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

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Presentation transcript:

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

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. Amalia Barone (UniNa) Dr. Francesca Cecchi (BIOGEN) Dr. Maria Luisa Chiusano (UniNa) Dr. Mara Ercolano (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)

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

CT211A TG CT19 A 6.4 TG CT CT TG CD6A 1.6 TG TG CT287A 6.3 TG28A 1.0 CT239B 2.0 TG CT CT80B 6.1 CT TG CD2 12 o TM14B Chromosome 12 Euchromatin portion = 11Mb Projected # of BACs 113

 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.

Restriction enzyme digestion and BAC end sequencing Fingerprinting analysis with the restriction enzyme BamHI SP6 and T7 end sequences alignament and comparison with SGN database

BAC sequencing with customized primer >SGN-E [] (trimmed) ATCTTGAAGCTTAAAAAAAACAAAAAAAAAAACAAACGATGTTGGCTATATTTCACAAG GCATTTGCTCATCCTCCTGAAGAATTAAATAGTCCTGCATCAAAAAAATGTTTACTTCCA CAACAAACGCTACAAAAATTCGTATCGACTCGTCCATTTGACACCTCTTATGTTACTTTT GGAGATGTTGCTGCTCTTGCTTTTGTTCGTCCTAATTGCACCTCCTTGCTCAATCACA AACAAAGGTATTTTTGTGGTTATGATGATATTTACTGTTTGTTCATGGGGAGTTTGAACA ATTTGTGTGCACAAATCAAACAATATGGGCTATCACCAAAAGGTACAAATGAAGCCATG CTAGTCATTGAAGCCTACAGGACACTTAGAGACAGGGGACCTTATCCAGCTGATCAAG TTATTAAGGATTTTGAAGGAAGNTTTGCTTTTGTTATCTATGA Primer design Amplification of BAC: LeHba 140M01 (A), LeHba 026c13 genotypes (B),LeHba 244c09 (C), LeHba 015A04 (D), LeHba 075C18( E), negative control (F). A B CE A BC D E F PCR markers development

BAC and marker sequences comparison LeHba026c13 TCCAATTTTGAGGCCAAGTATACATGCCAGAAGGGCAAAGATTACTGTGAAATTTCAAGA 314 cLPT-6E9 TCCAATTTTGAGGCCAAGTATACATGCCAGAAGGGCAAAGATTACTGTGAAATTTCAAGA 359 LeHba026c13 TCTGTATGGATTTACTGTTGAAGGGAATGTTGATGATGTTAATGTGTTGAATGAGGTTAG 374 cLPT-6E9 TCTGTATGGATTTACTGTTGAAGGGAATGTTGATGATGTTAATGTGTTGAATGAGGTTAG 419 LeHba026c13 GGAGAAAGTTAGGGAACAAGGGAAAGTTTGGTGGTCATTAGAAGCTAGCAAAGGTGCTAA 434 cLPT-6E9 GGAGAAAGTTAGGGAACAAGGGAAAGTTTGGTGGTCATTAGAAGCTAGCAAAGGTGCTAA 479

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

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

Summary… (1) ? One BAC end Contradictory data yes no may be Marker Bac analyzedBac end sequencing Cornell matching Specific amplicon Amplicon Sequence match IL mappingFish mappingSequencing status t1211LeHba021L02 in pipeline t989LeHba032k07 LeHba015A04 - finishing cLPT-E9 LeHba026c1 Pd LeHba026c13v LeHba026c13n (1) in pipeline C2At4g03280LeHba140m01-? t1045LeHba161H10 in pipeline t1487LeHba260c13- t1481 LeHba075c18? t1667 LeHba146I19 LeHba244C09 (1) (1) - ? in pipeline clet-8k4LeHba189E07 LeHba161b22 Poor - tg 68LeHba152M18-Difficult tg 263LeHba244J04-Difficult t0028LeHba163O04 ssr124LeHba059A05 LeHba061j21 (1) finishing

MarkerBACBac end sequencing Marker association p62LeHba in progress tg 394LeHba 107D15in progress t1676LeHba 115G22in progress t 1504LeHba 147G17in progress cd 2Le Hba 053L10in progress ….Summary

 Sequencing

-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 EUSOL tasks: setting up a distributed functional annotation system comparative genomics  Annotation

Genescan prediction analysis LeHba 059A05 LeHba032k07