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Cytological Architecture of Tomato Chromosome 2: Genome Sequencing Strategy

Explore the genetic map, markers, and BAC library of Tomato Chromosome 2 with detailed insights into cytological architecture and genome sequencing strategies for filling gaps. Discover contig information, physical maps, and gene predictions.

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Cytological Architecture of Tomato Chromosome 2: Genome Sequencing Strategy

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  1. Chromosome 2 Doil Choi, Sunghwan Jo KOREA

  2. Cytological architecture of chromosome 2 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) 540 kb/µm DAPI (4’-6-diamidino-2-phenylindole) stained pachytene chromosome

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

  4. Contig information • 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 NOR: nucleolar organizing region PH: pericentromeric heterochromatin CEN: centromere TEL: telomere 142 cM 13 cM 129cM C2_At2g34470 (38.5) CLER-5-N18 (86.0) U214677 (126.5) T1554 (142.0) cLET-1-A5 (79.0) cLEC-27-M9 (46.0) CT103 (63.7) cTOB-6-13 (140.0) cLEC-7-P21 (0) T1438 (70.0) T1395 (72.0) TM34 (73.0) T1535 (89.0) TG645R (90.0) U146494 (90.0) TG373 (94.0) T1537 (74.5) ? CT140 (16.0) T1706 (18.0) CT213 (24.0) SSR26 (77.5) T1480 (106.0) f.w.2. 2 (116.0) TG276 (14.0) T1668 (37.0) CT9 (101.0) d (108.0) T1158 (111.5) rcr3 (?) ? T1616 (1.0) Physical Map 13.0 40.0 48.0 CNR (?) 142.0 78.0 100.0 120.0 Markers (cM) Big GAP 1 Big GAP 2 Big GAP 3 Annotated regions 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. 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) Efforts for next BAC selection

  7. 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 Status of Chromosome 2 Sequencing

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

  9. Result of BAC filter screening with contig-end 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 Popular Arabidopsis GAP Tomato Probes for BAC library screening IL mapping Clone validation & Sequencing

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