BAC SEQUENCING AND ANNOTATION DRIVEN BY EXPERIMENTAL DATA: AN APPLICATION FOR A SERINE-THREONINE KINASE ON TOMATO CHROMOSOME 12

Through the International Tomato Sequencing Project, the first BAC (Bacterial Artificial Chromosome) of chromosome 12 (Le_HBa0032K07) has been sequenced and submitted to the Solanaceae Genomics Network (SGN) repository. The preliminary annotation of the BAC was based on comparisons with a collection of about 215,000 Expressed Sequence Tags (ESTs) from tomato species and on their tentative consensus sequences (TCs). Considering the ESTs/TCs-to-genome alignments, 11 gene loci were located on this BAC. One of these was detected at the SP6 end of the BAC where a partial match of a tentative consensus sequence, generated by assembling 24 ESTs, was found. In order to verify both the reliability of the computationally detected gene locus and of its expressed product, we designed primers on the complete sequence of the TC. Then polymerase chain reactions (PCRs) were carried out: a 5000 bp fragment was amplified on genomic DNA, while a 500 bp fragment was obtained from cDNA amplification, as expected. This fragment was sequenced and then compared to the TC. The result of the sequence comparison confirms both the reliability of the TC and the expression of the gene. Finally, using the SGN BAC-ends database, we selected four BACs in order to extend the seed BAC Le_HBa0032K07 and to cover the full length of the TC.