70 µm 0 µm International Tomato Genome Sequencing Project 123 456789101112 108.0 Mb 85.6 Mb 83.6 Mb 82.1 Mb 80.0 Mb 53.8 Mb 80.3 Mb 64.7 Mb 81.8 Mb 88.5.

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Presentation transcript:

70 µm 0 µm International Tomato Genome Sequencing Project Mb 85.6 Mb 83.6 Mb 82.1 Mb 80.0 Mb 53.8 Mb 80.3 Mb 64.7 Mb 81.8 Mb 88.5 Mb 64.7 Mb 76.4 Mb Mb T= BACs T=2276 Euchromatin Heterochromatin To sequence Chromosome Country USAKoreaChinaUKIndiaNLFranceJapanSpainUSA Italy

University of Delhi South Campus Akhilesh K. Tyagi J. P. Khurana 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 Indian Initiative on Tomato Genome Sequencing

Criteria for seed BAC selection 1.Selection of two candidate seed BACs on chromosome 5 specific marker kb size end sequence availability at SGN 4. BAC verification by direct sequencing using two marker-specific custom primers using vector-specific SP6 and T7 primers 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 5. Size estimation/confirmation of BAC clone by CHEF analysis of NotI digested BAC DNA

Selected BAC clones on chromosome LE_HBa070N08, LE_HBa191B01 LE_HBa298F18, LE_HBa051A13 LE_HBa224M24, LE_HBa261K11 LE_HBa042B19 LE_HBa257O04, LE_HBa179K09 LE_HBa232L14, LE_HBa179E24 LE_HBa003C20, LE_HBa027B05 LE_HBa169M21 LE_HBa166A02 LE_HBa040C21 LE_HBa131D04 LE_HBa108A18 LE_HBa239D11 LE_HBa251J LE_HBa245E05 T1252 CT101 C2-At1g60200 cLET-8-B23 T0876 cLED-8-G3 BS4 T1360 T1777 T1541 T1584 TG69 CT130 TG185 TG597 Centromeric Region Heterochromatic Region Euchromatic Region Telomeric Region Long Arm Short Arm UDSC & NCPGR NRCPB cMMarkerClones selected LE_HBa006N20

Single streak of BAC clones from seed BAC library DNA extraction 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 DNA fingerprinting (HindIII-digested) for BAC stock purity The path for genomic sequencing

2K library5K library Large scale 96 well format plasmid isolation Plasmid isolation

Clone assembly SP6 T7 C16C15C14C17C13C18 52H04 03E01 15A02 52H11 55F03 13C02 05B10 03G06 51F03 12G05 Physical Gap LE_HBa0179K09 (102 kb) C44C45C42C41C31C39C38C43 04B02 14B09 11D02 08C05 02C07 13B05 LE_HBa0042B19 (113 kb) C18C20C17C11C16C15C06C18C21C19C22C14 10D12 52A12 05F06 55F11 51H01 01F02 54F01 55D02 55F08 53H10 53E03 LE_HBa0179E24 (115 kb)

Sequencing status of selected BAC clones S. No.cMBAC cloneStatusAssembly 10LE_HBa191B01Library- 23LE_HBa051A13Library- 37LE_HBa261K11Library- 410LE_HBa042B19SequencingPhase I 512LE_HBa179K09SequencingPhase II 614LE_HBa179E24SequencingPhase I 721LE_HBa027B05Sequencing- 873LE_HBa169M21Library- 9105LE_HBa166A02Library LE_HBa040C LE_HBa131D04Library LE_HBa006N LE_HBa108A LE_HBa239D LE_HBa251J13Library LE_HBa245E05Library- UDSC NCPGR NRCPB

Important genes present on BAC clones S No.BAC cloneName of the geneOrganism 1HBa0042B19Beta fructosidase geneL. pennellii 2HBa0042B19Nematode resistance-like protein (Gro1-6)S. tuberosum 3HBa0042B19Glyceraldehyde-3-phosphate dehydrogenaseP. hybrida 4HBa0179K0910 kDa polypeptide precursor of PS IIL. esculentum 5HBa0179K09Salicylic acid-induced protein 19C. annuum 6 HBa0179K09 Light-inducible ST-LS1 geneS. tuberosum 7HBa0179K09Lateral suppressor gene (LS gene)L. esculentum 8HBa0179E24Tospovirus resistance protein C (Sw5-C)L. esculentum 9HBa0179E24Eukaryotic translation initiation factor 5A-3L. esculentum 10HBa0179E24ACS6 geneL. esculentum 11HBa0179E24UV-damaged DNA binding protein 1 (hp1)L. esculentum 12HBa0179E24Omega-3 fatty acid desaturase geneL. esculentum

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

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