Expansion and subfunctionalisation of flavonoid 3',5'-hydroxylases in the grapevine lineage
© Falginella et al; licensee BioMed Central Ltd. 2010
Received: 27 June 2010
Accepted: 12 October 2010
Published: 12 October 2010
Flavonoid 3',5'-hydroxylases (F3'5'Hs) and flavonoid 3'-hydroxylases (F3'Hs) competitively control the synthesis of delphinidin and cyanidin, the precursors of blue and red anthocyanins. In most plants, F3'5'H genes are present in low-copy number, but in grapevine they are highly redundant.
The first increase in F3'5'H copy number occurred in the progenitor of the eudicot clade at the time of the γ triplication. Further proliferation of F3'5'H s has occurred in one of the paleologous loci after the separation of Vitaceae from other eurosids, giving rise to 15 paralogues within 650 kb. Twelve reside in 9 tandem blocks of ~35-55 kb that share 91-99% identity. The second paleologous F3'5'H has been maintained as an orphan gene in grapevines, and lacks orthologues in other plants. Duplicate F3'5'H s have spatially and temporally partitioned expression profiles in grapevine. The orphan F3'5'H copy is highly expressed in vegetative organs. More recent duplicate F3'5'H s are predominately expressed in berry skins. They differ only slightly in the coding region, but are distinguished in the structure of the promoter. Differences in cis-regulatory sequences of promoter regions are paralleled by temporal specialisation of gene transcription during fruit ripening. Variation in anthocyanin profiles consistently reflects changes in the F3'5'H mRNA pool across different cultivars. More F3'5'H copies are expressed at high levels in grapevine varieties with 93-94% of 3'5'-OH anthocyanins. In grapevines depleted in 3'5'-OH anthocyanins (15-45%), fewer F3'5'H copies are transcribed, and at lower levels. Conversely, only two copies of the gene encoding the competing F3'H enzyme are present in the grape genome; one copy is expressed in both vegetative and reproductive organs at comparable levels among cultivars, while the other is transcriptionally silent.
These results suggest that expansion and subfunctionalisation of F3'5'H s have increased the complexity and diversification of the fruit colour phenotype among red grape varieties.
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.
F3'5'Hs and F3'Hs in grapevine: genomic location and phylogeny
Seven F3'5'H s on grapevine chr6 (F3'5'Hd, -f, -j, -l, -m, -n, -o) and F3'5'Hp on chr8 encode full-length proteins. In the haplotype of PN40024, the remainder gene models are either gene fragments without homology outside of conserved regions, or coding regions interrupted by transposable elements (TEs) or frameshift indels [see Additional file 3].
Grapevine contains two copies of F3'H (F3'Ha and F3'Hb) located in a 25-kb interval on chr17 [see Additional file 4a]. F3'H s reside in two blocks of ~5 kb, which share 93.5% identity over 4.3 kb of conserved sequence, separated by ~16 kb largely consisting of repetitive elements. Both F3'H s encode full-length proteins. F3'Ha and F3'Hb share 97% amino acid identity, but their genomic sequences differ extensively due to a large indel in the terminal intron [see Additional file 4]. Other genes surrounding the two F3'H copies on chr17 are not collinear with genes surrounding F3'H s on chr6 or on chr8.
The two copies of grapevine F3'H grouped tightly (Figure 2). F3'H s are consistently present in one or a few copies across fully sequenced plant species.
Evolution of the F3'5'H locus on chromosome 6
The pattern and mode of gene duplication were characterised through several approaches: (i) dot plot self-comparison of the entire locus, (ii) conservation of non-coding sequences, TE patterns, and sequence divergence between long terminal repeats (LTRs) of retrotransposons in duplicate blocks, (iii) level of identity between 10-kb windows around each F3'5'H, (iv) intron divergence between the most recent duplicated F3'5'H s, and (v) conservation of duplicate F3'5'H s across the family Vitaceae.
