Flavonoid 3',5'-hydroxylases (F3'5'Hs) and flavonoid 3'-hydroxylases (F3'Hs) are versatile enzymes that accept several phenylpropanoid substrates . Of particular interest for anthocyanin pigmentation is the 3',5'- or 3'-hydroxylation of naringenin and dihydrokaempferol. F3'5'Hs and F3'Hs compete for substrate recruitment and deliver their 3'5'- or 3'-OH products into the parallel synthesis of delphinidin and cyanidin , the precursors of blue and red anthocyanins in grape berries, respectively. Variation in anthocyanin profile within and between grape varieties is associated with differences in the ratio of F3'5'H to F3'H expression [3, 4].
Anthocyanin biosynthesis takes place over 8-10 weeks, from shortly after berry softening (~60 days after blooming) until harvest . F3'H s are expressed at comparable levels in both anthocyanin-pigmented and green-skinned varieties, before and after the onset of ripening [6, 4]. However, regulation of F3'5'H s is largely genotype-specific and responsive to environmental cues [3, 7]. The breadth of diversity in fruit colour among different grapevine accessions suggests a fine regulation of F3'5'H expression. Dark blue cultivars transcribe F3'5'H s at higher levels than light red cultivars, which nevertheless maintain traces of 3'5'-OH anthocyanins and barely detectable F3'5'H transcripts. In green-skinned cultivars, F3'5'H transcripts are completely absent [8, 9]. The invariant presence of some 3'5'-OH anthocyanins in red pigmented grapes contrasts with many other flowering plants such as roses, carnations, chrysanthemums, lilies, gerbera, and Arabidopsis, which accumulate anthocyanins but do not synthesise 3'5'-OH derivatives.
The lack of grapevines with F3'5'H loss-of-function genotypes could be explained either by selection, which acted against knockout mutations, or by gene redundancy, which obscured the effect of single-gene loss/silencing. The observation that an absence of 3'5'-OH anthocyanins is generally tolerated in plants disfavours the first hypothesis. Furthermore, gene redundancy of F3'5'H s is commonplace in grape genomes [10, 11], contrasting with most other species that have single or two-copy F3'5'H s, or none at all. We have previously shown that F3'5'Hs are highly duplicated, with multiple copies arrayed in clustered contigs of the 'Cabernet Sauvignon' physical map . The genome assembly of the nearly-homozygous line PN40024  allows a deeper investigation into the structure of the F3'5'H locus and into the evolutionary events that caused their proliferation in grapevine.
Expansion of gene families is common in plant genomes , and results from various mechanisms of duplication: whole-genome duplication (WGD), segmental duplication, tandem duplication, and transpositional duplication [14, 15]. WGDs have repeatedly occurred over evolutionary time in the common ancestor of eudicots and in specific lineages [12, 16]. Segmental duplications occur over chromosomal regions, which may undergo subsequent rearrangement. Tandem duplications generate nearby gene copies . Small-scale duplications may also cause transposition of one of the duplicate genes to an ectopic site. In this paper, local duplications of small fragments (<10 kb) containing a single gene are referred to as tandem duplications. Duplication of DNA blocks >10 kb are referred to as segmental duplications.
Retention of duplicate genes results from a stochastic process, in which the effect of the earliest mutation occurring after duplication governs the fate of extra copies. Deleterious mutations occur much more frequently than mutations resulting in novel and favourable functions . Following this assumption, gene disruption would largely prevail, with genomes populated by vestiges of ancient duplicates. This raises the question as to why intact duplicates are maintained and expressed much more frequently than expected by chance. According to the duplication-degeneration-complementation (DDC) model , degenerative mutations promote preservation of duplicate genes. Deleterious mutations in regulatory regions could eliminate different cis -elements in either duplicate, making both copies necessary to provide the full-complement of the expression profile of the ancestral single copy . This kind of partitioned expression among duplicate genes is referred to as subfunctionalisation, and includes differential expression among organs and developmental stages, or in response to environmental cues [20–25].
Duplicate genes involved in secondary metabolism or that are responsive to environmental stimuli appear to be more frequently maintained [26–28], and have more highly diverged transcriptional patterns and intraspecific variation in expression  than duplicate genes in other categories. The pioneering study of  provided a paradigmatic case of duplication and transcriptional diversification in members of the stilbene synthase gene family in grapevine. It is generally assumed that maintenance of duplicate genes provides a foundation for consolidation and refinement of established functions, particularly in secondary metabolism, by preserving extra copies that guarantee a gene reservoir for adaptive evolution, free from the constraints of purifying selection [31–33].
In this paper, we present (i) the evolutionary path that led to the structural architecture of the F3'5'H gene family in grapevine, (ii) the transcriptional sub-functionalisation of duplicate copies among organs and developmental stages, and (iii) the extent of variation of expression patterns in four cultivars with divergent anthocyanin profiles.