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THE FRENCH CONTRIBUTION TO THE INTERNATIONAL TOMATO GENOME SEQUENCING PROGRAM

This presentation discusses the selection and sequencing of "seed BACs" to cover the gene rich regions of Chromosome 7 in tomato. Various strategies including in silico search, genetic markers, collaboration, and 3D-DNA pool screening are used for BAC selection. The progress and state of the project are also presented.

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THE FRENCH CONTRIBUTION TO THE INTERNATIONAL TOMATO GENOME SEQUENCING PROGRAM

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  1. T1112 T1355 T1328 T1428 T1962 T1414 T1497 T1347 T0676 TM18 CT54 TG216 T1257 TG438 T0966 T0731 TM15 T0848 THE FRENCH CONTRIBUTION TO THE INTERNATIONAL TOMATO GENOME SEQUENCING PROGRAM 4th Solanaceae Genome Workshop 2007, September 09th-13th, Jeju Island, Korea

  2. T1112 T1355 T1328 T1428 T1962 T1414 T1497 T1347 T0676 TM18 CT54 TG216 T1257 TG438 T0966 T0731 TM15 T0848 Chromosome 7 Selection of seed BACs 4th Solanaceae Genome Workshop 2007, September 09th-13th, Jeju Island, Korea

  3. 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 (built from 2 BAC libraries). 4th Solanaceae Genome Workshop 2007, September 09th-13th, Jeju Island, Korea

  4. 43 BACs selected from the initial list of "overgo" seed BACs 20 BAC sequenced 12 Not on K7 11 Discarded (large overlapping, misannotation…) 27% of the « overgo » seed BAC are not on K7 Initial source of Seed BACs :overgo assay 4th Solanaceae Genome Workshop 2007, September 09th-13th, Jeju Island, Korea

  5. Initial strategy : FPC contig • Minimal tiling path strategy (FPC, USA and China): • 23 potential BACs selected • 9 sequenced • 11 not on K7 • 3 discarded (large overlapping, misannotation…) 47% of BACs selected on the basis of FPC contigs could not be used 4th Solanaceae Genome Workshop 2007, September 09th-13th, Jeju Island, Korea

  6. Expend2000 genetic map: 182 markers on group 7 27 potential seed BACs selected based on blastn of the marker sequence against BES database 18 sequenced 5 not on K7 4 discarded (large overlapping, misannotation…) 18% of seed BACs selected by in silico search are not on K7 New strategies for "seed BAC" selection:(1) in silico search using genetic markers 4th Solanaceae Genome Workshop 2007, September 09th-13th, Jeju Island, Korea

  7. New strategies for "seed BAC" selection:(2) Other sources • collaboration / bibliography : • Australie / David Jones (fusarium) 4 BACs • FISH / Steve Stack 3 BACs • Sun Locus (Steve Tanksley) 2 BACs • Sequences from Database (Genbank) 4 BACs • 13 seed BACs sequenced 4th Solanaceae Genome Workshop 2007, September 09th-13th, Jeju Island, Korea

  8. New strategies for "seed BAC" selection:(3) SYNGENTA markers • 26 gene markers and 48 SSR mapped on K7: After sequence analysis only 16 genes and 36 SSR were retained (sequences redondancy) • In silico search for BES sequence homology 6 seed BAC selected and sequenced • Macroarray hybridization with remaining gene markers (collab. CNRGV) 4 seed BAC selected and sequenced • The Syngenta SSR markers will be used when all Expend2000 markers will be tested 4th Solanaceae Genome Workshop 2007, September 09th-13th, Jeju Island, Korea

  9. "seed BAC" and "overlapping BAC" selection (4) 3D-DNA pool screening The 3D-DNA pools have been validated for selection of both: • seed BAC screens by genetic markers • and for overlapping BAC selection If markers are not found in the 3D-DNA pools of MboI and HindIII libraries, the EcoRI Macroarray filters are screened 4th Solanaceae Genome Workshop 2007, September 09th-13th, Jeju Island, Korea

  10. T1112 T1355 T1328 T1428 T1962 T1414 T1497 T1347 T0676 TM18 CT54 TG216 T1257 TG438 T0966 T0731 TM15 T0848 Chromosome 7 Check BAC location on K7 4th Solanaceae Genome Workshop 2007, September 09th-13th, Jeju Island, Korea