Sequence conservation in a 10-kb window surrounding each F3'5'H copy supports the hypothesis that most of the copies were generated by duplications of the entire segment in which they reside [see Additional file 7], with the following exceptions. Downstream of the segmental duplications, sequence similarity between the nearly identical copies F3'5'Hm and F3'5'Hn does not extend more than ~ 2 kb beyond each side of their coding regions. F3'5'Hk and -l are both located upstream of block 9. F3'5'Hl and its 5' non-coding region are dissimilar from the paralogous F3'5'H in duplicate blocks 4 and 9, as though F3'5'Hl originated from a small scale duplication of F3'5'Hg, -m, or -n. F3'5'Ho, the copy at the far extremity of the locus, shares low similarity only upstream of the coding region with F3'5'Ha, -b, -c, -d, -e, and -h. F3'5'Hp, the copy on chr8, has no similarity outside of the coding region with other F3'5'H s.
Intronic sequences of highly similar paralogous F3'5'H s reflect the relatedness of the entirety of the duplicated block in which each F3'5'H resides [see Additional file 5b]. The few F3'5'H s that lie in pairs at the forefront of a duplicate block (F3'5'Ha and -b; F3'5'Hc and -d) are less similar within the pair than with a member of a different pair. Thus, paired F3'5'H s at the forefront of blocks 1 and 2 originated from an ectopic duplication before the duplication of the corresponding segment. The absence of intronless F3'5'H s excluded a role for retroposition in the process of gene duplication.
Conservation of duplicate F3'5'H s in the family Vitaceae was assayed by PCR with copy-specific primers. The orphan F3'5'Hp gene on chr8 was detected in the genera Parthenocissus and Vitis, while it was faintly amplified in Ampelopsis, likely due to more divergent priming sites [see Additional file 8]. In contrast, only a few primer pairs that amplified the most recent duplicate genes in Vitis genomes yielded amplicons in Parthenocissus or Ampelopsis. A wide sample of cultivars and species within the genus Vitis bears the marks of that expansion [see Additional file 8], including wine and table cultivars of Vitis vinifera, Asian and American Vitis species, and the muscadine grape.
Prediction of functional domains among duplicate F3'5'H s
According to  and , six functional domains in the F3'5'H enzyme are important for the determination of substrate specificity and 3' vs. 3'5'-OH activity (substrate recognition sites, SRS; candidate region, CR1). F3'5'Ha, -c, -e, and -h are truncated in the PN40024 genome, and lack one or more functional domains [see Additional file 9]. All other grapevine F3'5'H s except F3'5'Ho have invariant amino acids specific for 3'5'-hydroxylation activity. In plants, F3'5'H s are conserved at three critical positions in the CR1 (positions 1, 3, and 10, which correspond to amino acids 178, 180, and 187 in the Osteospermum F3'5'H reference sequence used in ) and at two positions in the SRS6 (positions 5 and 8, which correspond to amino acids 484 and 487 in Osteospermum F3'5'H) [see Additional file 9]. All grapevine F3'5'H s that diverged less than 4DTV ~0.046 show complete amino acid conservation at the CR1 and SRS6 domains. F3'5'Hp on chr8 and F3'5'Ho, -m, and -n, the most divergent copies in the F3'5'H array on chr6, have a Met-to-Ile substitution at CR1 position 3 with respect to other paralogues. This substitution is shared with F3'5'H s in grasses. F3'5'Ho also has an Ala-to-Thr substitution at SRS6 position 8, which is shared with corn and sorghum F3'5'H s, as well as with most of the F3'H s. F3'5'Hp has an Ala-to-Val substitution at the same position, which is uniquely shared with F3'5'H s from orchids. F3'5'Ho has extensively diverged from all other F3'5'H s at SRS1 and SRS2, while F3'5'Hp has peculiar amino acid substitutions at SRS2, SRS4, and SRS5.
Variation in promoter regions of duplicate F3'5'H s
Deletions may have asymmetrically erased cis-elements from regulatory regions of duplicate F3'5'H s. Thus, the 2-kb promoter regions of duplicate F3'5'H s were searched for DNA-binding motifs (Figure 6). Segments that were alternatively maintained in either promoter contained binding sites for Myb-type transcription factors, light-responsive and drought-inducible cis-elements, motifs sensitive to ABA and methyl-jasmonate, and heat stress responsive motifs. Relatedness between the alignable regions of duplicate promoters was also evident from a phylogenetic tree [see Additional file 5c].
