International Tomato Genome Sequencing Project Country USA Korea

International Tomato Genome Sequencing Project Country USA Korea China 1 Mb 24 To sequence BACs 246 0 µm 2 26 268 Chromosome 3 26 274 UK 4 19 193 India 5 11 111 NL France Japan Spain USA Italy 6 20 213 12 11 T=219 113 T=2276 7 27 277 8 17 175 9 16 164 10 10 108 11 13 135 53. 8 Mb 64. 7 Mb 82. 1 Mb 80. 0 Mb 83. 6 Mb 85. 6 Mb 70 µm 108. 0 Mb 80. 3 Mb 64. 7 Mb 81. 8 Mb 76. 4 Mb 88. 5 Mb Euchromatin Heterochromatin

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

Criteria for seed BAC selection 1. 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 • Hind. III 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 SP 6 and T 7 primers 5. Size estimation/confirmation of BAC clone • by CHEF analysis of Not. I digested BAC DNA

Selected BAC clones on chromosome 5 Clones selected c. M CT 101 T 1252 C 2 -At 1 g 60200 c. LET-8 -B 23 T 0876 c. LED-8 -G 3 BS 4 0 3 7 10 12 14 21 Telomeric Region Euchromatic Region Short Arm LE_HBa 070 N 08, LE_HBa 191 B 01 LE_HBa 298 F 18, LE_HBa 051 A 13 LE_HBa 224 M 24, LE_HBa 261 K 11 LE_HBa 042 B 19 LE_HBa 257 O 04, LE_HBa 179 K 09 LE_HBa 232 L 14, LE_HBa 179 E 24 LE_HBa 003 C 20, LE_HBa 027 B 05 Marker UDSC & NCPGR Heterochromatic Region Centromeric Region Heterochromatic Region LE_HBa 166 A 02 LE_HBa 040 C 21 LE_HBa 131 D 04 LE_HBa 006 N 20 LE_HBa 108 A 18 LE_HBa 239 D 11 LE_HBa 251 J 13 LE_HBa 245 E 05 T 1360 73 T 1777 T 1541 T 1584 105 107 108 TG 69 111 CT 130 TG 185 TG 597 115 119 Euchromatic Region Telomeric Region Long Arm LE_HBa 169 M 21 NRCPB

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

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

Clone assembly Physical Gap 05 B 10 03 G 06 51 F 03 12 G 05 52 H 11 52 H 04 55 F 03 03 E 01 13 C 02 15 A 02 T 7 SP 6 C 15 C 14 C 17 C 13 C 18 LE_HBa 0179 K 09 (102 kb) 04 B 02 08 C 05 14 B 09 02 C 07 11 D 02 13 B 05 SP 6 C 44 C 45 C 42 C 41 C 39 C 38 C 43 T 7 LE_HBa 0042 B 19 (113 kb) 55 F 11 51 H 01 54 F 01 55 D 02 01 F 02 55 F 08 10 D 12 52 A 12 05 F 06 53 H 10 53 E 03 SP 6 C 18 C 20 C 17 C 11 C 16 C 15 C 06 C 18 LE_HBa 0179 E 24 (115 kb) C 21 C 19 C 22 C 14 T 7

Sequencing status of selected BAC clones S. No. c. M BAC clone Status Assembly 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 0 3 7 10 12 14 21 73 105 107 108 111 115 119 LE_HBa 191 B 01 LE_HBa 051 A 13 LE_HBa 261 K 11 LE_HBa 042 B 19 LE_HBa 179 K 09 LE_HBa 179 E 24 LE_HBa 027 B 05 LE_HBa 169 M 21 LE_HBa 166 A 02 LE_HBa 040 C 21 LE_HBa 131 D 04 LE_HBa 006 N 20 LE_HBa 108 A 18 LE_HBa 239 D 11 LE_HBa 251 J 13 LE_HBa 245 E 05 Library Sequencing Library Library Phase II Phase I - UDSC NCPGR NRCPB

Important genes present on BAC clones S No. BAC clone Name of the gene Organism 1 HBa 0042 B 19 Beta fructosidase gene L. pennellii 2 HBa 0042 B 19 Nematode resistance-like protein (Gro 1 -6) S. tuberosum 3 HBa 0042 B 19 Glyceraldehyde-3 -phosphate dehydrogenase P. hybrida 4 HBa 0179 K 09 10 k. Da polypeptide precursor of PS II L. esculentum 5 HBa 0179 K 09 Salicylic acid-induced protein 19 C. annuum 6 HBa 0179 K 09 Light-inducible ST-LS 1 gene S. tuberosum 7 HBa 0179 K 09 Lateral suppressor gene (LS gene) L. esculentum 8 HBa 0179 E 24 Tospovirus resistance protein C (Sw 5 -C) L. esculentum 9 HBa 0179 E 24 Eukaryotic translation initiation factor 5 A-3 L. esculentum 10 HBa 0179 E 24 ACS 6 gene L. esculentum 11 HBa 0179 E 24 UV-damaged DNA binding protein 1 (hp 1) L. esculentum 12 HBa 0179 E 24 Omega-3 fatty acid desaturase gene L. esculentum

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

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