BMC Genomics 2006, 7:12 http://www.biomedcentral.com/1471-2164/7/12 FNi-gJucrluest2ering of Vitis vinifera flavonoid 3',5'-hydroxylase genes N-J clustering of Vitis vinifera flavonoid 3',5'-hydroxylase genes. Nucleotide sequences VvF3'5'H-1a, -1b, -1c, -2a and -2b from 'Cabernet Sauvignon' (in bold) were aligned along with ESTs singletons and the resulting tentative consensus (TC45860) for grapevine F3'5'H held at the TIGR grape gene index. ESTs covered both the 5'-end of the VvF3'5'H gene fragments (left) and the 3'-end (right). The tentative consensus has been forced as an outgroup. For each EST, the GenBank ID is reported along with the source genotype and tissue. the 5' end of the VvF3'5'H gene fragments whilst four ESTs were alignable to the 3' end. All 17 ESTs, irrespective of their tissue-specific localisation and the source genotype, were aligned with the 'Cabernet Sauvignon' genomic sequences VvF3'5'H-1a, -1b, -2a, -2b (Figure 2). Six ESTs clustered with the 'Cabernet Sauvignon' VvF3'5'H-1a and VvF3'5'H-1b gene fragments. Those ESTs were detected in leaves of the white variety 'Chardonnay' and in petioles and berries of the red variety 'Cabernet Sauvignon'. Eleven ESTs grouped together with the 'Cabernet Sauvignon' VvF3'5'H-2a and VvF3'5'H-2b genomic sequences. The lat- ter ESTs were present in various tissues (leaves and ber- ries) of red ('Cabernet Sauvignon' and 'Syrah') and white ('Chardonnay') cultivars. Two major groups of F3'5'H transcripts including either VvF3'5'H-1 or VvF3'5'H-2 can be detected in different tissues of both red and white grapevines, suggesting that at least two genes encoding CYP75A cyt P450 hydroxylases are expressed in flavonoid- synthesizing tissues. Due to the putative presence of SNPs in ESTs from different cultivars, it was not possible to ascertain the number of functional copies belonging to each group, but together with the number of highly simi- lar sequences obtained from 'Cabernet Sauvignon' genomic DNA, this pointed to the presence of multiple gene members coding for different isoforms of flavonoid hydroxylases in the grape genome. In order to test the phylogenetic relationship between grapevine hydroxylases and the multi-functional family of plant monooxygenases, a Neighbor-Joining tree was con- structed using translated sequences of VvF3'H, VvF3'5'H- 1, VvF3'5'H-2 and 670 protein entries of plant cyt P450 monooxygenases (Figure 3). VvF3'H and VvF3'5'H grouped solely with P450 proteins specifically annotated as flavonoid hydroxylases belonging to the family-specific branch of CYP75 monooxygenases. VvF3'H and VvF3'5'H split into two terminal branches, each one hosting either CYP75B or CYP75A monooxygenases. Although most plant P450s are of polyphyletic origin and some P450 families are lineage-specific [19], CYP75 P450s are ancient genes that existed even before the separation between gymnosperms and flowering plants [20]. The ancient origin of CYP75 monooxygenases is also sup- ported by the finding that intron position of VvF3'H and VvF3'5'H was conserved when compared to other plant genes coding for flavonoid hydroxylases such as the Arabi- dopsis gene TT7 [21]. Genetic mapping Genetic loci harbouring genes that code for flavonoid hydroxylases were mapped using the single strand confor- mational polymorphism (SSCP) analysis of F3'H and F3'5'H sequences in an F1 pseudo-testcross mapping pop- ulation originated from the cross 'Cabernet Sauvignon' Page 5 of 17 (page number not for citation purposes)