Progress on sequencing tomato chromosome 12

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Presentation transcript:

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 1 Phase 3 6 Submitted 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 ANCHOR MARKER P2 P1 IL Mapping Sequencing comparison “BRIDGE” SEED BAC

Map position of overgo markers used for Chr. 12 (cM) Marker 12,5 C2_At4g03280 14,0 cLPT-6-E9 Map Position (cM) Marker 19,0 TG263 24,0 T1487 51,0 T1045 53,0 T1211 32,0 T1481 53,5 CT99 33,0 T0028 36,0 T0989 Near centromere 54,0 T1093 39,0 T1667 54,5 C2_At5g42740 41,0 cLET-8-k4 55,0 T1078 55,5 TG283 56,0 T1622 57,2 P62 57,5 T1451 65,0 T1947 57,7 cLET-8-E15 66,0 TG111 57,8 T1185 68,0 TG394 58,2 SSR20 68,5 TG367 59,0 CT189 71,0 T1266 60,0 SSR124 60,0 SSR44 60,0 cLET-8-G15 86,0 T1676 96,0 T1784 96,0 TG296 97,0 T0882 101,0 T0770 115,0 T1504 120,0 CD2

“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, SP6 end) Polymorphism detection and mapping of SP6 end Confirmation of T7 end on selected ILs

Results * Mapped on one end

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

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

SRG Database

Annotation results Le_HBa Centromero Telomero Marker BAC estension SP6 BAC estension T7 (SEED-BAC) 5000 10000 15000 20000 25000 30000 35000 40000 45000 50000 55000 60000 65000 70000 75000 80000 85000 90000 95000 100000 112743 3 esoni 5 esoni 2 esoni 10 esoni 2 esoni 4 esoni 3 esoni 5 esoni 14 esoni 8 esoni 3 esoni 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 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)