BMC Genomics 2006, 7:12 http://www.biomedcentral.com/1471-2164/7/12 assembled. A segmental duplication spanning the mark- ers F35-1int, F35-2int, BES40A22-FM, BES17K04-FM and BES38C24-FM that occurred within 2.2-3.0 cM on LG6 could explain the physical organization of flavonoid 3',5'- hydroxylase isogenes as it has been outlined in Figure 7. The contig ctg2373 that comprised only three BAC clones could not be unambiguously positioned in relation to the other contigs. BES markers from the other contigs did not match any BAC clones of ctg2373 but marker BES17K04- FM matched both ctg313 and ctg1320. Marker BES17K04- FM is therefore located within the segmental duplication. It remains uncertain whether ctg2373 partially overlaps the region of ctg313 and split from it due to high hetero- zygosity or if it actually covers an adjacent locus. Under the assumption of BAC alignment between BAC clones 9C17 and 17K04 that is supported by genetic co-segrega- tion of the corresponding BES and by co-amplification by marker BES17K04-FM, ctg2373 would carry the haplotype allelic to BACs 6H20, 9C17, 31P20 at the BES17K04-FM SSCP marker and the VvF3'5'H-1 gene fragment present in BAC17K04 would miss a counterpart on the homologous chromosome. Otherwise, ctg2373 would identify a forth different locus of VvF3'5'H-1, located in the surrounding region and under-represented among the BAC clones of the library. Based on the data available to us, we propose that the chromosomal region spanning the VvF3'5H gene cluster includes four copies of VvF3'5'H-1 and five to seven cop- ies of VvF3'5'H-2, all sharing a high sequence similarity. We were able to present a preliminary physical map of this cluster of genes by a combination of computational assembly of fingerprinted BACs and by PCR-based align- ment of BAC clones. The region has likely originated from both tandem duplications that generated nearby gene copies (i.e. paralogs within BACs 28B15 and 99I17 in the ctg313) and from segmental intrachromosomal duplica- tions involving a larger block of DNA (i.e. blocks of para- logs duplicated into ctg313 and ctg1320). Segmental duplications are an important source of gene evolution [25] and they have been estimated to cover 1.7–2.0% of the mouse genome [26] and 4% of the human genome [27]. In the rat genome, duplicated genes are largely con- fined into <1 Mb intrachromosomal duplications rather than widely interspersed through the genome [28]. A sim- ilar model of genome dynamics could also have shaped plant genomes as it is emerging for model species [29]. Expression analysis of VvF3'H and VvF3'5'H along with major structural genes of the flavonoid pathway in different grape tissues The genes encoding both flavonoid 3'-hydroxylase and flavonoid 3'-5'-hydroxylases are widely expressed in grapevine organs (Figure 8). Transcripts of gene VvF3'H-1 were detected in all tissues tested. VvF3'H-1 was strongly expressed in green apical leaflets and flowers but it was only slightly detectable in fully expanded leaves. It was also present in all berry tissues. The other sequence VvF3'H-2, which translated into a truncated flavonoid 3'- hydroxylase due to a 23-bp frameshift deletion, was apparently not expressed and is likely to be a pseudogene. Transcripts of VvF3'5'H-1 were present in all tissues. High levels of expression were detected in pigmented apical leaflets, flowers, seeds and berries but not in green fully expanded leaves and roots. Differential expression could be detected between green berry skin and red berry skin, the expression being higher in pigmented berry skin. The expression pattern of VvF3'5'H-1 mimicked the expres- sion profile of Chs2, Chs3 and the gene coding for fla- vanone hydroxylase (F3H). The gene VvF3'5'H-2 expressed transcripts of different size that could be distin- guished on agarose gel (VvF3'5'H-2-short, VvF3'5'H-2- medium and VvF3'5'H-2-long). The two shorter bands were invariantly present in all tissues, even though at a dif- ferent level of expression. The longest band was strongly expressed only in red berry skin, whilst it was slightly detectable in seeds and not detectable at all in other tis- sues. The expression of most flavonoid genes, including those coding for flavonoid hydroxylases, was more intensively activated in flowers and seeds than in other organs. Seeds expressed all isoforms of the flavonoid hydroxylase genes identified in this work, whilst all but VvF3'5'H-2-long were expressed in flowers. Among berry tissues, flesh poorly expressed the genes of the flavonoid pathway, except for BAN, VvF3'H-1, VvF3'5'H-1, VvF3'5'H-2-short and VvF3'5'H-2-medium. In the epicarp of berries col- lected at different developmental stages, Fls1 and BAN were more expressed at the pre-véraison stage; Chs3, F3H and VvF3'5'H-1 were induced in red skin compared to green skin; UFGT, VvF3'H-1 and VvF3'5'H-2-long were strongly expressed in red skin but were undetectable in green skin. The flavonoid pathway does not exclusively lead to the synthesis of anthocyanins and several flavonoid hydroxy- lase isogenes were therefore expressed also in non-pig- mented tissues. Berry flesh and seeds accumulate non- anthocyanin flavonoids such as proanthocyanidins and catechins. Their synthesis is catalysed downstream of the flavonoid hydroxylases by the genes DFR, LDOX and BAN. Gene BAN was strongly expressed in seeds, berry flesh and berry skin at pre-véraison, whilst was almost undetectable in post-véraison berry skin at the stage when the rate of accumulation of proanthocyanins and cate- chins is declining (Figure 9). Sunlight exposed tissues expressed the gene coding for flavonol synthase (Fls1). Flavonol synthase converts the products of flavonoid Page 11 of 17 (page number not for citation purposes)