Sequencing Chromosome 2

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Presentation transcript:

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

Est. Euchromatin size of chro. 2: 26Mb Est. number of BACs: 268 L. Telo S. Telo Cen Peri 26 Mb 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%)

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.)

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

BAC clone selection & verifying FISH IL mapping Known Information Seed BAC 38 23 2 Extension BAC 7 30 Total 45 53

11.5 Mb <- non-redundancy (44% ) = 26Mb 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 13 16 18 21 25 28 34 36.5 37 38 38.5 46 67 69.5 70 72 72.5 73 74.5 75 76 77.5 83.5 81.5 83 89 89.3 92 94 96 100 106 108/111 109 112 114 117 120 130 141 143 140 142 142/143 88 60.0 % 46.4% 31.3% 50.0% G1 G2 G3 G4 Marker-anchored seed BAC Extended BAC 6

New Markers (Nov. Dec. 2007) Total Unique marker IL mapping 3D pool Screening SSR KAZUSA Institute 62 28 Syngenta 44 38 106 66 - 3D pool is kindly provided by Tabata group

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

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

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

Genetic Map / Sequence Marker Protocol Map Position (kb) (cM) On sequence Confidence C2_At3g51510 CAPS Tomato-EXPEN 2000 24.50 X I SSR66 SSR 149 25.00 O I(LOD2) cLED-19-B18 RFLP 27.00 C2_At3g51520 28.00 C2_At4g20070 29.00 CT255 31.00 F(LOD3) T1768 CF(LOD3) cLET-5-A5 C2_At1g78690 32.00 cLPT-2-K20 157 33.00 cLPT-4-D23 194 T1361 207 34.00 C2_At2g35130 204 34.50 TG554 463 35.00 C2_At4g19830 35.20 T1698 303 36.00 SSR96 36.50 SSR104 C2_At5g08420 36.60 cLEC-27-E17 36.70 C2_At4g20410 36.90

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

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.

Inter-chromosomal Duplication Case 4 Inter-chromosomal Duplication or Chimera 77,419bp 38,184bp 15,607bp 320D08(ch. 4) 99.9% 155D20(ch. 2) C2_At4g37460 C2_At5g67370 T0634 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

H155D20-3’ – MspI IL mapping Pooling IL DNA Chromosome 2; 2-K 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 15

HBa0155D20 locates on chromosome 2

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

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