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4th Solanaceae Genome Workshop 2007, September 09th- 13th, Jeju Island, Korea THE FRENCH CONTRIBUTION TO THE INTERNATIONAL TOMATO GENOME SEQUENCING PROGRAM.

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Presentation on theme: "4th Solanaceae Genome Workshop 2007, September 09th- 13th, Jeju Island, Korea THE FRENCH CONTRIBUTION TO THE INTERNATIONAL TOMATO GENOME SEQUENCING PROGRAM."— Presentation transcript:

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

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

3 4th Solanaceae Genome Workshop 2007, September 09th- 13th, Jeju Island, Korea 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).

4 4th Solanaceae Genome Workshop 2007, September 09th- 13th, Jeju Island, Korea Initial source of Seed BACs : overgo assay 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

5 4th Solanaceae Genome Workshop 2007, September 09th- 13th, Jeju Island, Korea 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

6 4th Solanaceae Genome Workshop 2007, September 09th- 13th, Jeju Island, Korea New strategies for "seed BAC" selection: (1) in silico search using genetic markers 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

7 4th Solanaceae Genome Workshop 2007, September 09th- 13th, Jeju Island, Korea 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

8 4th Solanaceae Genome Workshop 2007, September 09th- 13th, Jeju Island, Korea 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

9 4th Solanaceae Genome Workshop 2007, September 09th- 13th, Jeju Island, Korea "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

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

11 4th Solanaceae Genome Workshop 2007, September 09th- 13th, Jeju Island, Korea 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 Screening of the multi-species introgression lines (ILs) for chromosome 7

12 4th Solanaceae Genome Workshop 2007, September 09th- 13th, Jeju Island, Korea ILs screening results Screened BACs125 BACs validated on K762 % BAC not on K715 % Unsuccessful tests 23 % * 78 BACs on chromosome 7 checked by screening the ILs * On 28 unusable BAC sequences, 18 BAC gave good result using FISH

13 4th Solanaceae Genome Workshop 2007, September 09th- 13th, Jeju Island, Korea BAC-FISH 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)

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

15 4th Solanaceae Genome Workshop 2007, September 09th- 13th, Jeju Island, Korea 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

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

17 4th Solanaceae Genome Workshop 2007, September 09th- 13th, Jeju Island, Korea Chromosome 7 BAC coverage –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 cM 0 cM 112

18 4th Solanaceae Genome Workshop 2007, September 09th- 13th, Jeju Island, Korea 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

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

20 4th Solanaceae Genome Workshop 2007, September 09th- 13th, Jeju Island, Korea Chromosome 7 The heterochromatin region M21D10 H30C22 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).

21 4th Solanaceae Genome Workshop 2007, September 09th- 13th, Jeju Island, Korea 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. HeterochromatinEuchromatin nb BAC 1015 total size (kb) 11241287 % repeats 161,3 nb gene / 100 kb 3,48,1 DNA sequence analysis in Hetero- and Eu-chromatin

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

23 4th Solanaceae Genome Workshop 2007, September 09th- 13th, Jeju Island, Korea 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 3D-DNA pool screening

24 4th Solanaceae Genome Workshop 2007, September 09th- 13th, Jeju Island, Korea 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) ROWS COLUMNS PLATES Block 1 (plates 1-24) Block 2 (plates 25-48) … 1 block : 64 wells

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

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

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

28 4th Solanaceae Genome Workshop 2007, September 09th- 13th, Jeju Island, Korea MboIHindIII 144 plates 2 macro-arrays5 macro-arrays 72 plates/filter = 27 648 clones 336 plates EcoRI 196 plates 3 macro-arrays spotting Macroarray filters generated by the French Plant Genomic Resource Centre (CNRGV, INRA-Toulouse)

29 4th Solanaceae Genome Workshop 2007, September 09th- 13th, Jeju Island, Korea CNRGV-INRA Chemin de Borde Rouge/ B.P. 52627 / 31326 Castanet-Tolosan Tél: 05 61 28 52 53 / Fax: 05 61 28 55 64 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/

30 4th Solanaceae Genome Workshop 2007, September 09th- 13th, Jeju Island, Korea CNRGV-INRA Chemin de Borde Rouge/ B.P. 52627 / 31326 Castanet-Tolosan Tél: 05 61 28 52 53 / Fax: 05 61 28 55 64 The French Genomic Ressources Center for Plant 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 Column Pools Plate Pools Row Pools - Production of different 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.

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