BMC Genomics 2006, 7:12 http://www.biomedcentral.com/1471-2164/7/12 FGirgaupreevin1e flavonoid hydroxylase gene fragments isolated from 'Cabernet Sauvignon' genomic DNA Grapevine flavonoid hydroxylase gene fragments isolated from 'Cabernet Sauvignon' genomic DNA. The posi- tion of the conserved sites among plant flavonoid hydroxylases, on which the degenerate primers were designed, and the region of the gene fragments sequenced in grapevine are referenced to the gene Ht1 of Petunia × hybrida [GenBank:AF155332] for the flavonoid 3'-hydroxylase (a) and to the Petunia × hybrida GenBank:Z22544 for the flavonoid 3',5'-hydroxylase (b) [41]. The consensus amino acid sequences of grapevine VvF3'H and VvF3'5'H gene fragments are shown along with the position of the intron and (line below the sequence) the variable sites among all the sequenced fragments. The symbol '-' stands for invaria- ble position, '+' underlines two functional domains invariantly found in plant flavonoid hydroxylases, '*' indicates the site of the 23-bp deletion in the VvF3'H-2 sequence. DQ298205]. Once the degenerate primer sites had been removed, the sequences VvF3'5'H-1a, -1b and -1c were 1,120 bp long with a 403 bp intron; VvF3'5'H-2a and VvF3'5'H-2b were 1,122 bp long with a 405 bp intron. The translated coding region matched several plant F3'5'H genes in GenBank. VvF3'5'H-1a, -1b and -1c shared the highest identity score at the amino acid level with the accession AAP31058 of Gossypium hirsutum (81%, E = e- 111) as well as VvF3'5'H-2a and -2b did with the accession AAM51564 of Glycine max (82%, E = e-112). Translated sequences of VvF3'5'H-1a and VvF3'5'H-1b diverged from each other for two out of 239 amino acids. The same occurred between the sequences VvF3'5'H-2a and VvF3'5'H-2b (Figure 1). The sequences VvF3'5'H-1a and VvF3'5'H-1b had 93–94% identity at the amino acid level with VvF3'5'H-2a and VvF3'5'H-2b. All sequences matched the tentative consensus TC45860 obtained from 19 ESTs at the TIGR grape gene index. The 'Cabernet Sau- vignon' gene fragments identified in this work spanned the 187–424 amino acid region of TC45860 over a pre- dicted ORF of 508 amino acids. The sequences VvF3'5'H- 1a and VvF3'5'H-1b had 97% and 98% amino acid iden- tity, respectively, with TC45860; VvF3'5'H-2a and VvF3'5'H-2b peptide sequences had 95% identity with TC45860. The VvF3'5'H gene fragments partially over- lapped also with a EST (ID CTG1028815) of a 'Cabernet Sauvignon' berry cDNA library already analysed by MPSS http://mpss.udel.edu/grape. In order to predict how many functional copies of the gene VvF3'5'H are present in the grape genome we looked at the number of different ESTs available at the grape gene databases. Twelve EST single- tons held at the TIGR and the EST CTG1028815 spanned Page 4 of 17 (page number not for citation purposes)