jan Bartos Jan Safar

J. Rogers M. Caccamo J. Wright

Natasha Glover

Valérie Barbe

K. Mayer M. Martis

K. Eversole (Eversole Associates)

María José Diéguez Nanda Pergolesi