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CURRENT STATUS ON SEQUENCING OF CHROMOSOME 12

CURRENT STATUS ON SEQUENCING OF CHROMOSOME 12. Mar a Ercolano Ischia, 2005. Italy: tomato chr. 12 Country Representative: Prof. Giovanni Giuliano. Principal Investigator s Prof. Luigi Frusciante (UniNa) Prof. Giovanni Giuliano (ENEA) Prof. Giorgio Valle (UniPd) Other Investigator s

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CURRENT STATUS ON SEQUENCING OF CHROMOSOME 12

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  1. CURRENT STATUS ON SEQUENCING OF CHROMOSOME 12 Mara Ercolano Ischia, 2005

  2. Italy: tomato chr. 12 Country Representative: Prof. Giovanni Giuliano Principal Investigators Prof. Luigi Frusciante (UniNa) Prof. Giovanni Giuliano (ENEA) Prof. Giorgio Valle (UniPd) Other Investigators Dr.AmaliaBarone (UniNa) Dr. Francesca Cecchi (BIOGEN) Dr.Maria Luisa Chiusano(UniNa) Dr.MaraErcolano (UniNa) Dr.Silvana Grandillo(IGV-CNR) Dr. Renato Rodrigues-Pousada (ENEA) Dr. Alessandro Vezzi (UniPd) Funding Agronanotech (Italian Ministry of Agriculture) FIRB (Italian Ministry of Research) EU-SOL (European Commission)

  3. HIGH-THROUGHPUT SEQUENCING CRIBI Unit Enea Unit BAC libraries repository Unina Unit Sequences assembling and Annotation setup according to the Sol Genome Pipeline Data repository ftp://tomato.unina.it

  4. o Chromosome 12 12 TM14B TG180 7.4 CT19A 6.4 TG68 14.4 CT7 4.3 CT211A 5.5 CT219 2.2 TG360 0.8 CD6A 1.6 TG283 4.5 TG394 13.4 CT287A 6.3 TG28A 1.0 CT239B 2.0 TG296 2.4 CT156 7.2 CT80B 6.1 CT276 5.8 TG473 1.8 CD2 Euchromatin portion = 11Mb Projected # of BACs 113

  5. Verification analysis • Single colony picking Identity confirmation • Digestion with restriction enzymes • BAC-end sequencing • Marker association - PCR markers development • Amplicon sequencing and sequence alignment • Localization • IL lines mapping confirmation. - FISH mapping.

  6. Restriction enzyme digestion and BAC end sequencing • SP6 and T7 end sequences alignament and comparison with SGNdatabase Fingerprinting analysis with the restriction enzyme BamHI

  7. Primer design >SGN-E314485 [] (trimmed) ATCTTGAAGCTTAAAAAAAACAAAAAAAAAAACAAACGATGTTGGCTATATTTCACAAGGCATTTGCTCATCCTCCTGAAGAATTAAATAGTCCTGCATCAAAAAAATGTTTACTTCCACAACAAACGCTACAAAAATTCGTATCGACTCGTCCATTTGACACCTCTTATGTTACTTTTGGAGATGTTGCTGCTCTTGCTTTTGTTCGTCCTAATTGCACCTCCTTGCTCAATCACAAACAAAGGTATTTTTGTGGTTATGATGATATTTACTGTTTGTTCATGGGGAGTTTGAACAATTTGTGTGCACAAATCAAACAATATGGGCTATCACCAAAAGGTACAAATGAAGCCATGCTAGTCATTGAAGCCTACAGGACACTTAGAGACAGGGGACCTTATCCAGCTGATCAAGTTATTAAGGATTTTGAAGGAAGNTTTGCTTTTGTTATCTATGA PCR markers development E D F B C A A Amplification of BAC: LeHba 140M01 (A), LeHba 026c13 genotypes (B),LeHba 244c09 (C), LeHba 015A04 (D), LeHba 075C18( E), negative control (F). C E B BAC sequencing with customized primer

  8. BAC and markersequences comparison LeHba026c13 TCCAATTTTGAGGCCAAGTATACATGCCAGAAGGGCAAAGATTACTGTGAAATTTCAAGA 314 cLPT-6E9 TCCAATTTTGAGGCCAAGTATACATGCCAGAAGGGCAAAGATTACTGTGAAATTTCAAGA 359 LeHba026c13 TCTGTATGGATTTACTGTTGAAGGGAATGTTGATGATGTTAATGTGTTGAATGAGGTTAG 374 cLPT-6E9 TCTGTATGGATTTACTGTTGAAGGGAATGTTGATGATGTTAATGTGTTGAATGAGGTTAG 419 LeHba026c13 GGAGAAAGTTAGGGAACAAGGGAAAGTTTGGTGGTCATTAGAAGCTAGCAAAGGTGCTAA 434 cLPT-6E9 GGAGAAAGTTAGGGAACAAGGGAAAGTTTGGTGGTCATTAGAAGCTAGCAAAGGTGCTAA 479

  9. IL mapping Multiple alignment between S. pennellii, M82, IL 12-2, BAC P161H10, EST T1045 and IL 8-1

  10. 1000 bp 300 bp FISH mapping  Dr. Stephen Stack (Colorado University) assistance LeHba026c13  Training in Prof. Hans de Jong Laboratory (Wageningen University)  Protocol set up

  11. Marker Bac analyzed Bac end sequencing Cornell matching Specific amplicon Amplicon Sequence match IL mapping Fish mapping Sequencing status t1211 LeHba021L02 in pipeline t989 LeHba032k07 LeHba015A04 - finishing cLPT-E9 LeHba026c1 Pd LeHba026c13v LeHba026c13n (1) (1) in pipeline C2At4g03280 LeHba140m01 - ? t1045 LeHba161H10 in pipeline t1487 LeHba260c13 - t1481 LeHba075c18 ? t1667 LeHba146I19 LeHba244C09 (1) (1) - ? in pipeline clet-8k4 LeHba189E07 LeHba161b22 Poor - tg 68 LeHba152M18 - Difficult tg 263 LeHba244J04 - Difficult t0028 LeHba163O04 ssr124 LeHba059A05 LeHba061j21 Summary… (1) (1) finishing (1) yes One BAC end (1) may be ? Contradictory data no

  12. ….Summary

  13. Sequencing

  14. Softwares for gene prediction are under evaluation and test • Implementation of automated analysis is under test • A secondary dbEST from NCBI primary data for Tomato was developed • Annotation EUSOL tasks: setting up a distributed functional annotation system comparative genomics

  15. Genescan prediction analysis LeHba032k07 LeHba 059A05

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