Spatial expression patterns of duplicate F3'5'H s and F3'H s
Expression analyses were conducted on nine out of the sixteen F3'5'H copies for which primer pairs could individually distinguish each paralogue and that passed the thresholds of PCR efficiency as set in the Methods section.
F3'Ha is widely expressed in many organs [see Additional file 4b]. In berry skins, F3'Ha expression increased 2-fold at full veraison, and then remained constant during the later stages of ripening [see Additional file 4d]. Transcripts of F3'Hb were never detected in the organs analysed in this study [see Additional file 4c] and weak expression of this copy was detected exclusively in adventitious roots of 'Cabernet Sauvignon' .
Expression of the F3'5'H gene family and variation of anthocyanin profiles across different cultivars
Berries of four cultivars were sampled at eight developmental stages in order to quantify cumulative expression of the F3'5'H gene family and relative contribution of individual F3'5'H copies, and to determine anthocyanin profiles. The accessions 'Aglianico', 'Grignolino', 'Marzemino', and 'Nebbiolo' were chosen for their contrasting phenotypes of fruit colour, based on literature reports [4, 9].
As a whole, expression of the F3'5'H gene family levelled off before veraison [see Additional file 12], in step with other genes of the flavonoid pathway . F3'5'H s became increasingly more expressed at 10% veraison, peaking at full-veraison and ten days after full-veraison. Expression then declined two weeks before harvest and at harvest, but remained at higher levels than those detected before the onset of ripening.
A three-way ANOVA was used to decouple and test the significance of three factors that contributed to the observed variation of expression patterns: gene-copy, cultivar, and developmental stage [see Additional file 12]. All three factors were significant, as well as the interactions: gene-copy × developmental stage, gene-copy × cultivar, cultivar × developmental stage, and gene-copy × cultivar × developmental stage (P < 0.00001).
Distinct temporal expression patterns of duplicate F3'5'H s during ripening
Expansion of the F3'5'H family in grapevine
Gene-copy number of F3'5'H s has increased in the grapevine lineage through recurrent cycles of duplication. The most ancient duplication resulted in two F3'5'H loci. One of these, F3'5'Hp, has been maintained as a single-copy gene on chr8 in grapevine and other Vitaceae but lost from other dicot genomes. The other was the founder of the present-day F3'5'H gene array on chr6, orthologous to the F3'5'H s expressed in other dicot species and syntenic with the F3'5'H loci found in poplar and papaya (Figure 1). The 4DTV distance between F3'5'Hp and other F3'5'H copies is close to the peak of 4DTV distances between grape paleologues observed by Tang and coworkers  (Figure 3). Timing of the earliest F3'5'H duplication is therefore coincident with the event of eudicot γ hexaploidy , and the chromosomes in which the duplicate genes reside are indeed paleologous chromosomes .
The orphan copy F3'5'Hp is predominantly expressed in grape vegetative organs, in contrast with the F3'5'H copies on chr6, which are predominantly expressed in fruit (Figure 7). Several amino acid substitutions in F3'5'Hp are shared with F3'H s and monocot F3'5'H s. For instance, F3'5'H s are present in many monocot species, but in all cases studied, their transcription is uncoupled from the expression of other genes in the anthocyanin pathway. As a result monocots seldom accumulate 3'5'-OH anthocyanins . For example, seed coats of rice varieties with dark red pigmentation contain exclusively 3'-OH anthocyanins, and the same holds true for sorghum and purple corn. 3'5'-OH anthocyanins are also absent in blue flowers of Dendrobium and Phalaenopsis orchids, albeit the detection of 3'5'-OH flavonols provides evidence for F3'5'H activity .
