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INDIAN INITIATIVE FOR TOMATO GENOME SEQUENCING Nagendra Singh National Research Centre on Plant Biotechnology Indian Agricultural Research Institute New.

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Presentation on theme: "INDIAN INITIATIVE FOR TOMATO GENOME SEQUENCING Nagendra Singh National Research Centre on Plant Biotechnology Indian Agricultural Research Institute New."— Presentation transcript:

1 INDIAN INITIATIVE FOR TOMATO GENOME SEQUENCING Nagendra Singh National Research Centre on Plant Biotechnology Indian Agricultural Research Institute New Delhi - 110012

2 INTERNATIONAL TOMATO GENOME SEQENCING CONSORTIUM

3 The NRCPB-IARI (New Delhi) is assigned the region between 73-119 cM on the long arm of chromosome 5. Initially 72 prospective seed BACs were received from Cornell for this region. These BACs were anchored by 13 genetic markers located at 9 distinct cM positions. All 72 BACs were screened using PCR primers designed from their respective markers to select confirmed seed BACs. OBJECTIVES

4 PHYSICAL MAPPING 1. HindIII-Fingerprinting for BAC purity 2. PCR with genetic markers 3. IL-mapping to chromosome 5 (Straight or CAPS) 4. FISH mapping of Seed BACs 3. PFGE size estimation BAC DNA Isolated from Single Colonies

5 S. No. MAP position (cM) MarkerBAC cloneSize (Kb) FPC contig 173T1360LE_HBa0169M21104Present 279cLEX-13-G5LE_HBa0334K2270No data 384T1746LE_HBa0138J03120Absent 4105T1777LE_HBa0166A02118Present 5656 107 T1541 LE_HBa0040C21 LE_HBa0168B11 111 107 Present 7108T1584LE_HBa0131D04102Present 8989 111 TG69 LE_HBa 0108A18 LE_HBa0006N20 202 123 Present 10115CT130LE_HBa 0239D11164Present 11119TG185LE_HBa0251J1398Present 12119CT138LE_HBa076P16105No data 13119TG597LE_HBa0245E05123Present Seed BACs Selected for Sequencing

6 STARTING SEED BACs FOR CHROMOSOME ARM 5 Q

7 1.Starting with confirmed seed BAC after: a) Marker PCR with “F” & “R” Sequences b) IL-mapping, c) FISH mapping 3.Identification of prospective extension BACs from North & South ends of a seed BAC by STC approach 4.Validation of the extension BACs by: a) PCR with 3 pairs of primers from the overlapping region b) Purity checking by HindIII-fingerprinting of 8 clones c) End sequencing for verification of the seed BACs c) PFGE size determination d) IL-Mapping 5.Provide DNA of confirmed and validated extension BACs to the Cloning lab for sequencing Genetic marker N1 N2 N3 Seed BAC S1 S2 S3 North Extn. BAC South Extn. BAC Identification of Extension BACs

8 M LE_HBa0166A02N4 LE_HBa0011D08N4 LE_HBa0089F22N5 LE_HBa0014E08N1 LE_HBa0138J03N3LE_HBa0009H01N3 SL_MBO0018L12N5 LE_HBa0011D08N5 LE_HBa0089F22N4 SL_MBO0018L12N4 LE_HBa0166A02N5LE_HBa0138J03N1LE_HBa0009H01N1 SL_MBO0082H02N1 LE_HBa0138J03N2LE_HBa0009H01N2 LE_HBa0014E08N3 M SL_MBO0093K24S3 LE_HBa0138J03S3 SL_MBO0093K24S2 LE_HBa0138J03S2 SL_MBO0093K24S1 LE_HBa0138J03S1SL_MBO0082H02N3SL_MBO0082H02N2LE_HBa0014E08N2 PCR PRIMER BASED VALIDATION OF EXTENSION BACs M = 100 bp ladder

9 S. NoSeed BACExtension sideExtension BACSize (kb) Overlap (kb) 1C05HBa0131D04NorthLE_HBa0025A19966.8 2C05HBa0239D11NorthLE_HBa0141A128917.4 3C05HBa0239D11SouthSL_EcORI0019P0310912.5 4C05HBa0251J13SouthLE_HBa0089M0612810.0 5C05HBa0076P16SouthLE_HBa0056N1012033.0 6C05HBa0141A12NorthLE_HBa0196G2310518.0 7LE_HBa0169M21North/SouthLE_HBa0201O2211016.0 8C05HBa0006N20NorthLE_HBa0100I0618524 9C05HBa0108A18SouthLE_HBa0019C241145.3 10C05HBa0245E05NorthLE_HBa0028M2010029.8 11C05HBa0138J03NorthLE_HBa0009H013012.4 12C05HBa0138J03SouthSL_MBO0093K2411535 13C05HBa0166A02NorthSL_MBO0018L1212557 IDENTIFICATION OF 13 VALIDATED EXTENSION BACs BY STC APPROACH

