Journal of Experimental Botany, Vol. 66, No. 15 pp. 4427–4440, 2015 doi:10.1093/jxb/erv159 Advance Access publication 12 June 2015 This paper is available online free of all access charges (see http://jxb.oxfordjournals.org/open_access.html for further details)

RESEARCH PAPER New candidate for the fine regulation of the colour of grapes

Laura Costantini1,*, Giulia Malacarne1, Silvia Lorenzi1, Michela Troggio1, Fulvio Mattivi2, Claudio Moser1 and Maria Stella Grando1 1 Department of and Biology of Fruit Crops, Research and Innovation Centre, Fondazione Edmund Mach, Via E. Mach 1,

38010 S. Michele all’Adige, Trento, Italy Downloaded from 2 Department of Food Quality and Nutrition, Research and Innovation Centre, Fondazione Edmund Mach, Via E. Mach 1, 38010 S. Michele all’Adige, Trento, Italy

* To whom correspondence should be addressed. E-mail: [email protected] http://jxb.oxfordjournals.org/ Received 19 December 2014; Revised 5 February 2015; Accepted 3 March 2015

Abstract In the last decade, great progress has been made in clarifying the main determinants of anthocyanin accumulation in grape berry skin. However, the molecular details of the fine variation among cultivars, which ultimately contributes to wine typicity, are still not completely understood. To shed light on this issue, the grapes of 170 F1 progeny from the cross ‘Syrah’×’Pinot Noir’ were characterized at the mature stage for the content of 15 anthocyanins during four grow- at Biblioteca Fondazione Edmund Mach on July 24, 2015 ing seasons. This huge data set was used in combination with a dense genetic map to detect genomic regions con- trolling the anthocyanin pathway both at key enzymatic points and at particular branches. Genes putatively involved in fine tuning the global regulation of anthocyanin biosynthesis were identified by exploring the predictions in the QTL (quantitative trait locus) confidence intervals and their expression profile during berry development in offspring with contrasting anthocyanin accumulation. New information on some aspects which had scarcely been investigated so far, such as anthocyanin transport into the vacuole, or completely neglected, such as acylation, is provided. These genes represent a valuable resource in grapevine molecular-based breeding programmes to improve both fruit and wine quality and to tailor wine sensory properties according to consumer demand.

Key words: Anthocyanin, berry, candidate gene, metabolic profiling, microarray, quantitative trait locus, Vitis vinifera.

Introduction Besides their direct role in the colour of grapes and wine, chemical diversity may be further increased by acylation (ali- anthocyanins and their derived compounds also contribute phatic or aromatic) in the C6-position of the glucose moi- to the sensory attributes of wine by interacting with mol- ety. These secondary modifications determine the chromatic ecules such as phenolics, polysaccharides, proteins, metals, properties of the pigments (Jackson, 2008). In particular, the and aroma substances (He et al., 2012). Moreover, in recent hue is affected by the pattern of hydroxylation and methyla- years they have been reported to provide numerous beneficial tion (a larger number of either free or methylated hydroxyl effects for human health (Pojer et al., 2013). groups causes a bathochromic shift in the visible spectrum Anthocyanins are present in the skins of coloured culti- of anthocyanins) and by aromatic acylation (stacking of aro- vars as 3-monoglucosides of five anthocyanidins that are dif- matic moieties of acylated anthocyanins plays a role in the ferently hydroxylated and O-methylated on the B ring. This blue colour shift). Colour intensity is promoted by acylation:

© The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. 4428 | Costantini et al. as a general rule, acylated anthocyanins are preferentially (Cardoso et al., 2012). Acylation of anthocyanins has never trapped in anthocyanic vacuolar inclusions (AVIs), which has been examined, although in some important cultivars the been proposed to result in further formation of AVIs and in acylated anthocyanins can account for >60% of the total dark coloration (Mizuno et al., 2006). anthocyanins. The proportion of the different types of anthocyanins Finally, the complexity of skin colour might be regulated also has a large effect on pigment extractability and colour at the level of anthocyanin vacuolar sequestration. Transport stability in , which in turn influence the wine into the vacuole is a prerequisite for anthocyanin biosyn- profile. In particular, the non-acylated glucosides and acetyl- thesis and as such it fulfils a regulatory function (Marinova glucosides are more readily extracted from the fruit than the et al., 2007), and it seems to be substrate specific (Mizuno p-coumaroyl-glucosides, whose extraction rate in a wine-like et al., 2006; Gomez et al., 2009; Francisco et al., 2013). Little solution is very low (Fournand et al., 2006). With respect to is known about the exact mechanism of anthocyanin trans- pigment stability in grapes, the adjacent hydroxyl groups of portation in grapes. In addition to vesicle-mediated transport o-diphenols in cyanidin, delphinidin, and petunidin enhance (Gomez et al., 2011), several types of transporters have been their susceptibility to oxidation. In contrast, due to methyla- proposed, most belonging to multigenic families which are tion, neither malvidin nor peonidin possesses the ortho-posi- little or not characterized in grapevine (for a review, see He tioned hydroxyl groups, which results in their comparatively et al., 2010). Up to now, a few proteins were demonstrated Downloaded from higher resistance to oxidation (Jackson, 2008), a very desir- to be involved in anthocyanin vacuolar sequestration in Vitis able feature in winemaking. The acylation of the sugar can vinifera: a bilitranslocase homologue (Braidot et al., 2008), also promote anthocyanin chemical stability (Mazza et al., a glutathione S-transferase (Conn et al., 2008), two MATE- 1995), as demonstrated in other plant species (Yonekura- type (multidrug and toxic compound extrusion) proteins