Expansion of F3'5'H s on chr6 occurred in the Vitaceae lineage after the separation from other dicots. Indeed, F3'5'H genes are present in low copy number in other fully sequenced plant genomes, if not lost. F3'5'H is absent from Arabidopsis, single-copy in rice and papaya, and dual-copy in poplar and sorghum. In poplar, the two copies of F3'5'H were generated by the Salicoid WGD . The presence of a single-copy gene in the syntenic locus of poplar and papaya (Figure 1), and molecular dating of grapevine paralogues favour the hypothesis of lineage-specific gene duplications. The estimated age of F3'5'H duplications based on transversion rate at four-fold synonymous third-codon positions predicts most duplicate copies having diverged by less than 4DTV ~0.046 (Figure 3). If the molecular clock in grape is approximately calibrated by comparing the evolutionary rates in perennial dicots, the 4DTV distance of ~0.046 in grape is roughly half of the median 4DTV distance (~0.091) observed in poplar between duplicate genes that arose from the 60-65 myr-old Salicoid duplication . However, grape has evolved more slowly than poplar, and the distances between paleologous genes that arose from the γ triplication are lower in grape (median Ks, 1.22) than in poplar (median Ks, 1.54), as estimated by . Thus, recalibrating the mutation rate in grape, the 4DTV distances between F3'5'H in the chr6 array suggest that most duplications occurred within the past ~40 myr.
Molecular dating based on rate of nucleotide divergence is consistent with the conservation of duplicate gene copies across lineages in the family Vitaceae. While most of the 4DTV ~0.046 copies are conserved among Vitis species, they failed to be amplified from the DNA of related genera Ampelopsis and Parthenocissus. Conversely, the paleologous F3'5'Hp was conserved among these genera. Fossil records from the Late Cretaceous dates the radiation of Vitis, Ampelopsis, and Parthenocissus genera back to ~65 mya , confirming that most of the F3'5'H expansion occurred in an ancestor of the Vitis lineage, after the separation from the related lineages Ampelopsis and Parthenocissus.
The founder of the array of F3'5'H s on chr6 was initially duplicated through tandem gene duplication. Subsequently, different F3'5'H copies were involved in reiterated segmental duplications of large DNA blocks in which they resided, generating 9 blocks that range in size from ~35 to 55 kb (Figure 4 and 5). This modular structure suggests that unequal crossing-over between mispaired blocks was the most likely force that shaped the locus. Subsequent reorganisation via TE insertion, deletion, etc., resulted in structural variation among blocks, which might have reduced illegitimate recombination between adjacent blocks, thus resulting in the maintenance of the number of duplicates within the current bounds. Although our data suggest that most of the F3'5'H copies are maintained across grape varieties, at least in a heterozygous state, the extent of structural variation among haplotypes remains to be determined.
Regulatory diversification within the F3'5'H family and anthocyanin profiles
Transcriptional subfunctionalisation has widely occurred within the F3'5'H family and is detectable even between some of the most recent duplicates that diverged less than 4DTV ~0.046. This is evident, for instance, among F3'5'Hf, -j, and -l, which have retained >94% amino acid identity, and among F3'5'Hf, -g, and -l, which show conservation at the CR1 and SRS6 domains for 3'5'-OH activity. Transcriptional subfunctionalisation is therefore one of the forces, if not the predominant one, that is responsible for the retention of the most recent duplicate F3'5'H s in grapevine. The extensive structural variation found in their 5' regulatory region, and the observed partitioned expression among organs and developmental stages might have promoted the diversification of duplicates shortly after their origination, and thus the preservation of both duplicates. These pieces of evidence fit well into the DDC model. Deletion of regulatory modules is expected to occur by chance in promoters of duplicate genes, eliminating different cis-elements in either duplicate and diversifying their expression profiles .
Alternatively, a gene dosage model may also explain retention of duplicate F3'5'H s , under the assumption that a fitness advantage is provided by extra F3'5'H copies. F3'5'H gene products compete with F3'H gene products for the enzymatic transformation of flavonoid substrates into delphinidin or cyanidin precursors. Copy number variation is a common cause of altered stoichiometry of concerted enzyme activities within metabolic pathways, which results in phenotypic variation . Unbalanced phenotypes with increased levels of 3'5'-OH anthocyanins might have increased fitness, due to dissipation of high-energy blue wavelengths, attenuation of UV-B radiation, or conspicuousness of fruits to seed dispersers [43–46].