10 BAC-BASED PHYSICAL MAP OF LONG ARM OF TOMATO CHROMOSOME 5 LE_HBa056N10 73 76 84 90 95 98.7 99.5 101 102.5 105 107 108 111 115 119 T1360 C2_At1g10500 T1746 C2_At3g55800 CT172 C2_At3g06440 C2_At3g17210 C2_At5g49510 C2_At2g31970 T1777 T1541 T1584 TG69 CT130 TG238 TG185 CT138 LE_HBa0169M21 LE_HBa0166A02 LE_HBa0040C21 LE_HBa0025A19 LE_HBa0131D04 LE_HBa196G23 LE_HBa0141A12 LE_HBa0239D11 SL_EcoRI0019P03(PCR) LE_HBa251J13 LE_HBa0089M06 LE_HBa076P16 LE_HBa0201O22 cM MARKER BACs SEED BACS EXTENSION BACS cLEX-13-G5 TG597 SL_MboI0093K24 LE_HBa0334K22 LE_HBa0138J03 SL_MboI0018L12 79 LE_HBa0009H01 LE_HBa0168B11 LE_HBa0028M20 LE_HBa0245E05

11 FISH MAPPING OF LE_HBa0169M21- First clone on chromosome arm 5Q

12 FISH MAPPING OF LE_HBa0251J13- Last clone on chromosome arm 5Q

13 BACs THAT ARE NOW REDUNDANT IN THE MTP OR MAP TO OTHER CHROMOSOMES S.NoMap Position (cM) BAC/PACChromosome Location MarkerSize (kb) StatusAccession No. 179.0Le_HBa0334K221070Phase I 2107.0Le_HBa0168B115 T1541 107Phase IIAC186291 3111.0Le_HBa0019C242114Phase IIAC196303 4111.0Le_HBa0100I062185Phase IIAC197889 5111.0Le_HBa0006N202TG69123Phase IIIAC182647 6111.0Le_HBa0108A182TG69202Phase IIAC189286 7119.0Le_HBa0028M209100Phase IIAC196875 8119.0Le_HBa0245E059TG597123Phase IIAC186297

14 PRESENT PHYSICAL MAP OF LONG ARM OF TOMATO CHROMOSOME 5 LE_HBa056N10 73 76 84 90 95 98.7 99.5 101 102.5 105 107 108 111 115 119 T1360 C2_At1g10500 T1746 C2_At3g55800 CT172 C2_At3g06440 C2_At3g17210 C2_At5g49510 C2_At2g31970 T1777 T1541 T1584 TG69 CT130 TG238 TG185 CT138 LE_HBa0169M21 LE_HBa0166A02 LE_HBa0040C21 LE_HBa0025A19 LE_HBa0131D04 LE_HBa196G23 LE_HBa0141A12 LE_HBa0239D11 SL_EcoRI0019P03(PCR) LE_HBa251J13 LE_HBa0089M06 LE_HBa076P16 LE_HBa0201O22 cM MARKER BACs SEED BACS SEED BACS MAPPED BY FISH EXTENSION BACS cLEX-13-G5 TG597 LE_HBa0009H01 SL_MboI0093K24 LE_HBa0138J03 SL_MboI0018L12 79

15 NEW SEED BACs IDENTIFIED FOR 5Q BY STC APPROACH S.No.MarkercMMethodBAC% Identity 1C2_At1g1050076Blast against BAC end database LE_HBa0135A02100% 2T073077Blast against BAC end database LE_HBa0135A02100% 3CT17295Blast against BAC end database LE_HBa0015E23100% 4C2_At2g31970102.5Blast against BAC end database LE_HBa0168F06 LE_HBa0065K15 100% 5TG60104Blast against BAC end database LE_HBa0065K1599%

16 MARKERS ANALYSED BY CORNELL BY OVERGO HYBRIDIZATION S. NoMarkerE XPEN 2000 map position (cM) Hits by over goBACs identified by STC and overgo at Hybridization Size (kb) 1T073077NoLE_HBa0135A02100 2C2_At3g5580090Yes (all weak hits) Marker based PCR was negative 3CT17295No hitsLE_HBa0015E23130 4C2_At3g0644098.7YesMarker Based PCR in Progress 5C2_At3g1721099.5YesMarker Based PCR in Progress 6C2_At5g49510101YesMarker Based PCR in Progress 7C2_At2g31970102.5YesLE_HBa0065K15145 8TG60104Yes (single weak hit) LE_HBa0065K15145 Total 7 new seed BACs (4 +3) identified using STC and BAC filter hybridization approaches

17 GENERATION OF FINGERPRINTS WITH 4 REs M1- 1Kb Ladder, M2- lambda HindIII / EcoRI1- EcoRI, 2- HindIII, 3- BamHI, 4- SmaI

18 Total 53 BACs Sequenced or being sequenced Library (5 clones); Phase 1 (7 clones); Phase 2 (23 clones); Phase 3 (18 clones)

19 119 cM Chromosome 5 0 cM 60 cM UDSC + NIPGR NRCPB Mapping of BAC clones on chromosome 5 using FISH 11 cM 37 cM 43.7 cM 46 cM 73 cM 84 cM ? cM

20 New BACs mapped on tomato chromosomes using CAPS markers 7 4 5 3 7 6 1 1 3 5 5 3 Total 50 BACs

21 Analysis of tomato Unigenes for mining disease resistance and defense response like genes Results Chr 1Chr 2Chr 3Chr 4Chr 5 Chr 6 Chr 7 Chr 8 Chr 9 Chr 10 Chr 11 Chr 12 Total Chromosome size (MB)10885.683.682.18053.880.364.781.888.564.776.4949.5 Number of BAC sequenced (Finished)9141092121524291514517616 Chromosome sequenced (%)6321090403937733571426 Disease Resistance genes mapped01094322401228 Defence Response genes mapped03022103000112 Total Mapped disease resistance and defense response-like genes40 Number of unigenes analyzed = 16945 Disease resistance and defence response like unigenes found =116

22

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