Progress on sequencing tomato chromosome 12 Mara Ercolano

Progress on sequencing tomato chromosome 12 Mara Ercolano

Summary BACs in sequencing pipeline SEED BAC EXTENDING BAC Library 5 13 Phase 0 2 3 Phase 1 10 9 Phase 2 3 1 Phase 3 6 6 Submitted 9 1 TOTAL 35 33

Chromosome 12 short arm sequencing status

Chromosome 12 pericentrometric area sequencing status

Chromosome 12 long arm sequencing status

Extending BAC validation protocol 5’ SEQUENCED SEED BAC 3’ POTENTIAL EXTENDING BACs P 2 ANCHOR MARKER Sequencing comparison “BRIDGE” SEED BAC P 1 IL Mapping

Map position of overgo markers used for Chr. 12 Map Position (c. M) Marker 12, 5 14, 0 19, 0 C 2_At 4 g 03280 c. LPT-6 -E 9 TG 263 24, 0 T 1487 32, 0 33, 0 36, 0 39, 0 41, 0 T 1481 T 0028 T 0989 T 1667 c. LET-8 -k 4 65, 0 T 1947 66, 0 TG 111 68, 0 TG 394 68, 5 TG 367 71, 0 T 1266 86, 0 T 1676 96, 0 97, 0 101, 0 T 1784 TG 296 T 0882 T 0770 115, 0 T 1504 120, 0 CD 2 Map Position Marker (c. M) Near centromere 51, 0 53, 5 54, 0 54, 5 55, 0 55, 5 56, 0 57, 2 57, 5 57, 7 57, 8 58, 2 59, 0 60, 0 T 1045 T 1211 CT 99 T 1093 C 2_At 5 g 42740 T 1078 TG 283 T 1622 P 62 T 1451 c. LET-8 -E 15 T 1185 SSR 20 CT 189 SSR 124 SSR 44 c. LET-8 -G 15

“De novo” BAC mapping Choice of BACs with good end sequences on SGN PCR primer design on BAC ends Amplification of S. lycopersicum and S. pennellii DNA Amplification and sequencing of 72 ILs (single pass, SP 6 end) Polymorphism detection and mapping of SP 6 end Confirmation of T 7 end on selected ILs

Results * Mapped on one end

Summary validation Overgo Chr 12 Syngenta Chr 12 De novo Validated Chr 12 43 (35 in pipeline) 23 (20 between 50 -60 c. M) 1 Validated other Chr 17 0 16 Total 60 23 17

Strategy used to identify genomic differences between two tomato genotypes Driver a a) b) c) Tester b Chromosome of S. lycopersicon Chromosome of S. pennelii IL line Fragment present only in the driver genotypes c

SRG Database

Annotation results 5 esoni 2 esoni 4 esoni 3 esoni 5 esoni 14 esoni 8 esoni 100000 T 7 95000 80000 75000 70000 65000 60000 550000 45000 40000 35000 30000 25000 2 esoni 10 esoni 90000 BAC estension (SEED-BAC) 15000 3 esoni Le_HBa SP 6 10000 BAC estension 0 Centromero Marker Telomero 3 esoni 112743 9 esoni 7 esoni 5 esoni 11 esoni 2 esoni 4 esoni 6 esoni 5 esoni 2 esoni 11 esoni 15 esoni 3 + 2 esoni 1 esoni 2 esoni 3 esoni 7 esoni 8 esoni 4 esoni SRG Fgenesh tabacco Fgenesh Arabidospis Database EST Risultati coincidenti

Summary A total of 68 BACs are currently at different phases of the sequencing pipeline. Nine seed BACs and 1 extending BAC have been submitted to NCBI database and SGN repository. A strategy that combine bionformatic analysis and molecular validation to identify extending BACs, has been set up. In order to select new starting points for sequencing a IL mapping effort has been being. A bioinformatic platform to provide an Italian resource for supporting the annotation of the tomato genome has been built up. Resources useful to analize data provided from sequencing project have been developed.

Participating Institutions Univ of Naples ‘Federico II’ Luigi Frusciante Mara Ercolano Amalia Barone Maria Luisa Chiusano ENEA, Rome Giovanni Giuliano Marco Pietrella Giulia Falcone CNR-IGV, Portici (Naples) Silvana Grandillo CRIBI and Univ. of Padua Giorgio Valle Alessandro Vezzi Funding Agronanotech (Italian Ministry of Agriculture - started October, 2004) FIRB (Italian Ministry of Research - started October, 2005) EU-SOL (European Commission - started May 2006)