Regulatory modules alternatively maintained in the promoter of either F3'5'H duplicate contain binding sites for Myb-type transcription factors, drought-inducible cis-elements, and motifs responsive to ABA, methyl-jasmonate, light, and heat stress (Figure 6). The nature of these putative cis-elements correlates well with those factors shown to regulate F3'5'H expression. Myb-type transcription factors are activators of anthocyanin biosynthetic genes, including F3'5'H s [47–50]. Light and water deficits promote F3'5'H expression in the grape berry [3, 7]. ABA and methyl-jasmonate are sucrose-dependent inducers of anthocyanin biosynthetic genes [51–53]. High temperatures restrict anthocyanin accumulation by promoting pigment degradation and transcriptional repression of anthocyanin genes [54, 55].
Transcriptional regulation of duplicate F3'5'H s in berry skin is largely dependent on genotype, consistent with the observation in other plants that tandem duplicates have highly variable expression patterns . In the present work, differential expression within the F3'5'H gene family between different cultivars was associated with the differential accumulation of 3'5'-OH anthocyanins. In the field, F3'5'H gene expression has a functional impact on anthocyanin biosynthesis that persists during fruit ripening. Different copies of duplicate F3'5'H s have also become temporally specialised for different developmental stages of berry ripening (Figure 10). The question remains as to why these nuanced expression patterns have been maintained evolutionarily. One hypothesis is that copy-specific cis-elements confer unique, adaptive patterns of expression and environmental responsiveness by increasing the ratio of F3'5'H/F3'H enzyme concentration (and thus 3'5'-OH anthocyanins) under circumstances when accumulation of this class of metabolites is advantageous.
Expansion in copy-number and transcriptional specialisation of F3'5'Hs have increased the regulatory complexity of anthocyanin biosynthesis and fruit colour among red grape varieties. Most duplications occurred rather recently within this gene family, long after the Vitaceae lineage had separated from other dicot lineages. Among duplicate copies, accumulation of structural variation in promoter regions was more significant than divergence in coding regions. Transcriptional subfunctionalisation across organs and along developmental stages in ripening fruit was commonplace among gene copies, in addition to the extensive variation in gene expression among different cultivars. Transcriptional differences within the F3'5'H gene family in different accessions were paralleled by significant changes in the major metabolites synthesised by the F3'5'H gene products. In berry skin, the abundance of different anthocyanins that modulate the pigmentation of red grapes and wines was greatly affected by these transcriptional variations.
F3'5'Hs and F3'Hs were identified in grapevine (on chr6, chr8, and chr17 sequence assemblies deposited under the NCBI accession no. FN597024, FN597027, FN597042 as of 25 November 2009), poplar (version 1.0, ), Arabidopsis, rice, papaya, and sorghum (version of the genome assemblies available at Phytozome  as of November 2009) by tBlastN homology, using cytochrome P450 monooxygenases of the CYP75A subfamily (accession no. AAP31058, AB078781, AJ011862, Z22544, BAA03439, BAA03440) and the CYP75B sub-family (AY117551, BAD00189, AF155332) as a query. Matches were retained at thresholds of E<e-20 and amino acid identity >50%. Each sequence was extended on each side until the next gene and annotated using GenScan, FgenesH, GeneMark, and Geneid. Sequence alignments were carried out using ClustalX. Exon-intron structure was predicted by comparison with ESTs and amino acid sequences from other plants. Trees were constructed using MEGA. Nucleotide substitution rate was calculated using DNAsp 4.0. 4DTV values were calculated and corrected for possible multiple transversions according to . Gene models other than F3'(5')H were given the predicted function of their best match in the NCBI protein database. Syntenic regions were identified using the Genome Evolution tool . Transposable elements were annotated according to the grape genome browser information . LTRs in Copia and Gypsy retrotransposons were identified by dot plot analysis. Global DNA alignments of chromosomal segments were performed using LAGAN  in a window of 100 bp with a minimum identity of 70%. Dot plots of segmental duplications were made using Dotter. Alignments of 2-kb promoter regions were performed with DiAlign2, using a minimum HSP length of 10 bp and visualised with GEvo. DNA binding motifs were predicted by PlantCARE .