Sakakibara et al., 2009). (Gomez et al., 2009), and an ABC (ATP-binding cassette) http://jxb.oxfordjournals.org/ Finally, the different chemical structures of anthocyanins transporter (Francisco et al., 2013). influence their bioavailability and bioactivity, with potential Very little is also known about anthocyanin degradation. consequences on their health-promoting effects (Passamonti Here, the identification of new candidates for the regula- et al., 2002; Rossetto et al., 2002). tion of anthocyanin accumulation in grape skin at technolog- In the last decade, great progress has been made in clarify- ical maturity is described. To this end, a segregating progeny ing the regulation of anthocyanin biosynthesis in grapevine derived from the cross of two divergent varieties was analysed (He et al., 2010; Hichri et al., 2011). The anthocyanin content during four growing seasons for their anthocyanic profile, in berry skin is mainly controlled by a cluster of MYB-type and an integrative approach combining metabolic, genetic, at Biblioteca Fondazione Edmund Mach on July 24, 2015 transcription factor genes on 2, with effects on and transcriptional sources of information was adopted. both the qualitative distinction between coloured and white cultivars and the quantitative variation in anthocyanin level within the coloured varieties (Fournier-Level et al., 2009; Materials and methods Huang et al., 2013). However, grape accessions, even those dis- Plant material playing the same haplotype at the MYBA locus, show a con- A population derived from ‘Syrah’×’Pinot Noir’ (clone ENTAV tinuous variation in anthocyanin content, which suggests the 115) consisting of 170 F1 individuals plus the parental lines was involvement of additional genes. For example, the products characterized at the biochemical, genetic, and transcriptomic level. of VvMYB5a, VvMYB5b, VvMYBPA1, and VvMYBPA2 All plants were grown at the experimental field Giaroni of FEM have been proposed to modulate several genes in the common (Edmund Mach Foundation, San Michele all’Adige, Italy). steps of the flavonoid pathway (Bogs et al., 2007; Terrier et al., 2009; Cavallini et al., 2014). Moreover, in all model systems, Sampling the regulation of anthocyanin accumulation, both spatially For anthocyanin analysis, two grape clusters were harvested from and developmentally, results from a co-operative interaction each plant at technological maturity (18 °Brix) in four different between WD-repeat proteins, basic helix–loop–helix (bHLH) vintages (2007, 2008, 2009, and 2011) and stored at –80 °C till use. transcription factors, and MYB transcription factors, which These samples were also analysed for flavonol content in a parallel experiment (Malacarne et al., 2015). The number of phenotyped F1 form the MBW regulation complex. Recently, two bHLH and individuals varied from 83 (in 2011) to 158 (in 2008). For micro- two WDR proteins were described in grapevine and, for one array analysis, three individuals having very low anthocyanin con- of them, VvMYC1, an involvement in the regulation of the tent (LAPs: codes 256, 260, and PN) and three individuals having flavonoid biosynthetic pathway was demonstrated (Hichri very high anthocyanin content (HAPs: codes 16, 56, and 63) were et al., 2010; Matus et al., 2010). selected from the progeny. These six individuals number among eight divergent individuals for flavonol content (Malacarne et al., A few studies have looked into the variation in the relative 2015). The selection was statistically supported by analysis of vari- abundance of specific anthocyanins, which is presumed to be ance (ANOVA) followed by Tukey HSD test (P-value <0.05) using finely tuned through tailoring reactions catalysed by flavonoid Statistica 9 software (StatSoft, Tulsa, OK, USA). A representative hydroxylases, glycosyltransferases, O-methyltransferases, and sample of 50 berries harvested from both sides of the canopy was acyltransferases. In these studies, generally only one pheno- collected for each individual during the 2007 season at three berry developmental stages: pre-véraison (33E-L, hard green berry, PV), type at a time was analysed (Jeong et al., 2006; Falginella véraison (35E-L, 50% coloured berries, VER), and maturity (38E- et al., 2010; Fournier-Level et al., 2011) or, when several traits L, 18 °Brix, MAT). From stage 35E-L, the collected berries were were considered, a limited number of genes were investigated dissected into skin, flesh, and seeds, and then the skins were stored Candidate genes of the grape anthocyanin pathway | 4429 at –80 °C till transcriptomic analysis. These stages were chosen as sequences of BAC (bacterial artificial chromosome) clones used véraison represents the starting point for anthocyanin accumulation for Pinot Noir physical map construction (Troggio et al., 2007) and in berry skin and maturity the stage at which anthocyanins were from electronic SNPs identified after the completion of Pinot Noir quantified in this work. genome sequencing (Pindo et al., 2008). All the SSR and SNP mark- ers were anchored to contigs included in the Pinot Noir physical map (Troggio et al., 2007). Anthocyanin analysis Markers were tested against the expected segregation ratio using a Anthocyanin extraction and HPLC-DAD (high-performance χ2 goodness-of-fit implemented in JoinMap 4.0 (Van Ooijen, 2006). liquid chromatography-diode array detection) analysis were per- Distorted markers were used for linkage analysis unless they affected formed as described in Mattivi et al. (2006). The choice of metha- the order of neighbouring loci. A consensus linkage map was gener- nol has a double justification: as a strong solvent it leads to the ated by selecting LOD (log of odds) independence as the parameter total extraction of the anthocyanins from the skins of grapes, in the grouping test (with a LOD threshold of 7.0) and regression which reduces the risk of differential extraction due to matrix mapping as the mapping algorithm. The Kosambi mapping function effects; moreover, it avoids the partial hydrolysis of anthocya- (Kosambi, 1944) was used to convert the recombination frequencies nin acetates that is observed with the widely used acidic solvents into map distances (cM, centiMorgans). (Castia et al., 1992). Fifteen anthocyanins were quantified by using Linkage groups (LGs) were graphically visualized with MapChart a calibration curve with malvidin 3-glucoside chloride, and their 2.2 (Voorrips, 2002). content was expressed as the concentration (mg kg–1 of berries). The accuracy of marker order estimated by meiotic methods was Statistical analyses of biochemical data were done with the soft- verified using the physical distance information from the BAC con- Downloaded from ware SPSS 11.0 (IBM SPSS Statistics). The normality of trait dis- tigs with three or more mapped markers. For the sake of simplic- tribution was checked by means of the Kolmogorov–Smirnov test. ity, base pairs (bp) were converted into cM by dividing by 300 000, If necessary, data were log-transformed with a ln(1+x) function. which is the approximative physical length corresponding to 1 cM in Correlations between traits within years and between years within grapevine (Velasco et al., 2007). traits were determined using the non-parametric Spearman cor- relation coefficient. http://jxb.oxfordjournals.org/ QTL analysis QTL mapping was performed on the consensus map each year sepa- Expression analysis rately by using the simple interval mapping and multiple QTL map- RNA isolation, chip hybridization, and microarray data analy- ping functions with the mixture model algorithm in MapQTL 6.0 sis were carried out as described at GEO (http://www.ncbi.nlm. (Van Ooijen, 2009). QTLs were declared significant if the maximum nih.gov/geo/query/acc.cgi?acc=GSE42909) and in Malacarne LOD exceeded the LG-wide LOD threshold (calculated using 1000 et al. (2015), where the rationale of the approach is described permutations) and mean error rate was <0.05. QTLs were consid- in depth. Briefly, a total of 18 hybridizations (6 individuals×3 ered reliable when consistently detected in at least two years. A non-

developmental stages) were performed on the custom Affymetrix parametric Kruskal–Wallis test was carried out to provide support at Biblioteca Fondazione Edmund Mach on July 24, 2015 GrapeGen GeneChip® (Peng et al., 2007). Probe sets with sig- to marker–trait associations. Confidence intervals corresponded nificant differential expression (DEPs) between LAPs and HAPs to a LOD score drop of one on either side of the likelihood peak. were selected in two steps under the assumption that the three Suffixes (a and b) were adopted to indicate different regions on the individuals of each group can be treated as biological replicates same LG where QTLs were found for anthocyanin (this work) and (pseudoreplicates): (i) within each group (LAPs and HAPs) the flavonol (Malacarne et al., 2015) content. The 12X reference genome data were analysed through pairwise comparisons of develop- (http://genomes.cribi.unipd.it/) was used to extract version 1 of the mental stages (PV versus VER, PV versus MAT, and VER versus gene predictions underlying QTLs. MAT). Differential expression analysis was performed with the A Fisher’s exact test was applied to evaluate the over-represen- Bayes t-statistics from LIMMA (Linear Models for Microarray tation of specific functional categories in QTL regions control- Data), where P-values were corrected for multiple testing using ling a given trait. Reference to the analysis was the distribution in the Benjamini–Hochberg method (Benjamini and Hochberg, the same categories of 30 748 12Xv1 gene predictions assigned to 1995). Three lists of DEPs for each group were thus obtained (V1+V1r-ChrUn) as derived from Additional file 1 [with a false discovery rate (FDR) <5% and a cut-off of 2-fold by Grimplet et al. (2012). Gene predictions annotated at the third change (FC) between each pair of stages]; (ii) the lists of DEPs level, as suggested by Mateos et al. (2002), were considered, with were then compared between LAPs and HAPs to provide a the exception of genes annotated at the first and second level if not final list of probe sets either exclusively modulated in LAPs or characterized at a deeper level and significantly represented in the in HAPs, or modulated in both groups but with low correlation genome. Adjusted P-values obtained applying the Benjamini and (−0.5 > (logFCLAPs – logFCHAPs)pairwise comparison > 0.5). Hochberg method (1995) for multiple testing correction were con- Probe set annotation updated according to the 12Xv1 predic- sidered significant when ≤0.05. tion (http://genomes.cribi.unipd.it/) and functional categorization were inferred from table S3 in Lijavetzky et al. (2012) and from the GrapeGen database (http://bioinfogp.cnb.csic.es/tools/GrapeGendb/). Candidate gene selection Real-time reverse transcription–PCR (RT–PCR) analysis, carried Candidate genes were selected among the 12Xv1 predictions included out as described in Malacarne et al. (2015), was used (i) to validate in the confidence interval of reliable QTLs according to at least one microarray results technically; and (ii) to confirm the applicability of the following pieces of functional evidence: (i) literature supporting of the pseudoreplicates approach by comparison with real biological the involvement in trait regulation; (ii) differential expression between replicates of each genotype. LAPs and HAPs (data from this study); (iii) expression detected in berry skin, possibly higher than in berry flesh (data from Fasoli et al., 2012; Lijavetzky et al., 2012), and consistent with the accumulation Map construction of anthocyanins during berry development; (iv) co-expression with Map construction was based on the segregation of 690 markers in genes involved in the regulation of anthocyanin biosynthesis or in 170 F1 individuals. Colour was mapped as a qualitative trait. SSR flavonoid metabolism (data from COLOMBOS and VTC databases: (simple sequence repeat) markers belonged to the series described Wong et al., 2013; Meysman et al., 2014); or (v) assignment to func- in Supplementary Table S1 available at JXB online. SNP (single tional categories significantly over-represented in QTL regions (data polymorphism)-based markers were obtained from end from this study). 4430 | Costantini et al.