  11. Screening of the multi-species introgression lines (ILs) for chromosome 7 • ILs (D. Zamir) for chromosome 7 • Lines 7.2 and 7.4 cover entire chromosome7, these 2 lines are used for detection of sequence polymorphism • For more precise location of the screened BAC, the polymorphism is tested on the 6 lines (7.1 to 7.5) separately 4th Solanaceae Genome Workshop 2007, September 09th-13th, Jeju Island, Korea

  12. ILs screening results *On 28 unusable BAC sequences, 18 BAC gave good result using FISH 78 BACs on chromosome 7 checked by screening the ILs 4th Solanaceae Genome Workshop 2007, September 09th-13th, Jeju Island, Korea

  13. BAC-FISH technology on pachyten meiotic chromosomes The Netherlands (H. de Jong) USA (Steve Stack / Colorado) China (Zukan Chen) BAC-FISH technology on mitotic chromosome France (INRA-Rennes / Olivier Coriton) BAC-FISH 4th Solanaceae Genome Workshop 2007, September 09th-13th, Jeju Island, Korea

  14. BAC-FISH on Chromosome 7 *On 20 unsuccessful BACs, 13 BACs gave good result using ILs 44 "seed BAC" assigned to K7 by FISH 4th Solanaceae Genome Workshop 2007, September 09th-13th, Jeju Island, Korea

  15. Present state of the progress Septrember 1st, 2OO7 • Total BAC sequenced 120 : 40 complete + 80 in progress (DACs finishing strategy) • "seed BAC": 31 complete + 57 in progress • "overlapping BACs": 9 complete + 23 in progress 4th Solanaceae Genome Workshop 2007, September 09th-13th, Jeju Island, Korea

  16. Chromosome 7 BAC coverage 0 cM 112 cM BACs in contigs Sequenced BAC phase 2 or 3 Sequenced BAC phase 1 BAC not yet sequenced 4th Solanaceae Genome Workshop 2007, September 09th-13th, Jeju Island, Korea

  17. BACs in contigs on chromosome 7 1 contig with 13 members : 1137 kb located at the most distal end of the long arm harbouring markers 80 cM to 110 cM of the F2-2000 genetic map 3 contigs with 5 members 2 contigs with 4 members 6 contigs with 3 members 12 contigs with 2 members Chromosome 7 BAC coverage cM 0 cM 112 4th Solanaceae Genome Workshop 2007, September 09th-13th, Jeju Island, Korea

  18. Contigs of BACs on chromosome 7 • The selection of overlapping BACs started only few months ago • 78 BACs are in contigs on chromosome 7 • 1 contig with 13 members 1137 kb located at the most distal end of the long arm harbouring markers 80 cM to 110 cM of the F2-2000 genetic map • 3 contigs with 5 members • 2 contigs with 4 members • 6 contigs with 3 members • 12 contigs with 2 members • 42 BAC remain single 4th Solanaceae Genome Workshop 2007, September 09th-13th, Jeju Island, Korea

  19. T1112 T1355 T1328 T1428 T1962 T1414 T1497 T1347 T0676 TM18 CT54 TG216 T1257 TG438 T0966 T0731 TM15 T0848 Chromosome 7 Gene content of euchromatic and heterochromatic BACs 4th Solanaceae Genome Workshop 2007, September 09th-13th, Jeju Island, Korea

  20. H30C22 M21D10 Chromosome 7The heterochromatin region • FISH mapping on meiotic chromosomes allowed the positioning of two BACs (H30C22 and M21D10) at the borders of a large heterochromatic region on chromosome 7. • 10 BACs lying in heterochromatin and 15 on euchromatin were sequenced. • The sequences were blasted for the presence of sequence repeats (blastn/SGN UniRepeats) and genes (blastx/RefSeq protein A.th). 4th Solanaceae Genome Workshop 2007, September 09th-13th, Jeju Island, Korea

  21. DNA sequence analysis in Hetero- and Eu-chromatin • heterochromatic BACs contain up to 1/3 of the total putative genes identified in the 25 BACs. • Coverage of heterochromatin might be required if the aim is to retrieve most of the gene-containing regions of chromosome 7. 4th Solanaceae Genome Workshop 2007, September 09th-13th, Jeju Island, Korea