Selective amplification of F3'5'Hs and F3'Hs paralogues
Selective primers were designed across dissimilar exonic DNA stretches or using a 3'-terminal SNP between the perfect match of the target gene-copy and the mismatched annealing site of paralogous sequences [see Additional file 13]. Absence of illegitimate cross-amplification of other paralogues was validated by amplification of genomic DNA, Sanger sequencing of the PCR products, and detection of variable sites inside of primer sequences that distinguished the target gene-copy from other paralogues. qPCR efficiencies in amplifying the DNA of PN40024 (from whose genome sequence gene-copy specific primers were designed) and of the mixed haplotypes of every heterozygous cultivar used in the present study were calculated using the equation E = 10-1/slope of the standard curve. The standard curve was constructed with five 10-fold serial dilutions, using cDNA from organs and developmental stages in which the specific gene-copy was expressed or, if not possible, genomic DNA. Paralogue-specific primers with a PCR efficiency comprised between 90 and 110% in PN40024 were considered acceptable, and were used for qPCR if the standard deviation of their PCR efficiencies among the accessions under study was less than 10%. PCR primers that distinguished individual paleologous copies, as well as highly similar paralogues, and passed the thresholds set for the qPCR experiment, could be developed for nine out of the sixteen F3'5'H copies. The remaining copies were either highly identical in sequence or contained only a few polymorphic sites within DNA segments unsuitable for primer design. The range of variation in average PCR efficiency of primer pairs among the accessions tested was within the bounds of 87% in 'Marzemino' and 102% in 'Nebbiolo', with a similar average efficiency of 93% in 'Aglianico' and 'Grignolino'. This excluded a substantial cultivar effect of the efficiency of primer annealing during qPCR on the estimation of transcript levels of the whole gene family among cultivars, caused by possible SNPs in the annealing sites across haplotypes.
Experimental design and statistics in expression and metabolite analyses
Variation in anthocyanin profile and in transcriptional level of duplicate genes among developmental stages and cultivars was studied using a complete randomized design, and tested for significance using ANOVA run by COSTAT statistical package (CoHort Software, Monterey, CA, USA). Each plot consisted of 10-in-a-row clonally replicated plants in north-south oriented rows.
Vines were grown at the germplasm repository of Vivai Cooperativi Rauscedo, northeastern Italy (46°04' N; 12°50' E; 110 masl). Vines were trained using the Sylvoz system. Three biological replicates of 20 berries per cultivar were collected at each developmental stage [see Additional file 14]. Berries of each replicate were collected in the vineyard on both sides of canopy by random sampling on every plant within each plot. Samples were frozen immediately in liquid nitrogen and stored at -80°C until processed. Skin of each biological replicate was peeled from frozen berries, powdered in liquid nitrogen, and split to obtain a 100 mg aliquot for RNA extraction and a 200 mg aliquot for anthocyanin extraction. A three-way ANOVA was used to partition the factors that contributed to expression divergence in ripening fruit: gene-copy, cultivar and developmental stage, and their interactions. A two-way ANOVA was used to assess the effect of gene-copy and developmental stage on expression level, regardless of the cultivar. A one-way ANOVA was used to assess the same effect in each cultivar, as well as the differences in metabolite content and composition among cultivars. Statistically significant differences were determined using the Student-Newman-Keuls test (P < 0.05).
Anthocyanins were extracted by sonication of 200 mg berry skin in 1.8 mL of 1:1 methanol-H2O for 30 minutes. After centrifugation at 13,000 × g for 15 min, samples were filtered with a 0.2 μm cellulose membrane (Phenomenex, Inc., Torrance, CA, USA). Anthocyanins were separated by an Agilent 1200 Series HPLC system (Agilent Technologies, Inc., Santa Clara, CA, USA) equipped with a C18 Purospher RP-18 (5 mm, 250 × 4 mm) column (Merck, Darmstadt, Germany), according to the procedure reported by , and detected at 520 nm by a UV-detector (Agilent Technologies, Inc., Santa Clara, CA, USA). Calibration curve was obtained with oenin-chloride (Extrasynthese, Genay, France). Total anthocyanins were expressed as malvidin 3-glucoside equivalents and included monoglucoside, acetyl-glucoside, and p-coumaroyl-glucoside fractions. The anthocyanin profile was calculated for the monoglucoside fraction as the percentage of 3'5'-OH derivatives.
