Transcript The Tomato Sequencing Project

The International Tomato
Sequencing Project: The first
Cornerstone of the SOL
Project
Lukas Mueller
on behalf of
International SOL Tomato Sequencing
Project
Overview
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Aims
Why sequence the tomato genome?
How to sequence the tomato genome?
Who is sequencing the tomato genome?
Resources for Sequencing the Tomato Genome
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Genetic Map
BAC libraries
Overgo mapping
BAC End Sequences
Minimal Tiling Path
Bioinformatics
• Summary
Mingsheng Chen
Zhukuan Cheng
Chuanyou Li
Hongqing Ling
Yongbiao Xue
Ramesh Sharma
Jiten Khurana
Akhilesh Tyagi
Doil Choi
Byung Dong Kim
Antonio Granell
Miguel A. Botella
Giovanni Giuliano
Luigi Fruciante
Steven D. Tanksley
Jim J. Giovannoni
Stephen Stack,
Joyce van Eck
Graham Seymour
Gerard Bishop
Willem Stiekema
P. Lindhout
Taco Jesse
Rene Klein Lankhorst
Daisuke Shibata
Satoshi Tabata
Mondher Bouzayen
Mathilde Causse
Aims
• Provide a high quality reference sequence for the
Solanaceae genomes
• Using mapping of other Solanaceae sequences
onto the tomato sequence, and comparative
genetic maps to derive “virtual” genomes for other
Solanaceae
• Prerequisite for studying natural diversity and
linking genotype to phenotype
• Build a Solanaceae bioinformatics platform to
integrate, analyze and distribute the information
WHY SOLANACEAE? Solanaceae is part of unique clade of
flowering plants. Genome research in Solanaceae will provide a
reference anchor and enable comparative genomics and systematic
throughout this clade
asterid I
Solanaceae Rubiaceae (coffee)
asterid II
Compositeae (sunflower, safflower, lettuce)
asterid III
asterid IV
asterid V
rosid I
Leguminosae (soybean,
Medicago
Rosaceae (apple, peach, cherry); Salicaceae (poplar)
rosid II
Malvaceae (cotton) ; Sterculiaceae (cocoa)
rosid III
Brassicaceae
caryophyllids
Chenopodiaceae (sugarbeet, spinach)
Arabidopsis
; Rutaceae (citrus)
hamamelid I
hamamelid II
ranunculids
paleoherb II
Magnoliales
monocots
Laurales
Gramineae (maize, wheat)
Liliaceae (onion)
Rice
; Musaceae (banana)
Why sequence tomato?
• Tomato is the most intensively researched
Solanaceae genome encoding approx. 35,000
genes euchromatic regions corresponding to
less than a 25% of the total DNA in the
tomato nucleus (220~250 Mb).
• Tomato provides the smallest diploid genome
for which homozygous inbreds are available.
• Its sequence will facilitate positional cloning
in tomato and other Solanaceae genomes (via
synteny maps).
How to sequence the tomato
genome?
• Whole Genome Shotgun
– Advantages: Fast, cheaper, ok with reference genome
– Disadvantages: Unordered contigs
• Methylation Filtering (Tobacco)
– Advantages: Selects for expressed genome, cheaper
– Disadvantages: unordered contigs
• Tiling Path (Arabidopsis, Drosophila, Rice)
– Advantages: Sequence and gene order; select gene rich
regions; easy to divide work
– Disadvantages: Relatively expensive, time consuming
• ORDER IMPORTANT FOR COMPARING
GENOMES
Tomato Genome Structure
• 12 chromosomes
• 950MB of total DNA
• 220MB contiguous, gene
rich euchromatin
• Sequence only gene-rich
euchromatin (>90% all
genes)
• Tiling path method
preferred
• Drosophila used and
Medicago is using
similar strategy
euchromatin
telomere
telomere
structure
pericentric
heterochromatin
centromere
162 bp subtelomeric repeat
pericentric
heterochromatin
euchromatin
7 bp telomeric
repeat
BAC libraries
• All libraries derived from Solanum lycopersicum
Heinz 1706.