Results and discussion QTL analysis Variation of anthocyanin content and composition in Thanks to the generation of an improved genetic map of Syrah, Pinot Noir, and their progeny Syrah×Pinot Noir and of chemical data collected over four growing seasons, several consistent QTLs for the content of A detailed description is presented in Fig. 1, and each compound, for their sums, and for the ratios between spe- Supplementary Text S1, Tables S2–S4, and Fig. S1 at JXB cific classes of anthocyanins could be identified, with differ- online. ent effects and stability among years (Fig. 2; Supplementary Table S5 at JXB online). Map construction By analysing all the progeny, a major QTL on LG 2 was detected for all the 15 anthocyanins and for their sums Out of the 690 scored markers, 36 were discarded as they (‘totals’), which explained between 31% and 74% of pheno- had too much missing data, they were identical to other typic variance averaged over years. When considering only the loci, or their genetic position was inconsistent with the coloured F1 individuals, the same QTL was found to regulate physical position. Out of the 654 remaining markers, 593 most of the above traits as well as their sums (‘derivatives’)

could be finally placed onto 19 LGs covering 1184 cM and ratios with a percentage of explained variance ranging Downloaded from with an average distance between markers of 2 cM (Fig. 2; from 6% to 45%. As a general trend, in the coloured progeny, Supplementary Table S1 at JXB online). QTL analysis the effect of this QTL was lower or lacking for traits involving is expected to benefit from it, as high-density maps were dihydroxylated anthocyanins. Candidate genes for this QTL recently demonstrated to improve the precision of QTL were previously identified in a cluster of MYB-type transcrip-

localization and effect estimation, especially for minor tion factors genes (Fournier-Level et al., 2009). http://jxb.oxfordjournals.org/ QTLs, as well as the power to resolve closely linked QTLs Several smaller effect QTLs were detected on LGs 1, 3, 4, (Stange et al., 2013). 5, 6, 7, 8, 9, 10, 11, 12, 17, 18, and 19, most of which were

A Cyanidin derivatives 0.90 (0.86-0.94) B Peonidin derivatives 0.89 (0.84-0.93) 0.71 (0.64-0.76) 0.68 (0.58-0.73) 0.40 0.40 0.35 0.35 0.30 0.30 2007 Sy-PN 2007 at Biblioteca Fondazione Edmund Mach on July 24, 2015 y y PN 0.25 0.25 nc nc 2008 2008 Sy ue ue 0.20 2009 0.20 2009 eq eq 0.15 2011 0.15 2011 Fr Fr 0.10 0.10 0.05 0.05 0.00 0.00 00.260.520.781.041.301.561.82 0< 1.00 1.25 1.50 1.75 2.00 2.25 2.50 log_conc (mg/kg) log_conc (mg/kg)

C Delphinidin derivatives 0.93 (0.90-0.96) D Petunidin derivatives 0.93 (0.91-0.95) 0.80 (0.74-0.83) 0.80 (0.76-0.82) 0.40 0.40 0.35 0.35 Sy 0.30 0.30 Sy 2007 2007 y y PN 0.25 0.25 2008 nc nc 2008 ue ue 0.20 PN 0.20 2009 2009 eq eq 0.15 0.15 2011 Fr Fr 2011 0.10 0.10 0.05 0.05 0.00 0.00 00.380.761.141.521.902.282.66 00.380.761.141.521.902.282.66 log_conc (mg/kg) log_conc (mg/kg)

E Malvidin derivatives 0.90 (0.88-0.92) 0.70 (0.68-0.74) 0.40 0.35

0.30 2007

y Sy 0.25 2008 nc PN

ue 0.20 2009 eq 0.15 2011 Fr 0.10 0.05 0.00 0< 2.05 2.25 2.45 2.65 2.85 3.05 3.25 log_conc (mg/kg)

Fig. 1. Variation of anthocyanin content in the Syrah×Pinot Noir progeny in four different vintages. For the two parents, averaged values across years are reported. In the right upper part of each plot, the Spearman rank-order correlation between years (mean value and range of variation in parentheses) is shown for the whole progeny (top) and the coloured progeny (bottom). Correlations are significant at the 0.01 level. Sy, Syrah; PN, Pinot Noir; conc, concentration. (This figure is available in colour at JXB online.) Candidate genes of the grape anthocyanin pathway | 4431

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t3 SNP7200SNP4102 W SNP7147 (3 29 48 SNP6092 H/ ) (2 11%(3) 54 SNP7127SNP7294 KW (3 M MA KW MA +K SNP6028SNP15058 W (2 24

SNP0050 ) 37 50 SNP7056SNP4153 (3 diOH 3) ) ) 56 SNP6124 (2

9%(2)+KW (2 26 SNP7268 ) 38 SNP7153 SNP6136SNP3046 W (2 ) c/Pet3M c/ De (2 52 57 SNP7075 ou/Tot3MAc 43 SNP0146 ) SNP7064 SNP5020SNP4027 ) ) 58 To 49 SNP6012 SNP4088 _Anth 8% 53 )

l3M SNP7238SNP7165 51 SNP7223 SNP7148 54 SNP4066SNP6099 t3 59 SNP6042SNP6160 54 SNP8028 SNP4162 SNP15009 SNP7003 MAc 10%(3) 11%(3)+ SNP6120SNP8055 SNP6057 55 56 SNP7067 60 SNP7257SNP7093 SNP6165 ) 57 6%(2 56 SNP13910 SNP5060 SNP4104SNP13149 (2) SNP4108 KW 58 57 SNP4109 64 SNP6105 65 SNP7276 SNP6156SNP4085 KW ) 58 (4) 68 SNP7314 SNP7149

59 SNP7205 (3

60 SNP4041 ) 69 SNP6107SNP4136 72 SNP3059 80 SNP6088 SNP7240SNP7210 91 VMC16F3 C10 C11

0 SNP4147 0 SNP6112 5 SNP0122 Peo3MC Meth/non_Meth 6%(3)+KW 3'5'Meth 1 SNP8074 De Pe

VRZAG25 Cya3MC 8 Cya3 2 SNP8165 SNP4057 Me 11 l3 t3 Ma Tot3 3 SNP4084 De MCou/D el3M

13 SNP1340 MC th M-

l3 MC 4 VVIP02 De To l3 SNP6157 Pe 14 De /n ou/Peo3M 16%(2) /3

MC SNP7225 ou/Pet3M 16 MCou KW Cya3M 6 t3 ou l3 M o3 18 SNP6064 on '5'OH 7%(3)+ l3

Mal3 M- SNP15015 SNP5041 Pe De MC DelD er To 22 SNP6017 ou ou/Tot3M 16 De De MAc- MA /C 7 SNP8143SNP10579 _Met t3 KW

VMC8D3 lD 23 Cya3 t3 /M ya3M Pet3MA Ma ou/T l3MA l3M-Pet3M Peo3MA

SNP7010 Cy Cya3M KW (2 MA Tot3M c/Peo3M 3% Ac M-

SNP6009 er KW 25 Mal3 MC al3M h-

(3)-Pet3M SNP7033 Pe 8 l3MC aD

26 SNP4101 Del3 -Pet KW 14%(3) To c/Pet3M MA 3' ot SNP7262SNP15037 (3)- t3 c/De

SNP5061 10%(2) 30 9 er KW (2 5'Meth/3'5 Del3 t_Anth %( 3M

16%(4)+KW SNP7108 Co MA c De SNP14068 SNP8019 c

31 MC ou Pe (2 KW (3)- c KW 13 SNP7312 2) KW Ac KW (3 ou/Mal l3 %( SNP7103SNP6114 u/Tot3MAc 25%(2)+KW (2

33 ) c KW r KW (3

t3 MA +K SNP4015

ou 14 KW KW M Pe 35 SNP4113SNP8027 (2) +K 4)+K (2) MCou KW KW (3 18 (2

SNP7030SNP8160

KW 15 W KW KW (2) c 50 %(

36 SNP4087 ) o3 KW ) (2 (3 ) W (2 %(4) 18 SNP7069 ) ) (3 (4 3M SNP4106SNP8015 'O (3 M (2) W ) ) (2 37 19 SNP7176 ) (2) ) (2 )

SNP8016 KW H ) KW 13%(2) (4 ) (4 25 SNP4002 ) 38 SNP6134 ) 2)+K ) SNP8018 ) 39 (2) (2

40 SNP7118 (3) ) 51 SNP6055 W (3

41 SNP4063 +K 42 SNP6072 W 43 SNP5007SNP8112 ) 45 SNP4111 62 VMC3E12 (2 ) ) never reported before. Only a few regulated a specific trait For example, it is likely that the QTL on LG 1a primarily (e.g. the peonidin level in the case of the QTL on LG 9), while regulates the methylation level of anthocyanins (25–31% many of them showed pleiotropic effects on several traits. of explained variance), in agreement with the findings of However, in some cases, a primary effect could be argued, Fournier-Level et al. (2011), who identified the underlying which then gives rise to a number of secondary effects. molecular factors in an anthocyanin-O-methyltansferase 4432 | Costantini et al.

