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Sequencing the gene-rich space of tomato chromosome 7 Current status of the French effort

Sequencing the gene-rich space of tomato chromosome 7 Current status of the French effort. Farid Regad Genomic and Biotechnology of Fruit UMR990 INRA/INP-Toulouse France regad@ensat.fr. Chromosome 7 project. Sequencing of gene-rich euchromatic region of chromosome 7 Genetic length: 112 cM

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Sequencing the gene-rich space of tomato chromosome 7 Current status of the French effort

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  1. Sequencing the gene-rich space of tomato chromosome 7 Current status of the French effort Farid Regad Genomic and Biotechnology of Fruit UMR990 INRA/INP-Toulouse France regad@ensat.fr

  2. Chromosome 7 project • Sequencing of gene-rich euchromatic region of chromosome 7 • Genetic length: 112 cM • Number of linked markers: 237 • Estimated number of BACs to be sequenced: 277

  3. Chromosome 7 project • Fundings: • INRA, allowing the start of the project • French National Research Agency, starting January 2006 • EU-SOL EU-SOL, starting September 2006

  4. Status of the project • Toulouse sequencing team • Sequencing pipeline • Validation of seed BACs • FISH localisation • IL validation • Sequencing status • Shotgun coverage optimized by clone selection (DACS)

  5. INRA Toulouse sequencing team • Mondher Bouzayen Project leader • Farid Regad Project management Seed BAC validation • Corinne Delalande BAC selection • Pierre Frasse Minimum Tiling Path Physical mapping • Mohamed Zouine Bioinformatics http://gbf.ensat.fr

  6. Main investigators Seed BACs, BAC libraries, genetic maps, IL lines Jim Giovannoni, Steve Tanksley (USA), Syngenta, Dani Zamir (Il) Farid Regad Mohamed Zouine Corinne Delalande Pierre Frasse Mondher Bouzayen Bioinformatics : C. Gaspin (Genopole Toulouse bioinformatic Plateforme) FISH : O. Coriton (DGAP Rennes) S. Stack (Colorado, USA) Z. Cheng (Pékin, Chine) BAC libraries management, BAC filters, hybridizations : Hélène Bergès (CNRGV Toulouse)

  7. Sequencing pipeline Seed BACs selection Location validation on chromosome 7 IL: GBF FISH: China / France / USA Sequencing Genome express Overlaping BAC selection GBF Data exchange and storage in local database Sequence analyses GBF NCBI SGN EUSOL Annotation INRA Toulouse Bioinformatics Platform

  8. FISH: Fluorescence In-Situ Hybridization BAC probes hybridised on pachytene chromosomes or on mitotic chromosomes IL (D. Zamir) BACs mapping on Introgressed Lines (ILs) Physical mapping of tomato BACs on chromosome 7

  9. FISH mapping of tomato BACs 195N01 telomere 232G04 T1112 213E05 T1355 euchromatin 230E07 T1328 T1428 pericentric heterochromatin T1962 T1414 centromere T1497 T1347 T0676 TM18 308M01 euchromatin CT54 241F16 TG216 chromomere T1257 130B18 TG438 059P18 T0966 euchromatin 167K07 T0731 309F18 TM15 T0848 309B15 telomere 215P04 pericentric heterochromatin Olivier Coriton (INRA Rennes) Song-Bin Chang Steve Stack (Colorado USA)

  10. Courtesy of Song-Bin Chang and Steve Stack (Colorado)

  11. LeHba 0027M11+Le-Hba0215P04

  12. FISH mapping status • BACs are in the process of FISHing • 10 BACs: Collaboration with Steve Stack, Colorado, USA (4 assigned on chr. 7) • 20 BACs: Collaboration with Zhukuan Cheng, China • Other BACs will be FISHed by HIS platform INRA Rennes, France

  13. Location confirmation - IL Hba0002M15

  14. Status of the project • 9 BACs shotgun libraries underway • 17 BACs, PHASE1 done • 3 BACs, PHASE2 done • 1 BAC finished • Shotgun coverage will be optimized by clone selection (DACS, patented by Genome-express)

  15. Sizing LE_HBa0037F23 7,5kb LE_HBa0002M15 LE_HBa0241F16 LE_HBa0309F18 LE_HBa0163O04 SL_MboI0031B19 LE_HBa0230E07 Estimations LE_HBa0023C09 LE_HBa0166A09 LE_HBa0002D20 120000 LE_HBa0095C18 LE_HBa0308M01 LE_HBa0215P04 SL_MboI0119A22 LE_HBa0188B22 100000 LE_HBa0059P18 LE_HBa0309B15 LE_HBa0325D07 SL_MboI0017L19 80000 LE_HBa0130B18 LE_HBa0001N06 60000 LE_HBa0130B18 LE_HBa0033O01 40000 40000 60000 80000 100000 120000 140000 160000 Phrap40

  16. Assembly • Validation of Waterman hypothesis • identification of 2 problematic BACs

  17. GE-DACS™ Technology • Single terminator, dye primer sequencing reaction • Pooling of 4 reaction products per capillary multiplexed signatures GENOME express

  18. GE-DACS™ Technology • Signature-to-sequence comparison • Alignment of pseudo-sequence against expected identity pseudo sequence expected identity adaptive thresholding GENOME express

  19. GE-DACS™ Technology correlogram • Signature-to-signature comparison • Segment comparisons by cross-correlation • ‘Correlogram’ based correspondence detection GENOME express

  20. BAC data summary

  21. Expected agenda • January 2006 • Official start of the project • June 2006 • Optimisation and validation of the sequencing pipeline • September 2006 • 21 BACs sequenced • January 2007 • 70 BAC completed • June 2007 • 150 BAC completed • March 2008 • 277 BAC completed • December 2008 • Chromosome 7 assembly and Finishing completed • 2009 • Chromosome 7 annotation

  22. Acknowledgements • Jim Giovannoni, Steve Tanksley, Joyce Van Eck • Mapping data, seed BACs and BAC libraries • Dani Zamir • IL lines • Syngenta • new markers on chromosome 7 • Steve Stack, Zhukuan Cheng, Olivier Coriton • BAC FISHing • INRA, French NRA, EU-SOL • Funding support • CNRGV INRA-Toulouse • BAC libraries storage and handling • SGN Consortium (Lukas Mueller, …) • Bioinformatics and n line access to all relevant data for the sequencing

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