INDIAN INITIATIVE FOR TOMATO GENOME SEQUENCING

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. OBJECTIVES 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.

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

5. Seed BACs Selected for Sequencing

6. STARTING SEED BACs FOR CHROMOSOME ARM 5 Q

7. Identification of Extension BACs • Starting with confirmed seed BAC after: • a) Marker PCR with “F” & “R” Sequences • b) IL-mapping, • c) FISH mapping • Identification of prospective extension BACs from North & South ends of a seed BAC by STC approach • 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 • Provide DNA of confirmed and validated extension BACs to the • Cloning lab for sequencing North Extn. BAC N1 N2 N3 Seed BAC S1 S2 S3 Genetic marker South Extn. BAC

8. SL_MBO0082H02N1 SL_MBO0082H02N2 SL_MBO0082H02N3 SL_MBO0093K24S1 SL_MBO0093K24S2 SL_MBO0093K24S3 SL_MBO0018L12N4 SL_MBO0018L12N5 LE_HBa0011D08N5 LE_HBa0166A02N4 LE_HBa0089F22N4 LE_HBa0011D08N4 LE_HBa0166A02N5 LE_HBa0009H01N1 LE_HBa0009H01N2 LE_HBa0009H01N3 LE_HBa0014E08N2 LE_HBa0014E08N3 LE_HBa0014E08N1 LE_HBa0089F22N5 LE_HBa0138J03N1 LE_HBa0138J03N2 LE_HBa0138J03N3 LE_HBa0138J03S1 LE_HBa0138J03S2 LE_HBa0138J03S3 M PCR PRIMER BASED VALIDATION OF EXTENSION BACs M M = 100 bp ladder

9. IDENTIFICATION OF 13 VALIDATED EXTENSION BACs BY STC APPROACH

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

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

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

15. NEW SEED BACs IDENTIFIED FOR 5Q BY STC APPROACH

16. MARKERS ANALYSED BY CORNELL BY OVERGO HYBRIDIZATION Total 7 new seed BACs (4 +3) identified using STC and BAC filter hybridization approaches

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

18. Sequencing status of BACs on chromosome 5 Status Clones selected Marker cM Phase III LE_HBa0191B01 CT101 0 Phase II SL_MboI0050C14 T1181 0 Phase I SL_MboI0004P04 Telomeric Region T1632 0 Phase II SL_EcoRI0101I15 Phase III SL_MboI0005B15 Phase III SL_EcoRI0086I08 C2_At1g60200 7 Phase III LE_HBa0261K11 cLET-8-B23 10 Phase III LE_HBa0042B19 T0564 11 Phase II LE_HBa0189E17 Euchromatic Region Short Arm Phase I SL_MboI0079D24 Phase I LE_HBa0115F01 Phase II SL_MboI0095J08 UDSC & NIPGR Phase III SL_MboI0037H06 Phase II cLED-8-G3 15.5 LE_HBa0179E24 Phase III CT242 LE_HBa0058L13 Phase III LE_HBa0060G21 T1592 16 Phase III TG441 SL_EcoRI0066O01 Phase III TG432 LE_HBa0145P19 Phase III BS4 44 LE_HBa0003C20 Heterochromatic Region Phase III LE_HBa0168M18 CT167 Phase III SL_MboI0118J18 Phase III SL_EcoRI0028N03 Phase III SL_EcoRI0122H05 C2_At2g01110 37 Phase III Centromeric Region LE_HBa0309L13 Phase II SL_EcoRI0037P02 C2_At3g55120 44 Phase II SL_EcoRI0082N07 Phase II LE_HBa0147F10 Phase II LE_HBa0023M16 Heterochromatic Region C2_At4g24830 51 Phase III SL_MboI0115G01 57 Phase III T1640 SL_MboI0079C22 Phase I LE_HBa0243O19 Phase II LE_HBa0057G22 60 TG96 Phase II LE_HBa0298C03 New CAPS Phase I LE_HBa0042L17 Long Arm Phase I 73 LE_HBa0169M21 T1360 Library LE_HBa0201O22 Phase II LE_HBa0135A02 C2_At1g10500 76 NRCPB Library LE_HBa0009H01 Phase II LE_HBa0138J03 T1746 84 Phase II SL_MboI0093K24 Euchromatic Region 105 Library SL_MboI0018L12 107 T1777 Phase II LE_HBa0166A02 Phase II T1541 LE_HBa0040C21 Phase II LE_HBa0025A19 108 Phase II LE_HBa0131D04 T1584 Phase II LE_HBa0196G23 Phase II LE_HBa0141A12 115 Phase II LE_HBa0239D11 CT130 Telomeric Region Phase I SL_EcoRI0019P03 119 Phase II LE_HBa0251J13 TG185 Library LE_HBa0089M06 119 Phase II LE_HBa0076P16 CT138 Library LE_HBa0056N10 Total 53 BACs Sequenced or being sequenced Library (5 clones); Phase 1 (7 clones); Phase 2 (23 clones); Phase 3 (18 clones)

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

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

22. THANK YOU