C12 C13 C14 C15 C16 De l3M-Pet3M-

0 SNP7089 Cya3M-Peo3MAc 0 SNP4107 0 SNP8118 6 SNP7090 CyaDer KW

2 SNP4069 8 SNP7043 Cya3MAc KW 12 SNP7022 7 SNP3037 Pe 0 SNP7313 3 SNP7057 Tot3MCou Tot_ 0 SNP4013 10 SNP7216 Mal3 M- tD 4 SNP7139SNP5028 To 22 SNP7236 6 VVMD5

Mal3 M SNP0047 SNP6074SNP5034 Peo3MC 10 er Ma lD 26 SNP0027 10 SNP7005 6 SNP7058 tM 12 Pet3MC An

Tot3 11 SNP0073 SNP0056 27 SNP8080 12 SNP7105 7 SNP8206 KW 15%(3)+K SNP6116 th 12 18 SNP6066 15 VMC8G6 29 SNP8024SNP4023 Pe 13 SNP4005 12 (2) er 12%( 22 SNP0138 19 SNP6063 MAc (2) 20%(2)+KW (2 SNP8106 17 SNP15007 32 SNP8145 t3 14 11%(3)+

ou 19%(3) SNP7221 23 SNP7251 25 (2 KW MC

ou SNP6006 19 SNP8135 %( 34 SNP4052 18

SNP7131 26 SNP15017 SNP6095 ) Pe 36 SNP4138SNP7102 30 20 SNP7269 7%(3

SNP15029 21 Tot3 4)+K ou KW (2 8%(2 32 SNP15018 SNP13396 28 SNP4061 (2 3) o3MCou 38 SNP8192 21 SNP6155 24 SNP15033 W (2 VVIP33 33 SNP4072 33 ) +K MalD 39 SNP4150VMC3D12 30 SNP6141 25 SNP15001 KW MC W ) 34 SNP7305 36 SNP7164SNP3039 ) SNP6093SNP7025 33 SNP7194 SNP4167 W 26 ) 41 (2 35 SNP0102 38 SNP6142 (2 SNP15011 ou 11% (3) SNP7208 35 SNP15051

29 er 12%(2 (2 ) ) SNP4103 40 SNP4049 36 ) 12% (2) SNP4037 ) 42 SNP7061 36 SNP7279SNP0054 30 ) SNP3060SNP13830 41 SNP15028 SNP6011 31 SNP15002 SNP7133SNP7040 37 SNP13204 44 38 SNP4064 SNP8095SNP7259 33 SNP15043 SNP12255 SNP8002 42 38 SNP7015SNP15039 39 SNP7145 SNP7183SNP6125 34 SNP6037 SNP6030SNP4115 40 SNP6126SNP7020 ) 45 43 SNP4159 41 SNP4081 SNP6076 47 SNP7193SNP6051 44 SNP13442 43 VMC1G3.2 46 SNP15020 VMCNG1E1 48 SNP4143 52 46 SNP7072 46 SNP6094SNP5043 48 SNP12309 49 SNP7184 47 SNP6149 47 SNP7062SNP6132 49 SNP4093SNP13339 51 SNP6163 48 SNP0124 48 SNP0149 51 SNP7247SNP7230 49 SNP6129 50 SNP15063 SNP7011 53 SNP6048 51 SNP5027SNP10053 56 SNP5019 53 SNP6044SNP7059 59 SNP6034 58 SNP4148 60 SNP7212SNP8069 66 SNP15024 SNP4024 65 SNP4029 71 SNP15064 72 SNP15041 SNP0039 Downloaded from

C17 C18 C19

0 SNP7081 3 SNP7074 SNP4042SNP7140 0 SNP8113 6 3 VMC2A3 SNP7252 8 SNP8170 10 SNP3036 10 SNP7110 11 SNP3030SNP0132 Pe 12 SNP7302SNP6036 0 SNP8061 13 SNP4051 http://jxb.oxfordjournals.org/ o3 SNP4026 13 SNP6031 4 SNP8204 15 MC SNP7126 SNP4129

To 16 VMC8B5 To

17 VVIV16 14 SNP5045SNP15036 Peo3MC ou/P t3

t3 SNP12534 SNP7028 triO

MC 23 MC 36 SNP7219 SNP14343 SNP7311 Tot_Anth 3%(2 20 SNP8108 eo3M 16 H/di ou/T SNP4034 SNP4116 SNP7217 SNP10784SNP7308 ou 39

SNP8199 SNP8128 ou 24 Pe SNP4016

De 44 SNP4082 /T 17 OH

ot SNP0142 12%(2)+KW (2

VVIM10SNP5025 7%

tDer 9%(3) tDer 49 Ma ot

SNP4039 SNP7041 l3 Ma lD 25 3M 13%(2) 50 SNP7201 24 SNP0085SNP4097 3M MCou 4%(2) SNP15022SNP15023 9%(2 l3M VVIN83 25 SNP8197 (2 30 SNP4096 51 Ac 14 To SNP7291SNP1144

26 ) SNP15006SNP6045 er 13%(2 60 SNP7048 31 5%(2) ) 61 SNP7055 t3 29 SNP13758 33 VVIB09SNP8048 ) 63 SNP3032 M- 31 SNP7256 37 SNP8071 %(3)+KW (2 +K SNP7254SNP4137 64 SNP7160SNP3010 T 32

38 SNP6008 ot3 Ma Tot3MCo ) 33 SNP6159 W SNP7161SNP7277 39 SNP8084 ) 65

Tot3MCou 8%(2) 34 SNP0069 MA 66 SNP3004 l3 MC Pe (2 43 SNP7085 Pe SNP6138 De 36

SNP5018 Mal

) 67 t3

SNP7177 Tot3MCo

45 c-Tot_Anth KW (2 70 SNP7220 o3MC 39 SNP5064 l3 MC ou 52 SNP8021 To 3M Pe ) 44 SNP4012

SNP8201 MCou/D u/To 73 Pe

57 SNP7270 at Biblioteca Fondazione Edmund Mach on July 24, 2015 t3 Pe

ou/Pet VMC9A2.1 11%(2)+KW (2 47 77 SNP0111 oD C o3MC ou SNP8194 Peo3MA 64 MA

ou/Mal 48 SNP8176 79 SNP0134 o3M t3 SNP4155 er 14%(2)+KW (3) 68 Cya3MC /Peo3M 14%(2) /Peo3M u/Tot3M Pe

VVIN16 MA 73 VMC3C11.1 80 c/Tot3M CyaDer KW (2) el 3M o3 81 SNP5058 ou triO 6%(2)+ 3M c 13%(

82 SNP4095 3M 16 MAc/

21%(3)+KW c 15%(3)+KW (3

83 SNP7226 H/di 4%(2)+ 10%(4)+KW (4 ou 17%(4)+KW (3) 84 SNP6024SNP6054 ) 88 SNP8181 SNP8216 11%(2)+KW (2 KW ) Pe /C %(3)+KW (4 2)+K 91 SNP0113 OH ya3M 92 SNP6162 o3M (3

KW KW +K

95 SNP0120SNP5053 W )

SNP7076 W (2 96 KW (3 (2 (2) (3 97 SNP6079 KW ) ) ) ) (2

SNP15040 ) ) 98 (2 ) ) 101 SNP8166 ) SNP7091SNP6025 ) 102 SNP7249

Fig. 2. Linkage map of the Syrah×Pinot Noir cross. Distances of markers from the top (in cM) are indicated on the left side of linkage groups. Bars on the right correspond to 1–LOD confidence intervals of QTLs identified in at least two seasons (or to regions where significant markers were found with Kruskal– Wallis analysis). For each trait, the average phenotypic variance explained by the QTL and the number of years (in parentheses) in which significants QTLs (or markers) were detected are reported. The marked portion of the linkage group corresponds to the overall confidence interval used for gene prediction extraction. Cya, cyanidin; Peo, peonidin; Del, delphinidin; Pet, petunidin; Mal, malvidin; 3M, 3-monoglucoside; Ac, acetate; Cou, p-coumarate; Tot, total; Der, derivatives (3-monoglucoside+3-monoglucoside-acetate+3-monoglucoside-p-coumarate); 3ʹMeth/3ʹOH, peonidin 3-monoglucoside/cyanidin 3-monoglucoside; 3ʹ5ʹMeth/3ʹ5ʹOH, malvidin 3-monoglucoside/delphinidin 3-monoglucoside; diOH, di-hydroxylated (cyanidin+peonidin 3-monoglucoside); triOH, tri-hydroxylated (delphinidin+petunidin+malvidin 3-monoglucoside). (This figure is available in colour atJXB online.) gene cluster. Similarly, it can be proposed that the QTL Candidate gene identification on LG 6 controls the hydroxylation level of anthocyanins (28% of explained variance) and that the QTLs on LGs 3, The number of genes within QTL confidence intervals varied 7b, 8b, 12b, and 18b contribute to the acylation level of from a minimum of 149 (LG 2) to a maximum of 861 (LG anthocyanins (6–21% of explained variance). In contrast, 8) (Table 1). the QTLs identified on LGs 4b, 5, 8a, 10, 11a, 12a, and Without claiming to be exhaustive, candidate genes for trait 17 might be related to a more general activity since they regulation were identified according to the criteria described seem to regulate flavonol content and composition as well in the Materials and methods (expression data and enrich- (Malacarne et al., 2015). Several QTLs (in particular those ment tests are reported in Supplementary Tables S6 and S7, identified on LGs 2, 6, 8a, and 17) also co-localize with respectively, at JXB online). Candidate functions based on regions controlling proanthocyanidin content and compo- previous knowledge were: regulation of transcription, enzy- sition (Huang et al., 2012). Finally, the QTL identified on matic activity, transport, and signalling/response to stimulus. LG 8b coincides with a QTL explaining up to 32% of vari- Among structural enzymes, those involved in phenylala- ance for total anthocyanin content in interspecific hybrid nine, phenylpropanoid, flavonoid, and anthocyanin metabo- grapes (Ban et al., 2014). lism were evaluated. Candidate enzymes for anthocyanin Candidate genes of the grape anthocyanin pathway | 4433

