PAA / Solanaceae July 23-27, Madison, Wisconsin, USA 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

PAA / Solanaceae July 23-27, Madison, Wisconsin, USA 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

PAA / Solanaceae July 23-27, Madison, Wisconsin, USA 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

PAA / Solanaceae July 23-27, Madison, Wisconsin, USA 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)

PAA / Solanaceae July 23-27, Madison, Wisconsin, USA 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 ZouineBioinformatics

PAA / Solanaceae July 23-27, Madison, Wisconsin, USA 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) Seed BACs, BAC libraries, genetic maps, IL lines Jim Giovannoni, Steve Tanksley (USA), Syngenta, Dani Zamir (Il) Main investigators BAC libraries management, BAC filters, hybridizations : Hélène Bergès (CNRGV Toulouse)

PAA / Solanaceae July 23-27, Madison, Wisconsin, USA Sequence analyses GBF Sequencing pipelineSequencing Genome express Genome express Annotation INRA Toulouse Bioinformatics Platform Seed BACs selection Data exchange and storage in local database NCBI SGN EUSOL Location validation on chromosome 7 IL: GBF FISH: China / France / USA Overlaping BAC selection GBF

PAA / Solanaceae July 23-27, Madison, Wisconsin, USA Physical mapping of tomato BACs on chromosome 7  FISH: Fluorescence In-Situ Hybridization –BAC probes hybridised on pachytene chromosomes or on mitotic chromosomes  IL (D. Zamir) –BACs mapping on Introgressed Lines (ILs)

PAA / Solanaceae July 23-27, Madison, Wisconsin, USA pericentric heterochromatin TG216 TG438 T1112 T1355 T1328 T1428 T1962 T1414 T1497 T0676 TM18 CT54 T0966 T0731 TM15 T1347 T1257 T0848 pericentric heterochromatin 309B15 euchromatin centromere telomere chromomere euchromatin 059P18 130B18 167K07 213E05 230E07 232G04 241F16 308M01 309F18 215P04 195N01 Song-Bin Chang Steve Stack (Colorado USA) Olivier Coriton (INRA Rennes) FISH mapping of tomato BACs

PAA / Solanaceae July 23-27, Madison, Wisconsin, USA Courtesy of Song-Bin Chang and Steve Stack (Colorado)

PAA / Solanaceae July 23-27, Madison, Wisconsin, USA LeHba 0027M11+Le-Hba0215P04

PAA / Solanaceae July 23-27, Madison, Wisconsin, USA  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 FISH mapping status

PAA / Solanaceae July 23-27, Madison, Wisconsin, USA Location confirmation - IL Hba0002M15

PAA / Solanaceae July 23-27, Madison, Wisconsin, USA 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)

PAA / Solanaceae July 23-27, Madison, Wisconsin, USA Sizing Phrap40 LE_HBa0325D07 LE_HBa0002D20 LE_HBa0095C18 LE_HBa0188B22 LE_HBa0215P04 LE_HBa0309B15 LE_HBa0309F18 LE_HBa0033O01 LE_HBa0130B18 LE_HBa0023C09LE_HBa0166A09 LE_HBa0163O04 SL_MboI0031B19 LE_HBa0308M01 LE_HBa0037F23 LE_HBa0059P18 LE_HBa0230E07 LE_HBa0130B18 SL_MboI0119A22 LE_HBa0002M15 LE_HBa0241F16 SL_MboI0017L19 LE_HBa0001N Estimations 7,5kb

PAA / Solanaceae July 23-27, Madison, Wisconsin, USA Assembly  Validation of Waterman hypothesis –identification of 2 problematic BACs

PAA / Solanaceae July 23-27, Madison, Wisconsin, USA  Single terminator, dye primer sequencing reaction  Pooling of 4 reaction products per capillary GE-DACS™ Technology multiplexed signatures GENOME express

PAA / Solanaceae July 23-27, Madison, Wisconsin, USA GE-DACS™ Technology  Signature-to-sequence comparison –Alignment of pseudo-sequence against expected identity adaptive thresholding expected identity pseudo sequence GENOME express

PAA / Solanaceae July 23-27, Madison, Wisconsin, USA GE-DACS™ Technology  Signature-to-signature comparison –Segment comparisons by cross-correlation –‘Correlogram’ based correspondence detection correlogram GENOME express

PAA / Solanaceae July 23-27, Madison, Wisconsin, USA BAC data summary

PAA / Solanaceae July 23-27, Madison, Wisconsin, USA 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

PAA / Solanaceae July 23-27, Madison, Wisconsin, USA 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