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Chromosome 2 Doil Choi, Sunghwan Jo KOREA. Cytological architecture of chromosome 2 540 kb/µm DAPI (4’-6-diamidino-2-phenylindole) stained pachytene chromosome.

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Presentation on theme: "Chromosome 2 Doil Choi, Sunghwan Jo KOREA. Cytological architecture of chromosome 2 540 kb/µm DAPI (4’-6-diamidino-2-phenylindole) stained pachytene chromosome."— Presentation transcript:

1 Chromosome 2 Doil Choi, Sunghwan Jo KOREA

2 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)

3 Strategy for Genome Sequencing of Chromosome 2 Genetic Map, Markers Overgo Probes BAC Filter Screening (X2) Fluorescent in Situ Hybridization Markers (Chr.2) Candidate BACs (Chr.2) BAC Library All BACs BAC-end sequences BACs (Chr.2), Seed BACs BAC sequences Annotation (Structural/ Functional) Database & Web Viewer Genome Sequencing Loop Next BACs To be sequenced Chr.2 Genome Annotation Minimum Tiling Path SGN Gene Bank Tomato whole-genome analysis BAC-end Sequencing BES BLAST BAC Sequencing IL mapping

4 Contig information # of Contig Length (bp) # of BAC # of Contig Length (bp) # of BAC # of Contig Length (bp) # of BAC 1 310,779311 209,692221 219,5212 2 169,963212 758,423922 308,5724 3 247,111213 161,420223 542,1869 4 221,251314 204,125224 214,7902 5 794,181915 779,9631025 545,2696 6 740,4151016 565,192526 185,4753 7 299,712417 554,4841027 736,3417 8 371,033418 443,805428 578,9417 9 309,991319 137,539229 497,1386 10 153,492220 571,1558301,143,26214  30 contigs  Total length – 12,9Mb  Average length – 432kb  Average # of BAC – 5.2  8 single  Total length – 849kb  Average length – 106kb

5 Chromosome contig information 142 cM 129cM CNR (?) 13.0 40.048.0 78.0100.0120.0 142.0 Physical Map Markers (cM) T1554 (142.0) cLEC-7-P21 (0) T1616 (1.0) TG276 (14.0) CT140 (16.0)T1706 (18.0)CT213 (24.0) T1668 (37.0) C2_At2g34470 (38.5) cLEC-27-M9 (46.0) ? rcr3 (?) CT103 (63.7) T1438 (70.0)T1395 (72.0)TM34 (73.0) T1537 (74.5) SSR26 (77.5) cLET-1-A5 (79.0) CLER-5-N18 (86.0) T1535 (89.0)TG645R (90.0)U146494 (90.0)TG373 (94.0) CT9 (101.0) T1480 (106.0) d (108.0)T1158 (111.5) f.w.2. 2 (116.0) U214677 (126.5) cTOB-6-13 (140.0) ? Annotated regions 13 cM NOR: nucleolar organizing region PH: pericentromeric heterochromatin CEN: centromere TEL: telomere Big GAP 1Big GAP 2Big GAP 3 Contig regions 1 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

6 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)

7 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

8 Strategy for Filling Gap 1 Marker IMarker II Chromosome assembly Contig 1Contig 2 I. Contig end Extending I. Contig end Extending GAP Gene prediction Chromosome assembly BAC library screening IL mapping Clone validation & Sequencing GAP Probe

9 Result of BAC filter screening with contig-end Total contig - ends to be screened On progressScreened probes Hit BACs 52510 HindIII – 16/107 EcoRI – 8/71 MboI – 3/41 BACBESPCR (no end seq.) BACBESPCR (no end seq.) H057H03-3’_CDS - 1 H136C06-5’_CDS -- H091J18-5’_CDS 24 H164H08-5’_CDS 2 - H090O01-5’_CDS -- H167J21-5’_CDS 1 - H073P13-3’_CDS 22 M020N15-3’_CDS 11 H066C13-3’_CDS 21 E010H16-3’_CDS 33 But none of clones meet the criteria (>60 kb overlap)

10 Sequencing of large overlapping clones (>60 kb overlap )  To make chance to find extendable BAC  GS FLX (~30X coverage)  BLAST against BES for extension

11 Strategy for Filling Gap 2 Synteny-based Extending: Find conserved gene from orthologous Grapevine GAP Popular Arabidopsis Tomato Probes for BAC library screening IL mapping Clone validation & Sequencing

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