70 µ m

1. International Tomato Genome Sequencing Project Country USA Korea China UK India NL France Japan Spain USA USA Italy 1 2 3 4 5 6 7 8 9 10 11 12 Chromosome Mb 24 26 26 19 11 20 27 17 16 10 13 11 T=219 To sequence BACs 246 268 274 193 111 213 277 175 164 108 135 113 T=2276 0 µm 53.8 Mb 64.7 Mb 64.7 Mb 80.0 Mb 82.1 Mb 80.3 Mb 81.8 Mb 76.4 Mb 83.6 Mb 85.6 Mb 88.5 Mb Euchromatin 108.0 Mb 70 µm Heterochromatin

2. National Research Centre for Plant Biotechnology Nagendra K. Singh T. Mohapatra T. R. Sharma K. Gaikwad Indian Initiative on Tomato Genome Sequencing University of Delhi South Campus Akhilesh K. Tyagi J. P. Khurana P. Khurana Arun Sharma National Centre for Plant Genome Research Debasis Chattopadhyay Sabhyata Bhatia

3. Criteria for seed BAC selection • Selection of two candidate seed BACs on chromosome 5 • specific marker • 80-100 kb size • end sequence availability at SGN • 2. Purity check of bacterial stock • HindIII fingerprint of DNA isolated from six independent colonies • 3. PCR amplification of genetic markers • two marker-specific primer pairs • 4. BAC verification by direct sequencing • using two marker-specific custom primers • using vector-specific SP6 and T7 primers • 5. Size estimation/confirmation of BAC clone • by CHEF analysis of NotI digested BAC DNA

4. Selected BAC clones on chromosome 5 Clones selected Marker cM Telomeric Region LE_HBa070N08, LE_HBa191B01 CT101 0 LE_HBa298F18,LE_HBa051A13 T1252 3 LE_HBa224M24, LE_HBa261K11 C2-At1g60200 7 LE_HBa042B19 cLET-8-B23 10 Euchromatic Region LE_HBa257O04, LE_HBa179K09 T0876 12 Short Arm UDSC & NCPGR LE_HBa232L14, LE_HBa179E24 cLED-8-G3 14 LE_HBa003C20,LE_HBa027B05 BS4 21 Heterochromatic Region Centromeric Region Heterochromatic Region LE_HBa169M21 T1360 73 Long Arm NRCPB LE_HBa166A02 T1777 105 Euchromatic Region LE_HBa040C21 T1541 107 LE_HBa131D04 T1584 108 LE_HBa006N20 TG69 111 LE_HBa108A18 LE_HBa239D11 CT130 115 LE_HBa251J13 TG185 119 Telomeric Region TG597 119 LE_HBa245E05

5. The path for genomic sequencing Single streak of BAC clones from seed BAC library DNA extraction DNA fingerprinting (HindIII-digested) for BAC stock purity PCR with genetic marker for re-confirmation CHEF-analysis for size estimation Shotgun cloning and sequencing Searching for STCs (Sequence Tag Connector) SGN end-sequence database

6. Plasmid isolation 2K library 5K library Large scale 96 well format plasmid isolation

7. Clone assembly 05B10 03G06 51F03 52H11 55F03 13C02 52H04 03E01 15A02 Physical Gap 12G05 T7 SP6 C16 C15 C14 C17 C13 C18 LE_HBa0179K09 (102 kb) 04B02 14B09 11D02 08C05 02C07 13B05 T7 SP6 C44 C45 C42 C41 C31 C39 C38 C43 LE_HBa0042B19 (113 kb) 54F01 55D02 55F08 10D12 52A12 05F06 55F11 51H01 53H10 53E03 01F02 SP6 T7 C18 C20 C17 C11 C16 C15 C06 C18 C21 C19 C22 C14 LE_HBa0179E24 (115 kb)

8. Sequencing status of selected BAC clones UDSC NCPGR NRCPB

9. Important genes present on BAC clones

10. Problems 1. Different fingerprints obtained of same BAC clone picked from seed BAC and main library Seed BAC library Main library

11. G BAC G BAC 1 2 1 2 1 2 1 2 Problems 3. No marker-specific amplification by PCR of few selected seed BACs 2. Contamination in few seed BAC stocks