Table 1. Genes with a potential role in the regulation of anthocyanin content and composition

No symbol is shown at the side of gene predictions that are not represented on the GrapeGen GeneChip, whereas * and # indicate the presence and absence of differential expression for gene predictions that are represented on the GrapeGen GeneChip, respectively. The column named ‘This study’ indicates whether a candidate gene is novel (i.e. no previous evidence was available).

Candidate gene Functional evidence This study

LG 1a: 0.0–18.8 cM=10 284 104–23 021 414 bp (636 gene predictions) Caffeoyl-CoA O-methyltransferase: Anthocyanin methylation affected by transcriptional regulation of VvOMT1 and by VIT_01s0010g03460, VIT_01s0010g03470 structural variation of VvOMT2 (Fournier-Level et al., 2011) (VvOMT3), VIT_01s0010g03490 (VvOMT2), VvOMT1 highly induced at véraison in both HAPs and LAPs, with a slightly higher VIT_01s0010g03510* (VvOMT1) fold change in HAPs; more expressed in the skin than in the flesh; co-expressed with VvMYBA1 Glutathione S-transferase: VIT_01s0026g01340#, AtGSTU17 and ZmBz2 co-expressed with the flavonoid biosynthetic genes and X VIT_01s0026g01370, VIT_01s0026g02400* responsible for the transport of anthocyanins into the vacuole (Marrs et al., 1995;

Yonekura-Sakakibara et al., 2008); VIT_01s0026g01340 more expressed in the skin Downloaded from than in the flesh VIT_01s0026g01370 induced by cluster thinning, in parallel with an increase in anthocyanins (Pastore et al., 2011) VIT_01s0026g02400 induced at maturity in both HAPs and LAPs; more expressed in the skin than in the flesh http://jxb.oxfordjournals.org/ Calcium-binding protein CML: VIT_01s0026g02590* Induced at maturity in both HAPs and LAPs, with a higher fold change in HAPs X LG 1b: 22.8–59.0 cM (not analysed) LG 2: 45.3–49.5 cM=14 047 004–18 251 419 bp (149 gene predictions) MYB domain protein: VIT_02s0033g00370 Polymorphisms in VvMYBA1, VvMYBA2, and VvMYBA3 associated with the (VvMYBA4), VIT_02s0033g00380, quantitative variation in anthocyanin content (Fournier-Level et al., 2009) VIT_02s0033g00390* (VvMYBA2), VvMYBA1, VvMYBA2, and VvMYBA3 induced at véraison in both HAPs and LAPs, VIT_02s0033g00410* (VvMYBA1), with a higher fold change in HAPs VIT_02s0033g00430, VIT_02s0033g00440, VvMYBA1 more expressed in the skin than in the flesh

VIT_02s0033g00450* (VvMYBA3) Co-expressed at Biblioteca Fondazione Edmund Mach on July 24, 2015 Alternative oxidase 1D: VIT_02s0033g01380* Involved in ABA signalling, induced at maturity in both HAPs and LAPs, with a higher X fold change in HAPs; more expressed in the skin than in the flesh Ankyrin repeat family: VIT_02s0087g00100# AtPIA2 (PHYTOCHROME INTERACTING ANKYRIN-REPEAT PROTEIN 2) mediating X the interaction between phytochrome A and partner proteins to regulate the expression of anthocyanin biosynthetic genes (Yoo et al., 2011) LG 3: 22.7–43.1 cM=3 433 730–6 688 642 bp (329 gene predictions) Serine carboxypeptidase: VIT_03s0063g00860# Co-expressed with VvMYBA1 X Glucosyltransferase: VIT_03s0091g00040# (VvGT1), At4g15480 belonging to an anthocyanin regulon (Vanderauwera et al., 2005) VIT_03s0180g00200# (VvGT2), VIT_03s0180g00320# Recombinant VvGT1–VvGT3 enzymes catalysing the formation of acyl-glucose esters (VvGT3) of phenolic acids, which could act as acyl donors in esterification reactions by SCPL proteins (Khater et al., 2012); more expressed in the skin than in the flesh; VvGT1 induced by cluster thinning, in parallel with an increase in anthocyanins (Pastore et al., 2011) Cinnamyl alcohol dehydrogenase: Induced at maturity only in HAPs VIT_03s0180g00260* LG 4b: 0.0–23.0 cM=16 020 906–23 862 496 bp (577 gene predictions) Calmodulin: VIT_04s0023g01100* Induced at maturity only in HAPs X Flavonone-3-hydroxylase: VIT_04s0023g03370* More expressed in the skin than in the flesh; up-regulated in VlMybA1-2-transformed hairy roots (Cutanda-Perez et al., 2009) and by low nitrogen supply, in parallel with an increase in anthocyanins (Soubeyrand et al., 2014); co-expressed with genes significantly enriched in the functional category ‘Phenylpropanoid biosynthetic process’ SEN1 (dark inducible 1): VIT_04s0023g03600* Involved in jasmonate signalling, induced at véraison only in HAPs X LG 5: 47.4–60.1 cM=745 680–4 176 378 bp (373 gene predictions) Receptor-like protein kinase: VIT_05s0020g00730* Reduced at maturity only in LAPs; more expressed in the skin than in the flesh X BZIP protein HY5-HOMOLOG (HYH): AtHY5/HYH involved in light signalling and regulating low temperature-induced X VIT_05s0020g01090# anthocyanin accumulation (Zhang et al., 2011) VIT_05s0020g01090 more expressed in the skin than in the flesh; up-regulated by UV radiation in berry skin, in parallel with an increase in some acylated anthocyanins (Carbonell-Bejerano et al., 2014a); co-expressed with genes significantly enriched in the functional category ‘Anthocyanidin 3-O-glucosyltransferase activity’ 4434 | Costantini et al.

Table 1. Continued

Candidate gene Functional evidence This study

LG 6: 12.2–25.6 cM=12 767 560–17 912 115 bp (265 gene predictions) Calmodulin-related protein: VIT_06s0009g01920* Reduced during development only in LAPs; potentially involved in the calmodulin- X dependent signal transduction pathways that mediate sugar induction of anthocyanin synthesis (Vitrac et al., 2000); up-regulated by higher sugar, in parallel with an increase in total anthocyanins and in the delphinidin/cyanidin derivative ratio (Dai et al., 2014) Flavonoid 3ʹ,5ʹ-hydroxylase: VIT_06s0009g02830*, Proportion of delphinidin- and cyanidin-based anthocyanins correlating with the VIT_06s0009g02840, VIT_06s0009g02910, ratio of F3ʹ5ʹH to F3ʹH expression (Jeong et al., 2006; Falginella et al., 2010); VIT_06s0009g03010* VIT_06s0009g02830 and VIT_06s0009g03010 induced at véraison in both HAPs and LAPs, with a slightly higher fold change in HAPs; VIT_06s0009g03010 more expressed in the skin than in the flesh; VIT_06s0009g02910 and VIT_06s0009g03010 induced by cluster thinning, in parallel with an increase in anthocyanins (Pastore et al., 2011); VIT_06s0009g02840 induced by low nitrogen supply, in parallel with an increase in anthocyanins (Soubeyrand et al., 2014); significant enrichment of the

functional category ‘Metabolism.Secondary metabolism.Phenylpropanoid metabolism’ Downloaded from Hexokinase 6: VIT_06s0061g00040 Potentially involved in sugar signalling transduction leading to anthocyanin X accumulation (Vitrac et al., 2000); up-regulated by higher sugar, in parallel with an increase in total anthocyanins and in the delphinidin/cyanidin derivative ratio (Dai et al. 2014) LG 7b: 46.6–69.3 cM=4 252 958–14 369 584 bp (500 gene predictions) ABC transporter C member 9: VIT_07s0005g04680 Induced by cluster thinning, in parallel with an increase in anthocyanins (Pastore et al., X http://jxb.oxfordjournals.org/ 2011); significant enrichment of the functional category ‘Transport overview. Macromolecule transport.Multidrug transport’ Glutathione S-transferase 25 GSTU7: Induced at maturity in both HAPs and LAPs; co-expressed with the VvMYBA genes on X VIT_07s0005g04890* chromosome 2; significant enrichment of the functional category ‘Transport overview. Macromolecule transport.Multidrug transport’ LG 8a: 11.5–24.1 cM=17 261 077–20 013 908 bp (283 gene predictions) Anthocyanin membrane protein 1 (Anm1): VIT_08s0007g03560 co-expressed with VvMYBA1 X

