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Chromosome 2Doil Choi, Sunghwan Jo KOREA
Cytological architecture of chromosome 2
540 kb/µm
DAPI (4’-6-diamidino-2-phenylindole) stained pachytene chromosome
Tomato Genome : 950Mb
Euchromatin :220Mb (23%)
Est. Euchromatin size of chro. 2: 22Mb
Est. number of BACs: 268
Number of Markers : 274(Tomato-EXPAN 13-143cM)
Strategy for Genome Sequencing of Chromosome 2
Genetic Map,Markers
Overgo Probes
BAC FilterScreening (X2)
Fluorescent in Situ
Hybridization
Markers (Chr.2)
CandidateBACs (Chr.2)
BAC Library
All BACs
BAC-endsequences
BACs (Chr.2), Seed BACs
BAC sequences
Annotation(Structural/ Functional)
Database &Web Viewer
Genome Sequencing Loop
Next BACsTo be sequenced
Chr.2Genome
Annotation
MinimumTiling Path
SGN
Gene Bank
Tomato whole-genome analysis
BAC-end Sequencing
BES BLAST
BACSequencing
IL mapping
Contig information
# of Contig
Length (bp)
# of BAC
# of Contig
Length (bp)
# of BAC
# of Contig
Length (bp)
# of BAC
1 310,779 3 11 209,692 2 21 219,521 2
2 169,963 2 12 758,423 9 22 308,572 4
3 247,111 2 13 161,420 2 23 542,186 9
4 221,251 3 14 204,125 2 24 214,790 2
5 794,181 9 15 779,963 10 25 545,269 6
6 740,415 10 16 565,192 5 26 185,475 3
7 299,712 4 17 554,484 10 27 736,341 7
8 371,033 4 18 443,805 4 28 578,941 7
9 309,991 3 19 137,539 2 29 497,138 6
10 153,492 2 20 571,155 8 30 1,143,262 14
30 contigs
Total length – 12,9Mb Average length – 432kb Average # of BAC – 5.2
8 single
Total length – 849kb Average length – 106kb
Chromosome contig information
142 cM129cM
CNR (?)
13.0 40.0 48.0 78.0 100.0 120.0 142.0
Physical Map
Markers (cM)
T15
54 (1
42.0
)
cLE
C-7
-P21
(0)
T16
16 (1
.0)
TG
276
(14.
0)
CT1
40 (1
6.0)
T17
06 (1
8.0)
CT2
13 (2
4.0)
T16
68 (3
7.0)
C2_
At2
g344
70 (3
8.5)
cLE
C-2
7-M
9 (4
6.0)
? rcr3
(?)
CT1
03 (6
3.7)
T14
38 (7
0.0)
T13
95 (7
2.0)
TM
34 (7
3.0)
T15
37 (7
4.5)
SSR
26 (7
7.5)
cLE
T-1
-A5
(79.
0)C
LER
-5-N
18 (8
6.0)
T15
35 (8
9.0)
TG
645R
(90.
0)U
1464
94 (
90.0
)T
G37
3 (9
4.0)
CT9
(101
.0)
T14
80 (1
06.0
)d
(108
.0)
T11
58 (1
11.5
)f.w
.2. 2
(1
16.0
)
U21
4677
(126
.5)
cTO
B-6
-13
(140
.0)
?
Annotated regions
13 cM
NOR: nucleolar organizing region PH: pericentromeric heterochromatin CEN: centromere TEL: telomere
Big GAP 1 Big GAP 2 Big GAP 3
Contig regions1 2 3 4 5 6 7 8 9 10 11 12 13/14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30
Contig validation : IL mapping- 131 regions, FISH – 35 BACs
Efforts for next BAC selection
Maximize marker-anchored seed BACs
- Tomato-Expen 2000- Kazusa Institute : 0 selected / total 60 SSR markers- Syngenta marker: 2 selected /total 43 markers- FISH or IL mapping (Dr. Zamir)
Chromosome contig extension
- Find candidates from BAC and cosmid-end sequence- Validate BAC overlap with BES, PCR, FISH, IL
mapping, Bridge BACs analysis- 3D pool screening (none) - Library filter screening (4 BACs with over 60kb overlap)
Status of Chromosome 2 Sequencing
Total Finished BAC clones -175 BAC clones in Phase 3 (ABI3730)
- 168 clones confirmed on chr.2
- 7 clones: not confirmed position
- 12 clones over 60kb overlap for GS FLX sequencing
Total sequence length
- 13.8 Mb non-redundancy /18.5 Mb
- 63 % done (22 Mb)
Average gap length
- 210 kb
Strategy for Filling Gap 1
Marker I Marker II
Chromosome assembly Contig 1 Contig 2
I. Contig end ExtendingI. Contig end Extending
GAP
Gene prediction Gene prediction
Chromosome assembly
BAC library screening BAC library screening
IL mappingIL mapping
Clone validation & SequencingClone validation & Sequencing
GAP
Probe Probe
Result of BAC filter screening with contig-end
Total contig -ends
to be screened
On progress Screened
probesHit BACs
52 5 10
HindIII – 16/107
EcoRI – 8/71
MboI – 3/41
BAC BES PCR (no end seq.)
BAC BES PCR (no end seq.)
H057H03-3’_CDS - 1 H136C06-5’_CDS - -
H091J18-5’_CDS 2 4 H164H08-5’_CDS 2 -
H090O01-5’_CDS - - H167J21-5’_CDS 1 -
H073P13-3’_CDS 2 2 M020N15-3’_CDS 1 1
H066C13-3’_CDS 2 1 E010H16-3’_CDS 3 3
But none of clones meet the criteria (>60 kb overlap)
Sequencing of large overlapping clones (>60 kb overlap )
To make chance to find extendable BAC GS FLX (~30X coverage) BLAST against BES for extension
Strategy for Filling Gap 2
Synteny-based Extending: Find conserved gene from orthologous Synteny-based Extending: Find conserved gene from orthologous
Grapevine
GAP
Popular
Arabidopsis
Tomato
Probes for BAC library screeningProbes for BAC library screening
IL mappingIL mapping
Clone validation & SequencingClone validation & Sequencing