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Sequencing Chromosome 2

Sequencing Chromosome 2. PAG 2008 meeting Jan. 2008 KRIBB/SNU, Korea Doil Choi. L. Telo. Peri. Cen. S. Telo. 26 Mb. Peri. NOR. Est. Euchromatin size of chro. 2: 26Mb Est. number of BACs: 268 Number of Markers : 274 (Tomato-EXPAN 2000: 13-143cM). Tomato Genome : 950Mb

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Sequencing Chromosome 2

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  1. Sequencing Chromosome 2 PAG 2008 meeting Jan. 2008 KRIBB/SNU, Korea Doil Choi

  2. L. Telo Peri Cen S. Telo 26 Mb Peri NOR Est. Euchromatin size of chro. 2: 26Mb Est. number of BACs: 268 Number of Markers : 274 (Tomato-EXPAN 2000: 13-143cM) Tomato Genome : 950Mb Euchromatin :220Mb (23%)

  3. BAC by BAC sequencing Strategy • Maximize marker-anchored seed BACs • Tomato-Expen 2000 • Syngenta • Kazusa Institute (Dr. Asamizu) • Contig extension & Validation • BAC end & fingerprint data • Validate BAC overlap (BES, PCR, fiber-FISH, Bridge BACs analysis, IL mapping, SGN info.)

  4. HK Identification of Euchromatin Border LG2 H007F24 0 NOR PH 10 13 H303I24 CEN H163K16 H190N21 PH 20 HK H101G09 30 13 cM H162I09 M045L06 H160F05 40 H072A04 50 60 M018P14 70 M019I01 H323A14 H204D01 H320D04 H198A03 H291P19 80 H060J03 H009K06 H164H08 H213A01 H134G09 90 H016A12 H011A02 M014P22 100 M042B19 H0461M08 110 H150M11 H210D10 120 H073P13 130 H155D20 142 cM 140 H064B17 TEL H194L19 142 H257H21

  5. BAC clone selection & verifying

  6. 13 16 18 21 25 28 G1 34 36.5 37 38 38.5 50.0% 46 G2 67 69.5 70 72 72.5 73 31.3% 74.5 75 76 77.5 83 81.5 83.5 88 G3 89 89.3 92 94 96 100 46.4% 106 108/111 109 112 114 G4 117 120 130 141 143 140 142/143 60.0 % 142 • Sequencing & assembly • 143 BACs phase 3 (143/268X100=53%) • 143 Genebank submit • 11 BACs in sequencing & assembly pipeline • 63 seed BACs • 48 Hind, 8 Eco, 6 Mbo • 80 extension BACs identified • 44 Hind, 7 Eco, 18 Mbo • 15.5Mb • 11.5 Mb <- non-redundancy (44% ) = 26Mb • 14 Singletons • 17 Contigs Marker-anchored seed BAC Extended BAC

  7. New Markers (Nov. Dec. 2007) 3D pool is kindly provided by Tabata group

  8. How to verify BAC extension • Sequence comparison • Bridge BAC analysis • IL mapping • FISH

  9. “BRIDGE” BACs Bridge BAC validation SEQUENCED SEED BAC EXTENEDBAC ANCHOR MARKER 1. Compare Overlap Sequence 2. BES BLAST 3. BES comparison, > 98%

  10. Hot Spot for Recombination Case 1 Hba0320M09(136,324bp) Hba0209K17(134,560bp) Hba0101G09(117,515bp) Hba0025A22(117,193bp) (27cM) (25cM) (36cM) 312,844bp 11cM

  11. Genetic Map / Sequence

  12. Cold Spot? Case 2 cLEC-27-M9 (46.0cM) Hba066C13 Hba167J21 Cold Spot? Hba323A14 T1395(72.0cM) Hba204D01 T1625(72.5cM) TM34(73cM)

  13. Case 3 T0702 (76.0 cM) C locus (75.0 cM) T1492 (77.0 cM) H044O16 (81,404bp) H165K22 (135,795bp) 2-H M049G16 (115,048bp) 35,695bp M021D12 (90,430bp) 16,904bp 2-I M073G04 (89,093bp) 40,085bp 2-I M049G16 and H044O16 have a marker ‘cLEX-13-I15’which is mapped on chromosome 7 with high confidence.

  14. Inter-chromosomal Duplication Case 4 or Chimera 77,419bp 38,184bp 15,607bp 320D08(ch. 4) 99.9% 155D20(ch. 2) T0634 C2_At4g37460 C2_At5g67370 CT59 T0769 Ch 2 137cM Ch 4 17.6cM Ch 4 94cM Ch 2 129cM Ch 2 130cM C04Hba0320D08 (ch. 4) -- T0769, 94cM -- 93025bp C02Hba0155D20 (ch. 2) -- T0634, 130cM -- 115597bp

  15. H155D20-3’ – MspI IL mapping Pooling IL DNA P M 1 2 3 4 5 6 7 8 9 10 11 12 Chromosome 2; 2-K P M 2-1 2-1-1 2-2 2-3 2-4 2-5 2-6 2-6-5

  16. HBa0155D20 locates on chromosome 2 HBa155D20

  17. To validate BAC extension • Multiple confirmation is required in some case! • Physical distance expectation is required comparing to genetic distance (FPC, FISH) • Need guidelines for contig validation

  18. Acknowledgements • Seoul National University Doil Choi Hae-Mi Park • KRIBB Chul-Goo Hur Sanghyeob Lee Hong-Seok Park Dal-Hoe Koo Sung-Hwan Jo Bo-Ra Kang Sang-Mi Kim • Kazusa DNA Research Institute Satoshi Tabata • Cornell University Lukas Mueller Jim Giovannoni • IL Resource Dani Zamir

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