BMC Genomics 2006, 7:12 http://www.biomedcentral.com/1471-2164/7/12 EFxigpurersesi1o0n of VvF3'5'H-2 during the progress of ripening Expression of VvF3'5'H-2 during the progress of ripen- ing. Length polymorphic transcripts of VvF3'5'H-2 were detected semi-quantitatively after cDNA samples had been normalised upon actin gene expression, at the sampling dates reported in Fig 9. The short, medium and long transcripts were named according to the fragment size amplified from genomic DNA. At sampling 5, green berries (5g) were kept separated from red berries (5r). PCR amplification on 'Mer- lot' genomic DNA was loaded in the right extreme lane. véraison, from the onset of colour transition till whole- bunch pigmentation, but remained roughly unmodified throughout the last four weeks of ripening. Although the anthocyanin profile of 'Merlot' was largely composed by malvidin and tri-substituted anthocyanins at harvest, derivatives of cyanidin were more promptly synthesised at the very early stage of véraison. The pattern of expression of VvF3'H-1, VvF3'5'H-1 and VvF3'5'H-2 is thus compati- ble with the hypothesis that they are strongly involved in the synthesis of the different types of anthocyanins and they affect anthocyanin composition in berry skin. Conclusion We have shown that genes coding for flavonoid 3'- and 3',5'-hydroxylases are present as low/medium copy mem- bers in the grapevine genome and are organised in com- plex gene clusters that most likely resulted from gene duplication and intra-chromosomal segmental duplica- tion. These genes are expressed in coordination with other genes of the flavonoid pathway in any tissues of the grape plant that are known to accumulate flavonoids, such as leaves, flowers, roots, seeds and, particularly, in skin of ripening berries. RT-PCR showed a strong relationship between the expression of VvF3'H-1 and VvF3'5'H and the kinetics of accumulation of cyanidin- and delphinidin- based anthocyanins in the red-skinned variety 'Merlot'. The presence of multiple putative 3',5'-hydroxylase iso- genes which emerged from fine structural analysis at phys- ical level, has first been shown for plant hydroxylases. This complexity was not unexpected for species like grape which synthesize large amounts of different classes of polyphenols and display a continuous variation of berry skin colour from brick red to dark blue among the so called red-skinned cultivars. Methods Plant material Genomic DNA was extracted from young leaves [30]. 'Cabernet Sauvignon' was used for cloning flavonoid 3'- and 3',5'-hydroxylase genes. Forty six full-sibs of the cross 'Cabernet Sauvignon' × Vitis 'breeding line 20/3' were used for genetic mapping. Gene expression was analysed in the red variety 'Merlot'. Apical leaflets of 1 cm2 surface area, fully expanded leaves (>10 cm2) and adventitious rootlets were collected from canes grown in hydroponic culture for two months. Flower buttons were collected one week before blooming, seeds and flesh were extracted from berries collected two weeks after véraison. Transcript and metabolite profiling in berry skin was performed on samples of 40 berries collected at ten ripening stages from pre-véraison (July 15th, 2004) till harvest (September 28th, 2004) from plots of twelve vines replicated four times. Cloning of flavonoid 3'- and 3',5'-hydroxylase genes with the help of degenerate primers Degenerate primers were designed on two specific and conserved regions of plant F3'H and F3',5'H genes (Table 1, Figure 1). PCRs were performed in a 40 µl volume con- taining 200 µM of each dNTP, 2.5 µM of each primer, 1 U of HotMaster Taq polymerase (Eppendorf) with 2.5 mM MgCl2 buffer, and 40 ng of template DNA. The denatura- tion step was 95°C for 1 min, followed by 40 cycles of 92°C for 45 s, 60°C for F3'H primers and 56°C for F3',5'H primers for 50 s, 65°C for 90 s. Amplified bands were extracted from agarose gel with Nucleospin Extract (Macherey-Nagel) and cloned into pGEM-T vector (Promega). Unique clones were identified by means of PCR with M13 universal primers and SSCP-fingerprinting of the insert. Selected clones were sequenced with BigDye terminator chemistry and a MegaBace500 sequencer (Amersham Biosciences). Sequence analysis Nucleotide and amino acid sequences were compared with GenBank accessions, with EST singletons and tenta- tive consensus sequences (TCs) held at the TIGR grape gene index. Conceptual translation was obtained with DNAclub software. Amino acid entries of 670 plant cyto- chrome P450 monooxygenases were downloaded from GenBank as of November 30th, 2004. Cluster analysis of plant cyt P450s and the candidate grape flavonoid hydroxylases was done with the Neighbor-joining method. Genetic mapping Nested primers were designed to amplify an exon of grapevine flavonoid 3'- and 3',5'-hydroxylase genes (F3, F3-1, F35-1, F35-2, see Table 1 and 2). Additional primer pairs (F3int, F35-1int and F35-2int) were designed in the exon sequences flanking an intron for detecting SNPs use- Page 14 of 17 (page number not for citation purposes)