  22. T1112 T1355 T1328 T1428 T1962 T1414 T1497 T1347 T0676 TM18 CT54 TG216 T1257 TG438 T0966 T0731 TM15 T0848 Chromosome 7 The built of 3-D DNA pools for BAC screening 4th Solanaceae Genome Workshop 2007, September 09th-13th, Jeju Island, Korea

  23. 3D-DNA pool screening • The 3D pools were generated in collaboration with the French Plant Genomic Resource Centre (CNRGV, INRA-Toulouse) • Half of the HindIII BAC library: • 168 plates 384 • 64512 clones • 7.8 X genome equivalent • The entire MboI BAC library : • 144 plates 384 • 52 296 clones • 7.5 X genome equivalent 4th Solanaceae Genome Workshop 2007, September 09th-13th, Jeju Island, Korea

  24. Block 1 (plates 1-24) Block 2 (plates 25-48) … COLUMNS ROWS 1 block : 64 wells PLATES 3D-DNA pool construction steps • Rows, columns and plates from 24 plates are pooled to generate a block. Cell cultures are then transferred in 384 wells plates. • HindIII library : 7 blocks generated (448 wells) • MboI library : 6 blocks generated (384 wells) 4th Solanaceae Genome Workshop 2007, September 09th-13th, Jeju Island, Korea

  25. 3D-DNA pool construction steps • DNA of the pooled cultures is amplified from the blocks using Phi29 enzyme Dean, F.B., Genome research, 2001 • After dilution a high amount of material is available for screening http://cnrgv.toulouse.inra.fr/ 4th Solanaceae Genome Workshop 2007, September 09th-13th, Jeju Island, Korea

  26. Identification of the positive BACs using qPCR 3D-DNA pools are screened by qPCR Control genomic DNA Column Pool 17 Positive clone : 4E17 Row Pool E Plate Pool 4 Identified clones are then validated by amplification on the BAC clone 4th Solanaceae Genome Workshop 2007, September 09th-13th, Jeju Island, Korea

  27. T1112 T1355 T1328 T1428 T1962 T1414 T1497 T1347 T0676 TM18 CT54 TG216 T1257 TG438 T0966 T0731 TM15 T0848 Chromosome 7 Macroarray DNA filters 4th Solanaceae Genome Workshop 2007, September 09th-13th, Jeju Island, Korea

  28. Macroarray filters generated by the French Plant Genomic Resource Centre (CNRGV, INRA-Toulouse) MboI HindIII EcoRI 144 plates 336 plates 196 plates spotting spotting spotting 2 macro-arrays 5 macro-arrays 3 macro-arrays 72 plates/filter = 27 648 clones 4th Solanaceae Genome Workshop 2007, September 09th-13th, Jeju Island, Korea

  29. The French Genomic Ressources Center for Plant CNRGV is both service provider and repository of plant genomes and data The missions are : - Centralize and maintain plant genomic resources - Distribute these resources at the international level - Provide high quality research material - Provide genomic tools for studying genomic collections - Propose genomics training court Hélène BERGES : INRA - CNRGV / email : hberges@toulouse.inra.fr http://cnrgv.toulouse.inra.fr/ CNRGV-INRA Chemin de Borde Rouge/ B.P. 52627 / 31326 Castanet-Tolosan Tél: 05 61 28 52 53 / Fax: 05 61 28 55 64 4th Solanaceae Genome Workshop 2007, September 09th-13th, Jeju Island, Korea

  30. The French Genomic Ressources Center for Plant - Production of different pools Superpool:mix of all of the clones from all the plates Plate Pools : mix of the 384 clones on each plate Row Pools :mix of the clones from each row for all the plates: 16 row pools Column Pools :mix of the clones from each column for all the plates: 24 column pools Plate Pools Row Pools Column Pools The CNRGV proposes pooling of genomic libraries. Pools of bacterial clones constitute a powerful tool for the screening of genomic libraries. Bacterial clones are mixed, with the aim of minimizing the number of reactions required to identify a clone containing a sequence of interest. The tomato HindII and MboI BAC libraries pools are available for the community. CNRGV-INRA Chemin de Borde Rouge/ B.P. 52627 / 31326 Castanet-Tolosan Tél: 05 61 28 52 53 / Fax: 05 61 28 55 64 4th Solanaceae Genome Workshop 2007, September 09th-13th, Jeju Island, Korea

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