The IWGSC: Strategies & Activities to Sequence

the Bread Wheat Genome

Kellye A. EversoleIWGSC Executive Director

&The IWGSC

Wheat Breeding 2014: Tools, Targets, & ProgressHarpenden, United Kingdom

29 January 2014

Launched in 2005 on the initiative of Kansas Growers Executive directorK. Eversole

SponsorsCoordinating CommitteeGeneral members

Co-chairsR. AppelsJ. DvorakC. Feuillet

B. GillB. Keller

Y. Ogihara

The International Wheat Genome Sequencing Consortium

Why

• Lay a foundation to accelerate wheat improvement• Increase profitability throughout the industry

Vision

• High quality annotated genome sequence, comparable to rice genome sequence

• Physical map-based, integrated and ordered sequence

Wheat Improvement is Complex

Yield potential and yield stability

Quality of grain and co-products

Adaptation to climate change

Durable resistance to biotic stress

Gene and QTL

mapping

Map-based cloning

Candidate genes

Perfect markers

Allele mining

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Improved wheat varieties

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

Dissection of the genome into single chromosomes (arms)

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

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Managing the 17 Gb, Hexaploid Genome

Chromosome genomics Chromosome specific BAC libraries and sequencing IEB

Faster and cheaper methods to tackle the wheat genome

Wetterstrand KA. DNA Sequencing Costs: Data from the NHGRI www.genome.gov/sequencingcosts.

http://flxlexblog.wordpress.com/2012/12/03/developments-in-next-generation-sequencing-a-visualisation/

A sequence for whom & for what purposes

Feuillet et al, TIPS, 2010

Genes

TEs

500 kb

BAC by BAC (physical map)

BAC by BAC vs WGS

Whole genome shotgun

√

1. BAC library construction

3. Contig assembly by FPC/LTC

4. MTP sequencing /Scaffold assembly

2. BAC fingerprinting (HICF/WGP)

GAATTCTTGGTCAGCAATAAGGACAAAGAAGAATTCCTCCTGTGCGGCCGTTTTAGAATTCATGCTCCAGCCAACTCATCCAAAAGAATTCAACAATATAAATATCACCTAAAGCGAATTCCATCAGTATGGAGTTTCTTCATGCGAATTCAAGCTTGAGTATTCTATAGTGTCAGAATTCAATATGTAGAAATTATAGGATGGCGAATTCACCGGAAGGGGTTCCGGGTGTTTCGAATTCCGGAACGCTTCCGGAGACCAAACAGAATTCTGGAATCACTTGATGATGAATCAGGAATTCTGGAGTCTTCCTTTCCGCAGCGAAGAATTCAACGAAATAGTTATGAAATAGAAGGAATTCTGGGGTAACGATGGAATCCTACACGAATTCTGTCAACGACACTATCATCAGGAAGAATTCTTCGATGCACTGAGTCGCACGGTTGAATTCTTGTGTGAGGTCCGCGTCCTAGAT

5. Pseudomolecule construction (meiotic/LD/RH mapping)

6. Automated and curated annotation

SNP1

SNP2

SNP3

SNP4

SNP5

SNP6

SNP7

SNP8

SNP9

SNP10

IWGSC Strategy for a Reference Wheat Genome Sequence

MTP sequencing

A reference sequence anchored to the genetic and phenotypic maps

Long term

Physical mapping of individual chromosomes

Short term

Gene catalogVirtual order

Markers

Survey sequencing of individual chromosomes

Roadmap to the Wheat Genome Sequence

Physical maps

Illumina Survey

sequences

Pseudo-molecules

Current status of individual projects

IWGSC Chromosome Survey Sequencing Initiative

Amplified DNA- sorted

individual chromosomes

~50X Illumina

sequence

>30X Illumina

sequence

Chromosome arm sequence

assemblies

Gene modeling, virtual ordering

(GZ), annotation, functional, structural analyses

Composition and Evolution

of the 21 Bread Wheat

ChromosomesA, S, D, &

AB genomes (7)

Read/assembly alignment to chromosome

arms

SummaryIWGSC Chromosome Survey Sequencing - Summary

• Almost full wheat gene complement identified and allocated to chromosome arms

• On average, 53% of genes virtually ordered along chromosomes

• High level of inter- and intrachromosomal duplication

• Over 3.5 M markers mapped to contigs (1.3M wheat markers + 2.3M SNPs) - SSR, EST, DArT, SNP (90k) markers...

