1. FINISHING WORKSHOP APRIL 2008 CHROMOSOME 7 THE FRENCH CONTRIBUTION TG216 TG438 T1112 T1355 T1328 T1428 T1962 T1414 T1497 T0676 TM18 CT54 T0966 T0731 TM15 T1347 T1257 T0848

2. FINISHING WORKSHOP APRIL 2008 INTRODUCTION Chromosome 7 : –27 Mbases of gene-dense euchromatin –The coverage of this region is expected to require the sequencing of more than 250 BACs BAC selection: –Selection and verification of the BACs on chromosome 7 by GBF (INPT) –Generation of new tools and resources allowing the selection and verification of the BACs to be sequenced BAC sequencing: –Private Company Cogenics (Genome Express) –From draft production to BAC finishing (phase 3)

3. FINISHING WORKSHOP APRIL 2008 TG216 TG438 T1112 T1355 T1328 T1428 T1962 T1414 T1497 T0676 TM18 CT54 T0966 T0731 TM15 T1347 T1257 T0848 Chromosome 7 SELECTION OF BACs

4. FINISHING WORKSHOP APRIL 2008 BAC selection strategy Selection and sequencing of 100 "seed BACs" to cover the gene rich regions of Chromosome 7 Check the location of each BAC on K7 by FISH and/or by polymorphism screening on ILs Selection of overlapping BACs by in silico approaches or 3D DNA pool screening.

5. FINISHING WORKSHOP APRIL 2008 3D-DNA pools from the BAC libraries The 3D pools were generated in collaboration with the French Plant Genomic Resource Centre (http://cnrgv.toulouse.inra.fr/) - Half of the HindIII BAC library: 168 plates 384 64 512 clones 7.8 X genome equivalent - The entire MboI BAC library : 144 plates 384 52 296 clones 7.5 X genome equivalent Available for the community

6. FINISHING WORKSHOP APRIL 2008 Seed BACs selection: Strategy of SelectionNumber of BACs Overgo assay20 in silico search using genetic markers (blast against the BES db) 28 Screening of 3D-DNA pools and macroarray filters from the BAC libraries 20 FPC contig :7 Collaboration / bibliography12 TOTAL87

7. FINISHING WORKSHOP APRIL 2008 Source of genetic markers anchoring the seed BACs: Sources of markersNumber of BACs SGN52 Syngenta10 SSR Japan3 Collaboration / bibliography12 56 markers from Japan on chromosome 7 30 new position (not present in BACs sequenced already)  3 markers have a hit in the BES database  3 markers were screened on the 3D DNA pools  24 more markers available for screening the 3D DNA pools

8. FINISHING WORKSHOP APRIL 2008 Chromosome 7 STATE OF THE PROGRESS TG216 TG438 T1112 T1355 T1328 T1428 T1962 T1414 T1497 T0676 TM18 CT54 T0966 T0731 TM15 T1347 T1257 T0848

9. FINISHING WORKSHOP APRIL 2008 Present state of the progress April 2008 14,9 Mb : 55 % of the total estimated euchromatin (27 Mbases) 1,4 Mb of phase 3 sequence

10. FINISHING WORKSHOP APRIL 2008 Contigs of BACs on chromosome 7 –127 BACs are included in 34 contigs on chromosome 7 –Our largest contig is 1.2 Mb long –28 BACs remain single

11. FINISHING WORKSHOP APRIL 2008 BACs coverage on chromosome 7 Total number of contigs: 34 Euchromatin short arm (19 BACs) : 3 contigs and 8 single BACs; 11 Gaps Euchromatin long arm (102 BACs): 20 contigs and 11 single BACs; 31 Gaps Heterochromatin (28 BACs) : 10 contigs and 4 single BACs Sequenced BACs BACs under sequencing Gap between BACs 0.2 2 3.5 6 15 22 23 28 30 35 39 43 44.5 52 69 78 104 112 Euchro Heterochromatin Euchro Centromere

12. FINISHING WORKSHOP APRIL 2008 Chromosome 7 BAC SEQUENCING AND FINISHING TG216 TG438 T1112 T1355 T1328 T1428 T1962 T1414 T1497 T0676 TM18 CT54 T0966 T0731 TM15 T1347 T1257 T0848

13. FINISHING WORKSHOP APRIL 2008 Sequencing Strategy BAC sequencing using capillary sequencers Finishing strategy : –Walking on shotgun clones when available clone links –Direct BAC sequencing between scaffolds 454 Assays on 4 BACs (shotgun + short PETs) : –results available –1 single scaffold 454 assays under progress on 16 BACs (Long PETs)

14. FINISHING WORKSHOP APRIL 2008 Technical issues Empty shotgun subclones : up to 40% Repeats : difficulty to obtain a stable assembly Example : HBa0117J06 (repetitions of more than 5kb conserved at more than 90%) Mono- or Di-nucleotide stretches : polymerase fails to pass throught Example : HBa0076O09 (polyC)

15. FINISHING WORKSHOP APRIL 2008 The very small BACs BAC nameSGN lengthfinal length SL_EcoRI0071O2211511,5 LE_HBa0103N0214917,9 LE_HBa0025K1113919 LE_HBa0129L059219,7 LE_HBa0073N225028,7 LE_HBa0033O014734,8 LE_HBa0174J0814836,7 LE_HBa0189M1017045 8 BACs are less than 50kb long. Discrepancy between the size estimated by SGN and the real size found after sequencing (confirmed by restriction digest) - In some cases we suspect a deletion during clone amplification, then the BAC sequence is not representative of the chromosome sequence - We will sequence other BACs encompassing these areas

16. FINISHING WORKSHOP APRIL 2008 Other points to be metionned/discussed Creation of Chromosome 13 We can upload the draft sequences of 4 BACs in Phase 1 or 2 not in chromosome 7

17. FINISHING WORKSHOP APRIL 2008 People Genomic and Biotechnology of the Fruits GBF (INRA/INP-ENSAT) Murielle Philippot Pierre Frasse Mohamed Zouine Farid Regad Mondher Bouzayen Cogenics-Genome Express Stéphanie Penaud Hervé Duborjal Marcel deLeeuw Diliana Dimova Réjane Beugnot François Pons