1. CHROMOSOME 12 Giovanni Giuliano PAG, 2006

2. Tomato chr. 12 Principal Investigators Luigi Frusciante (UniNa) Giovanni Giuliano (ENEA) Giorgio Valle (CRIBI) Other Investigators Amalia Barone (UniNa) Francesca Cecchi (BIOGEN) Maria Luisa Chiusano (UniNa) Mara Ercolano (UniNa) Silvana Grandillo (IGV-CNR) Renato Rodrigues-Pousada (ENEA) Alessandro Vezzi (UniPd) Funding Agronanotech (Italian Ministry of Agriculture - started October, 2004) FIRB (Italian Ministry of Research - started October, 2005) EU-SOL (European Commission - starting April 2006)

3. CRIBI SEQUENCING Unina Enea Sequence assembly and Annotation BAC repository Data repository

4. Validation at UniNA -Single colony picking -Medium scale prep -Restriction analysis to check E. coli contamination -BAC-end sequencing -PCR markers development -Amplicon sequencing and sequence alignment -Mapping on ILs -FISH on selected BACS

5. Results (1) ? One BAC end Contradictory data yes no may be Marker Bac analyzed Bac end sequencing Cornell matching Specific amplicon Amplicon Sequence match IL mappingFish mappingSequencing status t1211LeHba021L02 in pipeline t989LeHba032k07 LeHba015A04 - finished cLPT-E9 LeHba026c1 Pd LeHba026c13v LeHba026c13n (1) in pipeline C2At4g03280 LeHba140m01- in pipeline t1045LeHba161H10 in pipeline t1487LeHba260c13- in pipeline t1481 LeHba075c18 in pipeline t1667 LeHba146I19 LeHba244C09 (1) (1) - in pipeline clet-8k4LeHba189E07 LeHba161b22 Poor - tg 68LeHba152M18Difficult tg 263LeHba244J04-Difficult t0028LeHba163O04 in pipeline ssr124LeHba059A05 LeHba061j21 (1) finished

6. Validation at ENEA -Single colony picking -Medium scale prep (problems w minipreps) -Digestion with restriction enzymes to check E. coli contamination -End sequencing + sequencing with 2 internal oligos (passes test if 3 sequences out of 4 are good and matching)

7. Results marker offsetclone name End sequence Confirmation Cornell end seqs? Validation by internal sequencing T0770101LE_HBa0183M06Y(T7/SP6)Y(both)Yes (primer 5' to overgo ) T0770101LE_HBa0155J24Y(T7/SP6)Y(T7)Yes (primer 5' to overgo ) T167686LE_HBa0115G22Y(T7/SP6)Y(both)Yes (primer 3' to overgo ) T126671LE_HBa0193C03Y(T7/SP6)Y(both)Yes (primer 5' to overgo ) TG350120LE_HBa0053L10Y(T7/SP6)Y(both)To be rechecked T1504115LE_HBa0147G13Y(T7/SP6)Y(both)To be rechecked T126671LE_HBa0079O22Y(T7) No TG39468LE_HBa0107D15Y(T7/SP6)not possibleNo SSR12460LE_HBa0061J21Y(T7/SP6)Y(both)No P6257,2LE_HBa0104O23Y(T7/SP6)Y(both)No 3 BAC’s validated (3 markers); 5 BAC’s not validated (4 markers); 2 BAC’s to be rechecked (2 markers)

8. Summary LeHba021L02 LeHba059A05 2 finished LeHba032k07 LeHba026c13v LeHba140m01 LeHba161H10 LeHba260c13 LeHba075c18 LeHba244C09 LeHba163O04 8 in pipeline LE_HBa0183M06 LE_HBa0115G22 LE_HBa0193C03 3 validated

9. Sequencing and assembly (CRIBI)

10. Bioinformatics at UniNA -Annotated EST database from dbEST (NCBI)* for Tomato and Potato species (updated December 2005) -Gbrowse interface for BAC annotation ready. To be released to the SOL community. * D'AGOSTINO, AVERSANO, CHIUSANO ML,, “ParPEST: A PARALLEL PIPELINE FOR EST DATA ANALYSIS ”; BMC Bioinformatics 1 December 2005.