The document describes the sequencing of the wheat genome, specifically chromosome 3B. Key points: 1. An international effort led by the IWGSC sequenced individual wheat chromosomes including 3B using a physical map-based approach. 2. Sequencing of the 1Gb chromosome 3B generated over 1000 scaffolds covering 995Mb with an N50 of 463kb. Genes and markers were annotated. 3. The sequenced and ordered chromosome 3B provides a foundation for accelerating wheat improvement through map-based cloning, marker development, and integrating genetic and genomic resources.

1. ACTTGTGCATAGCATGCAATGCCAT
ATATAGCAGTCTGCTAAGTCTATAG The wheat genome
CAGACCCTCAACGTGGATCATCCGT sequence: a foundation
AGCTAGCCATGACATTGATCCTGAT
TTACACCATGTACTATCGAGAGCAG
for accelerating
TACTACCATGTTACGATCAAAGCCG improvement of bread
TTACGATAGCATGAACTTGTGCATA wheat
GCATGCAATGCCATATATAGCAGTC
TGCTAAGTCTATAGCAGACCCTCAA
CGTGGATCATCCGTAGCTAGCCATG
ACATTGATCCTGATTTACACCATGT
ACTATCGAGAGCAGTACTACCATGT Catherine Feuillet
TACGATCAAAGCCGTTACGATAGCA
TGAACTTGTGCATAGCATGCAATGC INRA Clermont-Ferrand, France
CATATATAGCAGTCTGCTAAGTCTA Genetics, Diversity & Ecophysiology of
TAGCAGACCCTCAACGTGGATCATC Cereals
CGTAGCTAGCCATGACATTGATCCT
GATTTACACCATGTACTATCGAGAG
CAGTACTACCATGTTACGATCAAAG BGRI 2012 Technical Workshop
CCGTTACGATAGCATGAACTTGTGC September 1-4, Beijing
ATAGCATGCAATGCCATATATAGCA
GTCTGCTAAGTCTATAGCAGACCCT
CAACGTGGATCATCCGTAGCTAGCC
ATGACATTGATCCTGATTTACACCA

2. The future is in an integrated toolbox
-> expertise and critical mass
Gene and
QTL
Training, capacity building
mapping
Map-based
cloning
Candidate
genes
Perfect
markers
Allele mining
Improved wheat varieties

3. 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
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)
Map-based cloning laborious and inefficient
3. Genome sequence: None but…. NGS sequencing revolution
opened perspectives

4. Sequencing Consortium
Launched
in
2005
on
the
ini0a0ve
of
Kansas
Growers
23
Sponsors
Funding or
Scientific
~
500
members
Contributors
40
countries
64
members,
22
countries
www.wheatgenome.org

5. An integrated and ordered wheat genome
sequence
Phenotyping Genetic mapping Physical mapping Sequencing

6. The Breadwheat genome is……
1. Big: 17Gb (5 x human genome, 40 x rice…)
2. Polyploid: 2n= 42 = 6x
1 MYA
T. urartu
T. turgidum
Ae. speltoides (?) 8-10KYA
T. aestivum
Ae. tauschii
3. Full of TEs (>90%)

7. A chromosome-based approach
Dissection of the genome to single
chromosomes (arms) representing
AA BB Sheath fluid
individual (sub)genomes
Triticum aestivum
(2n = 6x = 42) Flow D
Laser chamber
1C ~ 17,000 Mbp Fluorescence
emission B
Excitation
DD light
A
Deflection
plates Scattered
light
;
Waste
Doležel et al., Chromosome Res. 15: 51, 2007
§ Chromosomes: 605 - 995 Mbp
(3.6 – 5.9% of the genome)
§ Chromosome arms: 225 - 585 Mbp
(1.3 – 3.4% of the genome)
• Chromosome specific BAC libraries (End 2012)
• Amplified DNA for chromosome survey (June 2012) IEB

8. Combined strategies to establish a wheat
reference genome sequence
Physical mapping of
individual chromosomes
Survey sequencing of
MTP sequencing
individual chromosomes
Gene catalog Anchored and ordered sequence
Virtual order Intergenic regions
Markers Markers
Short term Long term
A reference sequence anchored to the
genetic and phenotypic maps