VIT_08s0007g03560, VIT_08s0007g03570* VIT_08s0007g03570 induced at véraison in both HAPs and LAPs, with a higher fold at Biblioteca Fondazione Edmund Mach on July 24, 2015 change in HAPs; more expressed in the skin than in the flesh 4-Coumarate-CoA ligase: VIT_08s0007g05050 Induced by cluster thinning, in parallel with an increase in anthocyanins (Pastore et al., 2011) LG 8b: 24.1–48.5 cM=11 523 362–17 250 890 bp (578 gene predictions) AUX1 protein: VIT_08s0007g02030* AtAUX1 involved in anthocyanin accumulation in response to UV light X VIT_08s0007g02030 reduced during development only in LAPs; more expressed in the skin than in the flesh ATAN11 (ANTHOCYANIN11): VIT_08s0007g02920# PhAN11 involved in the post-translational regulation of the MYB-domain transcriptional X activator PhAN2 (de Vetten et al., 1997) VIT_08s0007g02920 more expressed in the skin than in the flesh P-coumaroyl shikimate 3ʹ-hydroxylase isoform 1: Induced at maturity only in HAPs; more expressed in the skin than in the flesh; VIT_08s0040g00780* co-expressed with genes significantly enriched in the functional category ‘Phenylpropanoid biosynthetic process’ Serine carboxypeptidase: VIT_08s0040g01040, Specifically up-regulated around véraison (Sweetman et al., 2012) X VIT_08s0040g01060#, VIT_08s0040g01110 Phenylalanine ammonia-lyase: VIT_08s0040g01710* Induced at maturity in both HAPs and LAPs, with a slightly higher fold change in HAPs; more expressed in the skin than in the flesh Calcium-transporting ATPase 1, endoplasmic Induced at maturity only in HAPs; Ca2+ ATPases involved in anthocyanin accumulation X reticulum-type ECA1: VIT_08s0040g02830* in carrot callus cultures (Sudha and Ravishankar, 2003) Glutathione S-transferase GSTO1: Induced at maturity (one probe exclusively in HAPs); induced by cluster thinning, in X VIT_08s0040g03040* parallel with an increase in anthocyanins (Pastore et al., 2011) TTG2 (TRANSPARENT TESTA GLABRA 2): AtTTG2 regulating proanthocyanidin and possibly anthocyanin production (Ishida VIT_08s0040g03070# (VvWRKY19) et al., 2007) VvWRKY19 proposed as an intermediate regulator of flavonoid biosynthetic pathway acting downstream of VvMYB5a and VvMYB5b and in concert with the regulatory complex MBW, with an effect on anthocyanin accumulation among others (Cavallini, 2012); more expressed in the skin than in the flesh Candidate genes of the grape anthocyanin pathway | 4435

Table 1. Continued

Candidate gene Functional evidence This study

LG 8c: 44.9–57.0 cM (not analysed) LG 9: 19.0–26.1 cM=13 924 117–19 956 861 bp (202 gene predictions) WRKY DNA-binding protein 40: VIT_09s0018g00240* Induced at maturity in both HAPs and LAPs, with a slightly higher fold change in X HAPs; more expressed in the skin than in the flesh LG 10: 0.0–30.7 cM=17 991–9 015 334 bp (605 gene predictions) Squamosa promoter-binding protein 3 (SPL3): LeSPL-CNR residing at at the tomato Colorless non-ripening (Cnr) locus and showing X VIT_10s0003g00050* reduced expression in the colourless mutant (Manning et al., 2006) VIT_10s0003g00050 reduced at maturity only in LAPs; more expressed in the skin than in the flesh Protein kinase: VIT_10s0003g01420*, VIT_10s0003g01420 induced at maturity only in HAPs; VIT_10s0003g03330 X VIT_10s0003g03330 co-expressed with genes significantly enriched in the functional category ‘Flavonoid metabolic process’; significant enrichment of the functional category ‘Signaling. Signaling pathway.Protein kinase’

Heat shock transcription factor A4A: Associated with berry colour (Ageorges et al., 2006) Downloaded from VIT_10s0003g01770# Multidrug resistance-associated protein 5: Induced by cluster thinning, in parallel with an increase in anthocyanins (Pastore et al., X VIT_10s0003g03470 2011) Jasmonate ZIM domain-containing protein 8: Induced during development only in LAPs, with a high fold change X VIT_10s0003g03790* Early-responsive to dehydration stress protein (ERD4): Induced at véraison only in HAPs X http://jxb.oxfordjournals.org/ VIT_10s0003g04180* WRKY DNA-binding protein 6: VIT_10s0116g01200* Induced at maturity in both HAPs and LAPs, with a slightly higher fold change in X HAPs; more expressed in the skin than in the flesh Auxin-responsive protein: VIT_10s0405g00040* Induced at véraison only in HAPs, with a high fold change; more expressed in the skin X than in the flesh LG 11a: 25.1–51.0 cM=12 199–5 477 250 bp (558 gene predictions) MYB domain protein: VIT_11s0016g01300 Regulating proanthocyanidin (PA) biosynthesis through activation of general flavonoid

(VvMYBPAR), VIT_11s0016g01320# (VvMYBPA2) pathway genes and of PA-specific genes (Terrier et al., 2009; Koyama et al., 2014); at Biblioteca Fondazione Edmund Mach on July 24, 2015 VvMYBPA2 more expressed in the skin than in the flesh Clavata1 receptor kinase (CLV1): Induced during development in both HAPs and LAPs, with a higher fold change in X VIT_11s0016g03080* HAPs; more expressed in the skin than in the flesh LG 12a: 7.3–26.2 cM=1 682 201–5 318 476 bp (317 gene predictions) Ethylene-responsive transcription factor 9: Induced during development only in HAPs; more expressed in the skin than in the X VIT_12s0028g03270* flesh; co-expressed with VvMYBA1 LG 12b: 26.2–40.5 cM=5 320 497–9 268 609 bp (358 gene predictions) Auxin-responsive protein AIR12: VIT_12s0057g00420* AtAIR12 involved in positive regulation of the flavonoid biosynthetic process X VIT_12s0057g00420 induced at maturity in both HAPs and LAPs, with a higher fold change in HAPs; more expressed in the skin than in the flesh; co-expressed with genes significantly enriched in the functional category ‘Ammonia-lyase activity’ Protein kinase: VIT_12s0057g00520, VIT_12s0057g00520 co-expressed with genes significantly enriched in the functional X VIT_12s0134g00450* category ‘Regulation of anthocyanin metabolic process’, including the VvMYBAs on chromosome 2 VIT_12s0134g00450 induced at véraison only in HAPs; more expressed in the skin than in the flesh; co-expressed with genes significantly enriched in the functional category ‘Flavonoid metabolic process’ Significant enrichment of the functional category ‘Signaling.Signaling pathway.Protein kinase’ Prephenate dehydratase: VIT_12s0059g00750# More expressed in the skin than in the flesh; co-expressed with VvMYBA1 Acyltransferase: VIT_12s0134g00580 (VvAT1), VIT_12s0134g00590 up-regulated specifically in the berry skin during the daytime X VIT_12s0134g00590 (VvAT2), VIT_12s0134g00600 near maturity (Carbonell-Bejerano et al., 2014b) (VvAT3), VIT_12s0134g00620 (VvAT4), VIT_12s0134g00620 induced by cluster thinning, in parallel with an increase in VIT_12s0134g00630 (VvAT5), VIT_12s0134g00640 anthocyanins (Pastore et al., 2011) (VvAT6), VIT_12s0134g00650# (VvAT7), VIT_12s0134g00670 more expressed in the skin than in the flesh VIT_12s0134g00660 (VvAT8),VIT_12s0134g00670# Significant enrichment of the functional category ‘Metabolism.Secondary metabolism. (VvAT9) Phenylpropanoid metabolism’ LG 17: 30.1–57.5 cM=4 031 362–9 700 367 bp (430 gene predictions) COBRA-like protein 4: VIT_17s0000g05050* AtCOB proteins playing a role in anthocyanin synthesis (Ko et al., 2006) VIT_17s0000g05050 reduced at véraison only in LAPs; proposed as a regulator of proanthocyanidin total quantity (Carrier et al., 2013) 4436 | Costantini et al.