• 13.2 million SNPs from POPSeq aligned to contigs

• New phylogenetic analyses of wheat genome evolution

• Definition of core and pan gene sets for Triticeae by comparing di-tetra- and hexaploid genomes

• Homoeologue-specific gene expression analyses• Wheat haplotype map – E. Akhunov • TILLING projects in T. durum and bread wheat –

UC, Davis, JIC, Rres• Establishment of WheatNet – UC, Davis

IWGSC CSS Resources are Facilitating

Chr 3B physical map 1,282 BAC-contigs8,441 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)

3B pseudomolecule assembly and analyses

3B SEQuencing Project

454 scaffolds

Illumina contigs

Super-scaffolds

BAC pool

BAC-ends

(~35X)

Catherine Feuillet & Fred Choulet

An integrated and ordered 3B reference sequence MetaQTL analysis 3B consensus map (5000 markers) 3B Physical map 3B pseudomolecule

Map-based cloning projects on 3B

Disease resistance genes (Sr, Lr, Yr, Stb…) Solid stem (saw fly) Yield Drought tolerance Boron transporter Flowering time NUE Chromosome pairing…

40 genes and QTL mapped on 3B 4722 markers on 3B consensus map, 3102 in 964 SC (679 Mb =82 % of the

sequence)

13 map-based cloning projects

A Breeder’s PerspectiveCurtis Pozniak

1 year-1 EST marker, not closer- ascertainment bias

Xgwm114XBE200774XPSP3001SSt1Xgpw4513Xgwm340Xgwm247Xgwm181

SSt13BL

3 weeks-12 cosegregating markers (SNPs)- no phenotyping in the fields- No ascertainment bias

gwm1140.09K_0808_ck3.2PSP30016.5EI_02_115946EI_01_1458347.2

EI_05_466579.3EK_02-210770EK_02-276963EK_03-83684EK_03-59414SSt1EK_08-5169EK_02-239361EK_02-21792EK_03-83026BF200774

10.7

gwm34014.1gpw451315.1gwm24716.2Xgwm18121.5

SSt1

Xgwm114Xfba217XBE200774ctg580ctg854Xgwm4703ctg668Xwmc274ctg165Xgwm247Xgwm340Xtam63Xfba310Xfba133.2Xfbb293Xdarts149Xgwm181Xgwm547Xbarc68.2Xgpw4513

3B Physical

Speed Gene Discovery: Towards Cloning SSt1

A High-density map

EI_05-46657

EK_08-5169XBF200774

EK_02-239361EK_02-292495

Xgwm247

SSt1

B Fine map C 3B Pseudomolecule

4000 Kbp

EI_05-46657 10.6

EK_08-5169XBF200774

EK_02-239361 12.6

Xgwm247 16.6

EK_02-292495

SSt1

15.3Ctg1109-90254

2.8 cM

Approx. 2.2 Mb

13.3

Power of A High Quality Reference Sequence

Feuillet et al, Trends in Plant Sciences, 2010; Rey et al, unpublished update)

Number of QTLs Cloned

RiceWheat

CS WGS 5x(2012)

A and D genome related ancestors

(2013)

CS chromosome survey sequences

(2013)

CS 3BSEQ(2013)

completeness of information

94% real order

Wheat Sequences Available Now

Gold standard

Max 50% using synteny and genetic mapping (GZ): « virtual order »

WGS WCS MTP

Applications5X CS Ae.

tauschii

T. urartu

CS CSS

MTP based (3B)

« blind » SNP marker development

x x x x

Targeted SNP marker development

x x

Conversion rate in genotyping for breeding

45% 95%

Map-based cloning time (years)

10 10 10 8 2

Fundamental studies limited to genes complete

IWGSC Projects

The Annotated Reference Sequence of the Bread Wheat Genome

Pseudomolecules(1 of 21 completed)

Chromosome Shotgun Sequences and marker alignment

(completed)

Physical Maps with markers(12 of 21 completed)

IWGSC Resources in Breeding

• Unlimited source of DNA markers for MAS/Genomic Selection

• Candidate genes for traits = perfect markers

• Reduce the time and improve the success of resolving QTL

• Discovery and exploitation of new alleles

BAC Libraries

Physical Maps

Wheat Survey Sequence

3B Sequence

Curtis Pozniak

272 institutions 40 countries

http://wheat-urgi.versailles.inra.fr/Seq-Repository/• All resources accessible at URGI, Versailles

• Raw sequence reads in the SRA• IWGSC CSS assemblies available at URGI and EBI• IWGSC CSS assemblies & gene models integrated into EnsemblPlants

52,000 BLAST searches 2.500 downloads

IWGSC Resource Access

http://plants.ensembl.org/Triticum_aestivum

Thank you for your attention!

www.wheatgenome.org

@wheatgenome