TG216 TG438 T1112 T1355 T1328 T1428 T1962 T1414 T1497 T0676 TM18 CT54 T0966 T0731 TM15 T1347 T1257 T0848 THE FRENCH CONTRIBUTION TO THE INTERNATIONAL.

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TG216 TG438 T1112 T1355 T1328 T1428 T1962 T1414 T1497 T0676 TM18 CT54 T0966 T0731 TM15 T1347 T1257 T0848 THE FRENCH CONTRIBUTION TO THE INTERNATIONAL TOMATO GENOME SEQUENCING PROGRAM PAG XVI, January 2008

INTRODUCTION Chromosome 7 : Estimated length of 27 Mbases of gene-dense euchromatin The coverage of this region is expected to require the sequencing of more than 250 BACs. To increase the number of BACs assigned to our chromosome Generation of new tools and resources allowing the selection and verification of the BACs to be sequenced. PAG XVI, January 2008

Chromosome 7 Selection of BACs TG216 TG438 T1112 T1355 T1328 T1428 TM18 CT54 T0966 T0731 TM15 T1347 T1257 T0848 PAG XVI, January 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 (built from 2 BAC libraries). PAG XVI, January 2008

Source of Seed BACs : Strategy of Selection Number of BACs Overgo assay 19 FPC contig (from China): 9 in silico search using genetic markers (Expend2000 / Genbank) 22 Collaboration / bibliography 13 SYNGENTA markers (26 genes and 48 SSR) 11 3D-DNA pools of the BAC libraries screening 7 PAG XVI, January 2008

Other source of "seed BAC" and "overlapping BAC" 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: 7.8 X genome equivalent The entire MboI BAC library : 7.5 X genome equivalent Available for the community (Spain, India, USA, Israel…) http://cnrgv.toulouse.inra.fr/ PAG XVI, January 2008

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 PAG XVI, January 2008

Screening of the multi-species introgression lines (ILs) for chromosome 7 97 BACs on chromosome 7 validated by screening the ILs Screened BACs 154 BACs validated on K7 62 % BAC not on K7 15 % Unsuccessful tests 23 % PAG XVI, January 2008

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) PAG XVI, January 2008

FISH hybridization on mitotic chromosomes O. Coriton, INRA of Rennes Double BAC FISH hybridization on mitotic chromosomes BAC LE_HBa 0104E22 + 309B15 PAG XVI, January 2008

FISH hybridization on meiotic chromosomes M141H03 H33O01 H12N15 H de Jong’s lab, Wageningen, the Netherlands 4 to 5 different dyes permit the hybridization of 4 to 5 BACs at the same time Analysis of chromomosome structure (hetero and euchromatin location, sequence repeats…) PAG XVI, January 2008

BAC-FISH on Chromosome 7 51 "seed BAC" assigned to K7 by FISH FISH in progress for 35 BACs in the Netherlands (delimitation of eucgromatin and heterochromatin regions of K7) PAG XVI, January 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 PAG XVI, January 2008

State of the Progress 12,833 bp : 47 % of the total estimated Euchromatin (27 Mbases) 32 additional BACs are now in progress among which 16 are used to test 454 strategy PAG XVI, January 2008

Contigs of BACs on Chromosome 7 97 BACs are in contigs on chromosome 7 25 BACs are singlets PAG XVI, January 2008

Chromosome 7 BAC coverage U146437 cM 0,1 cLET-6-N21 cM 22 TG418 cM 2 cLED-21-J7 cM 4 CT52 cM 2 T1940 cM 2,3 T1328 cM 13 SSR276 cM 18 T1974 cM 23 T1962 cM 25 C2_At4g26680 cM 38,4 C2_At3g26060 cM 28 C2_At2g42750 cM 44,3 TG252 cM 31 TM10 cM 33 T1329 cM 43 U242881 cM 29,5 C2_At1g17200 cM 46 C2_At3g14770 cM 56,5 TM18 cM 44,5 C2_At1g16900 cM 62,5 C2_At3g15430 cM 68 C2_At4g26750 cM 80 C2_At1g55670 cM 110 T1257 cM 72 U216327 cM 52 + 10 BACs are not anchored to a mapped marker but lay in Euchromatin (FISH Results) Sequenced BAC phase 2 or 3 Sequenced BAC phase 1 BAC not yet sequenced phase 0 PAG XVI, January 2008

Chromosome 7 BAC coverage BAC phase 2 or 3 BAC phase 1 BAC in progress or phase 0 PAG XVI, January 2008

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 PAG XVI, January 2008

Chromosome 7 The 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. M21D10 H30C22 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). PAG XVI, January 2008

DNA sequence analysis in Hetero- and Eu-chromatin Heterochromatin Euchromatin nb BAC 10 15 total size (kb) 1124 1287 % repeats 16 1,3 nb gene / 100 kb 3,4 8,1 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. PAG XVI, January 2008

Chromosome 7 TG216 TG438 T1112 T1355 T1328 T1428 T1962 T1414 T1497 TM18 CT54 T0966 T0731 TM15 T1347 T1257 T0848 PAG XVI, January 2008

More seed BAC to be selected Strategy of Selection Results 3D-DNA pools of the BAC libraries screening 12 BACs under validation Markers SSR/EST from Japan Japon 14 markers under screening 16 markers to be analysed SYNGENTA markers 36 SSR to be analysed PAG XVI, January 2008