Table 1. Continued

Candidate gene Functional evidence This study

Myb domain protein 94: VIT_17s0000g06190* ZmMYB31 overexpression leading to a large increase in the anthocyanin level through X the up-regulation of anthocyanin biosynthetic genes (Fornalé et al., 2010) VIT_17s0000g06190 induced at véraison only in HAPs; more espressed in the skin than in the flesh; paralleling the kinetics of anthocyanin accumulation during ripening (Ziliotto et al., 2012) Flavonoid 3ʹ-hydroxylase: VIT_17s0000g07210# More expressed in the skin than in the flesh; induced by cluster thinning, in parallel with higher relative levels of dihydroxylated anthocyanins (Pastore et al., 2011) EDS1 (Enhanced disease susceptibility 1): Involved in jasmonate signalling, induced at maturity only in HAPs; more expressed in X VIT_17s0000g07560* the skin than in the flesh LG 18b: 64.0–97.4 cM=16 285 312–28 018 133 bp (566 gene predictions) UDP-glucose:anthocyanidin VIT_18s0041g00710 more expressed in the skin than in the flesh; X 5,3-O-glucosyltransferase: VIT_18s0041g00710#, VIT_18s0041g00740 induced at maturity in both HAPs and LAPs; VIT_18s0041g00740*, VIT_18s0041g00840, VIT_18s0041g00840 and VIT_18s0041g00950 induced by cluster thinning, in parallel

VIT_18s0041g00950 with an increase in anthocyanins (Pastore et al., 2011); significant enrichment of the Downloaded from functional category ‘Metabolism.Secondary metabolism.Phenylpropanoid metabolism’ Peroxidase 12: VIT_18s0072g00160* Induced at maturity in both HAPs and LAPs, with a higher fold change in HAPs; more expressed in the skin than in the flesh Leucine-rich repeat protein kinase: Induced at maturity only in HAPs; more expressed in the skin than in the flesh X VIT_18s0072g00990* LG 19: 25.4–30.8 cM=4 694 353–9 176 259 bp (397 gene predictions) http://jxb.oxfordjournals.org/ Avr9/Cf-9 rapidly elicited protein 20: At4g27280 up-regulated after PAP1 overexpression and probably involved in the X VIT_19s0014g04650* downstream regulation of anthocyanin biosynthesis by PAP1 (Tohge et al. 2005) VIT_19s0014g04650 induced at maturity only in HAPs ABC transporter C member 9: VIT_19s0015g00010 VIT_19s0015g00010 and VIT_19s0015g00040 induced by cluster thinning, in parallel X (VvMRP2), VIT_19s0015g00040 (VvMRP4), with an increase in anthocyanins (Pastore et al., 2011); VIT_19s0015g00050 more VIT_19s0015g00050# (VvMRP5 in Cakir et al., 2013) expressed in the skin than in the flesh; significant enrichment of the functional category ‘Transport overview.Macromolecule transport.Multidrug transport’ at Biblioteca Fondazione Edmund Mach on July 24, 2015 degradation, such as peroxidases, laccases, polyphenol oxi- auxin, cytokinins, ethylene, and salicylic acid are typically dases, and β-glucosidases (Oren-Shamir, 2009), were also responsible for an increase in anthocyanins, whereas gibber- considered. Among transport-related molecules, priority was ellic acid has the opposite effect. Methyl jasmonate, which is given to GST (mainly of class phi; Dixon et al, 2010), ABC a stress signalling molecule, has been shown to elicit a higher (ABCC subfamily; Cakir et al., 2013), and MATE proteins, level of anthocyanins, whereas JASMONATE-ZIM domain based on the crucial role they play in anthocyanin vacuolar (JAZ) proteins repress anthocyanin accumulation (He et al., sequestration in Arabidopsis and several crops, including 2010; Qi et al., 2011). grapevine (Zhao and Dixon, 2009). These proteins are abun- In-house expression data for progeny individuals with con- dant in plants, especially in grapevine, for which 87, 26, and 65 trasting anthocyanin content (LAPs and HAPs) were used to genes were predicted for the most important classes of GST, support the choice of candidates genes. The reliability of these ABC, and MATE proteins, respectively (Gomez et al., 2009; data was confirmed by real-time RT–PCR (Supplementary Zenoni et al., 2010; Cakir et al., 2013). Therefore, their co- Fig. S2 at JXB online; Malacarne et al., 2015). Comparative localization with QTLs is expected to help in the selection of analysis of the transcriptome at three developmental stages candidates for functional characterization. Indeed, sequence for each triplet of extreme individuals revealed the differ- similarity has not always proved to be sufficient to predict ential expression of 3755 and 2933 probe sets in LAPs and functional similarity (Alfenito et al., 1998). Similarly, a strict HAPs, respectively. Of these, 1841 were exclusively modu- correlation between the expression of transport-related pro- lated in LAPs and 1019 in HAPs, while 1914 were commonly teins and anthocyanin accumulation during berry ripening is modulated but with low correlation (Supplementary Table not expected in view of gene redundancy in plant transporter S6). This approach was preferred to the direct comparison families on the one hand and their multifunctionality and between LAPs and HAPs at each developmental stage in up-regulation effect by their substrates on the other hand. order to minimize the possible differences due to non-perfect Finally, genes involved in response to stimuli which are known phase sampling. to affect anthocyanin content and composition were taken The selected candidate genes are shown in Table 1. Some into account. In particular, anthocyanin accumulation is previous findings have been confirmed, with the addition considered a common response to stress conditions (Teixeira of new information. For example, the co-localization of et al., 2013). Sugars also induce anthocyanin biosynthesis the QTLs for 3ʹ and 5ʹ methylation (Fig. 2; Supplementary through the participation of hexokinases, Ca2+, calmodulin, Table S5 at JXB online) and the high correlation between protein kinases, and protein phosphatases (Vitrac et al., 2000; the 3ʹMeth/3ʹOH and 3ʹ5ʹMeth/3ʹ5ʹOH ratios in the Lecourieux et al., 2014). As regards hormones, abscisic acid, Syrah×Pinot Noir progeny (Suppelmentary Table S4A) and Candidate genes of the grape anthocyanin pathway | 4437 in the grapevine germplasm (Mattivi et al., 2006) suggest JXB online), are a cluster of UDP-glucose:anthocyanidin that the candidate enzymes are able to perform both reac- 5,3-O-glucosyltransferases, mostly expressed in V. vinifera tions, as biochemically demonstrated for the methyltrans- berry skin (Fasoli et al., 2012). It is not known whether ferase that best matches with VvAOMT1 (Hugueney et al., these enzymes really perform two sequential glucosyla- 2009). The identification of a QTL regulating the hydroxyla- tions as suggested by their annotation, considering that tion level on LG 6 supports the involvement of the flavonoid 3-O-glucosylation is generally required and sufficient for 3ʹ,5ʹ-hydroxylases that have been previously suggested based anthocyanidin stabilization in V. vinifera. However, it can be on expression analysis (Jeong et al., 2006; Falginella et al., speculated that they glucosylate anthocyanins, and gluco- 2010), providing a possible genetic determination for trait sylated anthocyanins are the substrates for acyltransferases. variation (in contrast, gene expression might be the result of The presence on LG 18b of a gene cluster comprising 29 a cascade). Most of the candidate genes shown in Table 1 are predictions for laccase is also noteworthy. Most of them reported for the first time and represent a valuable resource were reported to be expressed in berry skins, especially at for functional characterization. post-harvest (Fasoli et al., 2012), which is in agreement with the higher degradation rate observed at over-ripening for Anthocyanin acylation coumaroylglucoside derivatives than for non-acylated glu- coside derivatives or acetylglucoside derivatives (Fournand Downloaded from One of the objectives of this work was a better understand- et al., 2006). Finally, a significant enrichment of QTL ing of the acylation step, which is, at the moment, com- regions related to the content of p-coumaric derivatives was pletely obscure in grapevine. Unfortunately, no QTL could observed in gene predictions belonging to the functional be found for the presence/absence of acylated anthocyanins, subcategories ‘Response to stimulus.Stress response.Biotic as expected by lack of segregation for this Pinot Noir trait stress response’ and ‘Diverse functions.Gene family with http://jxb.oxfordjournals.org/ in the progeny. However, several minor QTLs, which might diverse functions.NBS-LRR superfamily’ (for the QTLs on regulate the quantitative variation in the acylation level, were LG 18b) and ‘Response to stimulus.Stress response.Abiotic identified (Fig. 2; Supplementary Table S5 at JXB online). stress response’ (for the QTL on LG 12b) (Supplementary The strongest candidate genes code for enzymes responsible Table S7). This observation is in agreement with the integra- for anthocyanin decoration with acyl groups, which can be tive analysis of Zamboni et al. (2010) showing a correlation classified within the BAHD and serine carboxypeptidase- of coumarated anthocyanins, that are accumulated mainly like (SCPL) families (St-Pierre et al., 2000; Milkowski and during withering, with stress- and pathogenesis-induced Strack, 2004). For example, in the confidence interval of transcripts and proteins. at Biblioteca Fondazione Edmund Mach on July 24, 2015 the QTL on LG 12b, which explains ~12% of the pheno- typic variance for peonidin-3-monoglucoside-p-coumarate and malvidin derivatives, a cluster of nine BAHD acyl- Conclusions transferases was found. After screening for the presence of In this work, a huge amount of data has been generated to conserved motifs, two of them (VvAT1 and VvAT9) were characterize the genetic bases of anthocyanin content and retained for phylogenetic analysis and were grouped in composition in ripe grapevine berries. A dense map was clade Ia with plant acyltransferases involved specifically in obtained from the segregation of ~600 markers, and the the modification of anthocyanins. In contrast, the remain- amount of 15 distinct anthocyanins was measured dur- ing grapevine gene predictions considered for phylogenetic ing four growing seasons in the Syrah×Pinot Noir progeny. analysis were attributed to clades Ib–Vb, whose members are Several consistent QTLs were identified and special atten- specific to different substrates (Supplementary Fig. S3). As tion was paid to the minor ones, which might be responsible for methylation and hydroxylation, a QTL controlling sev- for the fine regulation of berry and wine colour. Based on eral acylation-related subtraits, which co-localizes with the expression data from this study and on functional evidence cluster of MYBA transcription factors, was detected on LG from the literature, reliable candidate genes are proposed, 2 (Fig. 2; Supplementary Table S5). The MYBA locus might which represent a valuable resource for further validation. exert its pleiotropic effect on these traits indirectly (as a con- sequence of its effect on total anthocyanin accumulation); however it is tempting to speculate that grapevine anthocya- Supplementary data nin acyltransferase genes are regulated by MYB factors, sim- ilar to what was reported in Arabidopsis and Gerbera hybrida Supplementary data are available at JXB online. (Tohge et al., 2005; Laitinen et al., 2008). In agreement with Text S1. Variation of anthocyanin content and composi- this hypothesis, a very high proportion of coumaroylated tion in Syrah, Pinot Noir, and their progeny. anthocyanins (70%) was found in Syrah roots overexpressing Figure S1. Variation of anthocyanin content in the VlMYBA1-2, while ~30% of this kind of anthocyanins are Syrah×Pinot Noir progeny. usually found in mature red berries from the same cultivar Figure S2. Validation of the pseudoreplicates approach in (Cutanda-Perez et al., 2009). the gene expression study. Alternative candidates for the QTL on LG 18b, which Figure S3. Evolutionary relationships of 108 BAHD pro- putatively explains from 10% to 21% of the phenotypic vari- teins from grapevine and other plant species. ance related to acylation (Fig. 2; Supplementary Table S5 at Table S1. Main features of the Syrah×Pinot Noir map. 4438 | Costantini et al.