Total RNA was extracted as described in , treated with RNase-Free DNase I Set (Qiagen S.p.A., Milan, Italy), and purified with RNeasy MinElute Cleanup (Qiagen S.p.A., Milan, Italy) according to manufacturer's instructions. Complete removal of gDNA was assessed by direct use of treated RNA as a template for PCR reactions using the gene VvUbiquitin1. Absence of PCR products was visually inspected in 1% agarose gel stained with ethidium bromide. Absence of gDNA in reverse-transcribed samples was further confirmed by the melting curve performed during qPCR cycling using the intron-flanking primers for the normalisation gene VvUbiquitin1. The integrity of treated RNA was verified by electrophoresis in 1% agarose gel stained with ethidium bromide. RNA purity (A260/A280 nm) and quantification were estimated using a Nanodrop 1000 spectrophotometer (Thermo Fisher Scientific Inc., Wilmington, DE, USA). cDNA was synthesised using 2 μg of treated RNA, 0.5 μM (dT)18 primer, 0.5 mM dNTPs (Promega, Madison, WI, USA Cat# U1240), and 100 U of M-MLV Reverse Transcriptase (Promega, Madison, WI, USA Cat# N1701) in a 20 μL reaction volume supplemented with 20 U of RNasin Plus RNase inhibitor (Promega, Madison, WI, USA Cat# N2611) and incubated at 37°C for 90 min. Quantitative RT-PCR was carried out on a DNA Engine Opticon2 (MJ Research, Waltham, MA, USA) in a 20 μL reaction volume containing 5 μL of 20-fold diluted cDNA, 0.4 U of HotMaster Taq polymerase, 4.0 mM Magnesium acetate, 0.4 mM dNTPs, 1X SYBR solution (5 PRIME GmbH, Hamburg, Germany, Cat# 2200800), and 200 nM of each forward and reverse primer. Thermal cycling parameters were: initial denaturation at 95°C for 3 min, followed 40 cycles of 94°C for 15 s, 61°C for 20 s, and 68°C for 30 s, plate read at 78-82°C depending on each primer pair for 1 s, melting curve from 65°C to 95°C, read every 1°C, hold 1 s, and a final extension at 68°C for 5 min. Threshold cycle (Ct) was determined using the Opticon Monitor analysis software (version 2.02, MJ Research, Waltham, MA, USA) with a threshold level of fluorescence signal detection of log -1.7. Aliquots from the same cDNA were run in duplicate in the qPCR assay. Intra-assay repeatability between technical replicates was below 1 Ct. All assays included no-template controls. Relative gene expression of the target gene was calculated with the 2-ΔΔCt method, using the constitutive expression of the housekeeping Ubiquitin gene (VvUbiquitin1) . VvUbiquitin1 has been widely used in qPCR experiments conducted in grapevine across various organs by several research groups, in particular for berry samples. Semi-quantitative PCR was performed upon cDNA normalisation based on VvUbiquitin1 expression and visualised in a 1% agarose gel stained with ethidium bromide, or on SSCP gel stained with silver nitrate.
The authors thank Elisa De Luca and Francesco Anaclerio (Vivai Cooperativi Rauscedo) for providing grape samples, Enrico Braidot for help in HPLC analyses, Alberto Stefan and Dario Copetti for assistance in bioinformatics analysis, and Courtney Coleman for proofreading. This research was funded by the Italian Ministry of Agriculture (VIGNA Project), the Regional Government of Friuli Venezia Giulia (Grape Breeding Project and LR26/2005art17 "Methods for rapid determination of polyphenolic and aromatic profiles in must and wine").
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