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