9. An international effort
1A
2A
3A
4A
5A
6A
7A
1B
2B
3B
4B
5B
6B
7B
1D
2D
3D
4D
5D
6D
7D
T.
aes/vum
cv
Chinese
Spring

10. Physical map of the 1Gb chromosome 3B
chr 3B
1000 Mb
Paux
et
al,
Science
2008;
Rustenholz
et
al,
Plant
Physiol
2011
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

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

13. Combined strategies to establish a wheat
reference genome sequence
Physical mapping of
individual chromosomes
Survey sequencing of
MTP sequencing
individual chromosomes
Gene catalog Anchored and ordered sequence
Virtual order Intergenic regions
Markers Markers
Short term Long term
A reference sequence anchored to the
genetic and phenotypic maps

14. Sequencing
Survey
IniPaPve
Assembly
of
gene
catalog
ComparaPve
Virtual
Gene
Amplified
~50X
Survey
for
each
–
“Genome
Order
of
the
Sorted
DNA
sequence
of
all
chromosome Zipper”
21
Bread
individual
(IEB)
/arm
Wheat
chromosomes
(MIPS)
Chromosomes
(TGAC)

15. Chromosome Survey Sequencing
Amplified DNA/sorted chromosomes
IEB
Illumina reads (2*108 bp/) PE 0.5 kb
Min 50 x
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

16. 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

17. An unlimited source of markers
Low
copy
fracPon:
10%
RepePPve
fracPon:
90%
Gene
density:
1
/
104
kb
ISBP
density:
1
/
5
kb
Candidate
genes
Anonymous
markers
Paux
et
al
Plant
J
2006;
Plant
Biotech
J
2010
IWGSC
chromosome
X
Resequencing
4
European
arm
survey
wheat
elite
lines
(Premio,
Renan,
Robigus
and
Xi19)
sequences

18. An unlimited source of markers
Gene-derived SNPs: 670,000 ISBP-derived SNPs: 3 millions
Average gene density: 1 / 104 kb Average polymorphic ISBP density: 1 / 20 kb
Average SNP density: 2.9 SNPs / gene Average SNP density: 1.8 SNPs / ISBP
è Low density blocks of "candidate" SNPs è High density isolated anonymous SNPs
Intergenic region-derived SNPs: 2,1 Millions
variable density
Average SNP density: 2.2 SNPs / kb
è Low density blocks of anonymous SNPs
And integration of 12’175 ESTs, 1181 DArTs, 38’905 GBS
and 7000 gene SNPs from the 9K infinium array

19. Combined strategies to establish a wheat
reference genome sequence
Physical mapping of
individual chromosomes
Survey sequencing of
MTP sequencing
individual chromosomes
Gene catalog Anchored and ordered sequence
Virtual order Intergenic regions
Markers Markers
Short term Long term
A reference sequence anchored to the
genetic and phenotypic maps

20. 3B SEQuencing Project (1Gb)
Chr 3B physical map 1282 BAC-contigs Sorted chr. 3B
8448 BACs
Pool of 10 BACs
(Roche 454 GSFLX Titanium, 8 Kb MP)
922 pools
BAC pool
Illumina (82X)
454 scaffolds
Sanger Illumina contigs
BAC-ends
(2*108 bp)
(2*600 bp) PE 0.5 kb
Super-scaffolds
ü Annotation (TriAnnot)
ü Anchoring/orientation (ISBP SNPs)
ü Resequencing and polymorphisms analyses
ü Transcription map (15 RNASeq)

21. 3B sequence automated annotation
RNASeq data
from 15
Assembly v2 samples
•
5109
scaffolds
•
995
Mb
• N50
=
463
Kb
(Max
Leroy et al, Frontiers in Plant Science 2012)
1,6
Mb)
7975
non
redundant
genes
with
expression
profiles