• HindIII library (Rod Wing, Clemson U)
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~120,000 clones, 120kB average size
~15x coverage
FPC contigged
Overgo analysis
75,000 clones BAC end sequenced
• MboI library
– 50,000 clones, 140kb average size
– Will be BAC end sequenced
• EcoRI library (being prepared)
– Will be BAC end sequenced
F2-2000 Genetic Map
• Parents:
– Solanum lycopersicum x Solanum
pennellii
• Mapping population of 80 F2
individuals
• # Markers: 1579
• Total cM: 1453
• Density: 1 marker/0.92cM
• SGN http://sgn.cornell.edu/
Marker-Types:
rflp
ssr
tm
p-mrkr
cos
est-by-clone
unknown
caps
cosii
kfg
Total
345
149
43
39
576
265
8
21
98
35
1579
Tying the Genetic Map to the Physical
Map: Overgos
• Overgos are “overlapping oligos”, short,
very hot probes, developed from genetic
markers of the F2-2000 map
• Overgos are organized in 96 well plates,
analyses are carried out with row and
column pools
• Pools are hybridized to BAC filters, raw
pool results are deconvoluted
• A total of 1536 overgos developed (16
plates)
• Analyses of all plates is complete
Overgo Anchoring Results
Anchors:
• 652 anchor markers are involved in plausible non-conflicted
associations with BACs.
• 4857 good marker--BAC associations
FPC contigs:
• 1880 BACs in 705 plausible contigs
• 2166 BAC singletons
• 652 seed BACs ==> 1/3 of euchromatic
genome sequence
Distribution of Anchor Markers on Chromosomes
92
165
1.8
79
67
143 171
1.8 2.6
62
137
2.2
40
119
3.0
63
101
1.6
51
112
2.2
34
87
2.6
40
116
2.9
41
87
2.1
43
103
2.4
39 # anchors
120 cM chr length
3.1 cM per anchor
+ 1000 markers from Keygene AFLP map
Verification of overgo
mappings
• Fluorescence In-Situe Hybridization (FISH)
– BAC probe on pachytene chromosomes
• IL lines (Zamir lab)
– Map BACs to IL lines
– CAPS assays
(Hans de Jong)
Summary of FISH verification
• Song-Bin Cheng, Hans de Jong (Holland, chromosome 6):
– 9 BACs analyzed
– 8 mapped to chromsome 8 in right order
– 1 BAC gave signals on centromere of chromosome 1
• Sangheob Lee, Doil Choi (Korea, chromosome 2):
– 27 BACs analyzed with FISH
– 25 confirmed to specific location, same order as F2-2000 map
– 2 match to other chromosomes
• Chuanyou Li (China, chromosome 3)
– >30 BACs being analyzed
• Steven Stack (USA):
– Telomere and heterochromatic boundary determination
– FISH service for countries without FISH capability
BAC end
sequences
• Total of 400,000 reads (200,000 BACs from both ends)
selected from the 3 BAC libraries
• Batch of 75,000 BACs in process (HindIII library)
• ~45,000 BAC end sequences already obtained
(ftp://ftp.sgn.cornell.edu/tomato_genome/)
• Average read length 655bp
• Annotation in progress
• SeqWright Inc, Houston, TX
• SeqWright is sponsoring a happy hour after this session.
Obtaining the Tiling Path
A
B
“seed BAC”
anchored bacs
overgos
C
genetic map
“seed BAC”
US Korea
BACs
finished:
in process:
China
4
16 20
UK
India
NL
14
9
France Japan
5
Spain
US
US
Italy
5
Overview: sgn.cornell.edu -> About -> tomato sequencing
Building a Bioinformatics
Platform for the Solanaceae
• Project-wide standards for quality, gene naming,
annotation (http://sgn.cornell.edu/solanaceae-project/)
• Create a unified web presence for the entire project
• Develop distributed model for annotation, web
presentation, involving different centers in SOL countries
• All data and programs developed in the project are shared
in an open source format
• Integrate all data into the SOL bioinformatics platform,
facilitating a systems approach to explore diversity and
adaptation and the complex interactions that occur on all
levels of biological organization
CAS
Genome India
SGN
Agronanotech
Kazusa
VIB Ghent
Annotation Phases
1. First pass annotations of sequences and
gene models on BAC basis, available
immediately
2. BAC based, common, distributed
platform, stable BAC-based identifiers
3. Chromosome based, stable identifiers
Summary
• Sequencing of tomato is under way by a consortium
of 10 countries
• High quality, ordered sequence using BAC tiling
path
• BAC ends available, overgo results verified by
FISH analyses
• Sequence will be tied to other Solanaceae and
closely related species (coffee and beyond)
• Provide a foundation for shared biology for this
economically important clade of plants
Acknowledgments
SOL community
Tomato Sequencing Project
Funding
National Science Foundation
Other National Funding Sources
Keygene NV
Seqwright Inc. (Happy Hour)
Colleagues
Steven Tanksley, Jim Giovannoni, Joyce van Eck , Steven Stack
SGN:
Teri Solow, Beth Skwarecky, Nick Taylor, Robert Buels, John Binns, Chenwei Lin