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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
Complexity Requires an Integrated ToolboxTr
ain
ing
, ca
pac
ity
bu
ild
ing
->
exp
erti
se a
nd
cri
tica
l m
ass
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
Sheath fluid
Deflectionplates
Excitationlight
Waste
Laser
Scatteredlight
Fluorescenceemission
Flowchamber
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/
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