22. An integrated and ordered wheat genome
sequence
Phenotyping Genetic mapping Physical mapping Sequencing

23. An integrated and ordered wheat genome
sequence
Ø Integration of all known markers into the ordered sequence
RFL DAr AFL Other
SSR ISBP SNP SFP GBS STS Tot.
P T P s
Nb 348 99 88 373 114 790 108 30 4 19 1973
Se
293 40 88 373 114 96 108 0 0 0 1112
q
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

24. 3B physical map and sequence utilization
• 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…
-> 343 scaffolds accounting for 29 Mb targeting 74 BAC-
contigs sequences provided to collaborators

25. Map-based cloning
1-2 CM
A
C E D B
7-­‐8
years
C D
E
E YFG D
A
C E D B
1-­‐3
years
C E YFG D

26. R locus: a multiple disease resistance region
R locus Sn2 Stagonospora nodorum
20 Mb
Sr2 Puccinia graminis
Sv2 Puccinia triticina
Stb2 Septoria tritici
Fhb1 Fusarium graminearum
Yr Puccinia striiformis
3B

27. Map-based cloning of LrSv2
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
María
José
Diéguez
LrSV2:
dominant
race-­‐specific
Adult
Plant
Resistance
(APR)
subtelomeric
3BS

28. 3BS physical (Kb) 3BS genetic (cM) cross
0 swm13 cfb3417 swm
50
• 2 physical contigs (ctg 11 and 10 0
15 0
344 of >1 Mb ) identified with 20 0
25 0
markers flanking SV2 30 0
wmm1104 wm
35 0 cfp1410 cfp1
40 0
45 0
0.88 cM
50 0
• 48 new markers developped and 55 0
cfb5008
cfb5021
cfb5
cfb5
cfb5018 cfb5
tested on parents and populations 60 0
65 0
FMO
cfb5006
FMO
cfb5
cfb5009 cfb5
70 0 cfb5023 cfb5
• 15 new markers at the SV2 75 0
80 0
nw1821
gpw7080
SCAR 40/42
nw1
gpw
SCA
locus 85 0
cfb5000
cfb5007
cfb5010 swm13
cfb5
cfb5
cfb5
90 0 cfb5025 cfb5
cfb5026 cfb5
95 0
cfb5015
0.22 cM
cfb5010 cfb5
10 00 cfb5011 SV2 cfb5
cfb5013 wmm1 104-cfp 41 cfb5
10 50 cfp5222
0.04 cM
cfp37 cfp5
• a high resolution genetic map 11 00
cfb5019
cfb5061
ger9
cfb5
cfb5
ger9
11 50
Sinvalocho x G6 (1308 F2s = 2616 12 00
Sr 2-CA PS
D10F-C5
csSr 2RK
0.26 cM
Sr2
D10
csS
gametes) 12 50
13 00
CoA
cfb5014 gwm533
stm559
CoA
cfb5
cfb5060 cfb5
stm560
13 50
• Crossover detection Sinvalocho x 14 00
G6 (3403 F2s = 6806 gametes)
14 50
15 00
cfp41 cfp4
15 50
cfp37 cfp3
16 00
16 50
17 00
cfp5231 cfp5
17 50 cfp5243 cfp5
18 00
18 50 gwm533 gwm

29. IWGSC MTP sequencing
www.wheatgenome.org

30. Some challenges remain…..
Polygenic traits
Sequencing
Genotyping Phenotyping
Monogenic traits

31. Acknowledgments
K. Eversole (Eversole Associates)
Frédéric Choulet Adriana Alberti Michael Alaux
Etienne Paux Institute of Experimental Botany
Julie Poulain Hadi Quenesville
Jaroslav Dolezel
Pierre Sourdille Hana Simkova
Arnaud Couloux
Jan Bartos
Philippe Leroy Jan Safar
Valérie Barbe
Nicolas Guilhot
Patrick Wincker J. Wright
Sébastien Theil M. Caccamo
J. Rogers
Lise Pingault
Josquin Daron K. Mayer
M. Martis
Natasha Glover
Delphine Boyer
María José Diéguez
Catherine Feuillet Nanda Pergolesi