Code F3'H F3'5'H F3 F3-1 F35-1 F35-2 F3int F35-1int F35-2int 38C24-FM 38C24-RM 17K14-FM 9C17-FM 40A22-FM 5A23-FM Sense primer F3'H_S F3'5'H_S VvF3'H_S VvF3'H-indel_S Vv3'5'H-1A_S Vv3'5'H-2A_S VvF3'H-intron_S Vv3'5'H-1A-intron_S Vv3'5'H-2A-intron_S 38C24-FM_S 38C24-RM_S 17K4-FM_S 9C17-FM_S 40A22-FM_S 5A23-FM_S F3'H_AS F3'5'H_AS VvF3'H_AS VvF3'H_AS VvF3'5'H-1A_AS Vv3'5'H-2A_AS VvF3'H-intron_AS Vv3'5'H-intron_AS Vv3'5'H-intron_AS 38C24-FM_AS 38C24-RM_AS 17K4-FM_AS 9C17-FM_AS 40A22-FM_AS 5A23-FM_AS BMC Genomics 2006, 7:12 http://www.biomedcentral.com/1471-2164/7/12 Table 2: Primers pairs and PCR conditions used for the amplification of grapevine flavonoid hydroxylase genes. Anti-sense primer Expected size (bp) Annealing temp. (°C) 825* 1020* 196 166 156 152 255 589 593 312 330 327 358 327 218 60 56 58 58 58 65 58 58 58 58 58 58 58 58 54 * in absence of introns. plants Antirrhinum majus [11], Campanula medium [12], Dianthus caryophyllus [13], Gentiana triflora [14], Ipomoea purpurea, [15], Petunia hybrida [16], Phalaenopsis [17] and Vinca major [18]. In all these species, F3'H and F3',5'H are found as low/medium copy number genes and have been genetically manipulated to produce novel flower pigmen- tation. In grapevine, only tentative consensus (TC) sequences for F3'H and F3',5'H genes are available in the TIGR grape gene database http://www.tigr.org. TCs were built based on partially overlapping ESTs from cDNA libraries obtained from different genotypes and tissues. This makes it unfeasible to predict or rule out the presence of different isogenes. This paper reports (1) the isolation of flavonoid 3'- and 3',5'-hydroxylase gene members in grapevine via sequence homology, (2) the assessment of their chromosomal loca- tion by genetic and physical mapping, (3) their transcript profiles in different tissues, (4) the time-course of expres- sion in berry skin of the red cultivar 'Merlot' from pre- véraison to harvest compared to the curves of accumula- tion of anthocyanins, catechins and total polyphenols. Results and discussion Isolation of flavonoid 3'- and 3',5'-hydroxylase genes in grapevine Gene isolation was accomplished via sequence homology with the help of newly designed degenerate primers. PCR bands that were amplified from genomic DNA of Vitis vin- ifera 'Cabernet Sauvignon' were longer than predicted on the basis of plant flavonoid hydroxylase transcripts (Table 1 and 2). VvF3'H-2 [GenBank:DQ298196-DQ298200]. Once the primer sites had been removed, the sequences VvF3'H-1a to -1d were 1,034 bp long with a 95 bp intron; VvF3'H-2 was 1,011 bp long with a 95 bp intron. Sizing of genomic amplicons performed by capillary electrophoresis con- firmed that the two classes of amplicons diverge from each other for a 23-bp indel. Sequence identity was between 99.8 and 99.9% (99.7–99.8% for the coding region) among the sequences VvF3'H-1. The presence of mutations introduced by Taq polymerase during the PCR process and immortalised through the cloning of single PCR-amplified molecules could not be ruled out as the origin of differences among the four VvF3'H-1 sequences. The sequences VvF3'H-1a to -1d translated into protein fragments of 313 amino acids with 311 positions con- served among the sequences when pair-wise comparisons were performed (Figure 1). VvF3'H-2 contained a frame shift mutation due to a 23-bp deletion in the coding region. Function was putatively assigned to VvF3'H-1 based on its highest identity match in GenBank at the pro- tein level (76% identity, E = 8e-134) that corresponded to a flavonoid 3'-hydroxylase of Petunia × hybrida [Gen- Bank:AAD56282]. The nucleotide coding sequence of VvF3'H-1 matched the grapevine tentative consensus sequence TC42042 that has been predicted from three partially overlapping ESTs sequenced in 'Cabernet Sauvi- gnon' and 'Chardonnay' (TIGR grape gene index). The VvF3'H-1 protein fragments spanned the 1–245 amino acid region of TC42042 and extended it on the 5' end of the gene by 67 amino acids. Amino acid sequence identity between the four VvF3'H-1 protein fragments and TC42042 was 95–96%. Five different sequences were isolated with the F3'H degenerate primers following SSCP analysis and were given the designation VvF3'H-1a, -1b, -1c, -1d, and The degenerate primers targeting F3'5'H yielded five sequences that were given the designation VvF3'5'H-1a, - 1b, -1c, VvF3'5'H-2a, -2b [GenBank:DQ298201- Page 3 of 17 (page number not for citation purposes)