Table S2. Anthocyanin profile of the parental varieties. Cavallini E. 2012. Unravelling the regulatory network putatively controlling Table S3. Anthocyanin variation in the coloured individu- flavonoid biosynthesis in grapevine . PhD thesis, Verona University. Cavallini E, Zenoni S, Finezzo L, Guzzo F, Zamboni A, Avesani L, als of the Syrah×Pinot Noir progeny. Tornielli GB. 2014. Functional diversification of grapevine MYB5a and Table S4. Correlations between metabolites in the coloured MYB5b in the control of flavonoid biosynthesis in a petunia anthocyanin progeny. regulatory mutant. Plant and Cell Physiology 55, 517–534. Table S5. List of QTLs for anthocyanin content and com- Conn S, Curtin C, Bézier A, Franco C, Zhang W. 2008. Purification, molecular cloning, and characterization of glutathione S-transferases position identified in the Syrah×Pinot Noir progeny. (GSTs) from pigmented Vitis vinifera L. cell suspension cultures as putative Table S6. List of the GrapeGen Chip probe sets differen- anthocyanin transport proteins. Journal of Experimental Botany 59, tially expressed during development in high and low antho- 3621–3634. cyanin producers. Cutanda-Perez MC, Ageorges A, Gomez C, Vialet S, Terrier N, Romieu C, Torregrosa L. 2009. Ectopic expression of VlmybA1 in Table S7. Test for over-representation of specific functional grapevine activates a narrow set of genes involved in anthocyanin categories in QTL regions. synthesis and transport. Plant Molecular Biology 69, 633–648. Dai ZW, Meddar M, Renaud C, Merlin I, Hilbert G, Delrot S, Gomès E. 2014. Long-term in vitro culture of grape berries and its application to Acknowledgements assess the effects of sugar supply on anthocyanin accumulation. Journal of Experimental Botany 65, 4665–4677. Downloaded from We thank the staff of the Grapevine Breeding Platform for assistance in the de Vetten N, Quattrocchio F, Mol J, Koes R. 1997. The anll locus field, Domenico Masuero and Urska Vrhovsek for metabolic analysis, Juri controlling flower pigmentation in petunia encodes a novel WD-repeat Battilana for his contribution to QTL analysis, Emanuela Coller for microar- protein conserved in yeast, plants, and animals. Genes and Development ray analysis, and Francesco Emanuelli for critical discussion. 11, 1422–1434. Dixon DP, Skipsey M, Edwards R. 2010. Roles for glutathione transferases in plant secondary metabolism. Phytochemistry 71, 338–350. http://jxb.oxfordjournals.org/ References Falginella L, Castellarin SD, Testolin R, Gambetta GA, Morgante Ageorges A, Fernandez L, Vialet S, Merdinoglu D, Terrier N, Romieu M, Di Gaspero G. 2010. Expansion and subfunctionalisation of flavonoid C. 2006. Four specific isogenes of the anthocyanin metabolic pathway 3ʹ,5ʹ-hydroxylases in the grapevine lineage. BMC Genomics 11, 562. are systematically co-expressed with the red colour of grape berries. Plant Fasoli M, Dal Santo S, Zenoni S, et al. 2012. The grapevine expression Science 170, 372–383. atlas reveals a deep transcriptome shift driving the entire plant into a Alfenito MR, Souer E, Goodman CD, Buell R, Mol J, Koes R, Walbot maturation program. The Plant Cell 24, 3489–3505. V. 1998. Functional complementation of anthocyanin sequestration in the Fornalé S, Shi X, Chai C, et al. 2010. ZmMYB31 directly represses vacuole by widely divergent glutathione S-transferases. The Plant Cell 10, maize lignin genes and redirects the phenylpropanoid metabolic flux. The 1135–1149. Plant Journal 64, 633–644. Ban Y, Mitani N, Hayashi T, Sato A Azuma A, Kono A, Kobayashi Fournand D, Vicens A, Sidhoum L, Souquet JM, Moutounet M, at Biblioteca Fondazione Edmund Mach on July 24, 2015 S. 2014. Exploring quantitative trait loci for anthocyanin content in Cheynier V. 2006. Accumulation and extractability of grape skin tannins interspecific hybrid grape (Vitis labruscana×Vitis vinifera). Euphytica 198, and anthocyanins at different advanced physiological stages. Journal of 101–114. Agriculture and Food Chemistry 54, 7331−7338. Benjamini Y, Hochberg Y. 1995. Controlling the false discovery rate: a Fournier-Level A, Hugueney P, Verriès C, This P, Ageorges A. 2011. practical and powerful approach to multiple testing. Journal of the Royal Genetic mechanisms underlying the methylation level of anthocyanins in Statistical Society 57, 289–300. grape (Vitis vinifera L.). BMC Plant Biology 11, 179. Bogs J, Jaffé FW, Takos AM, Walker AR, Robinson SP. 2007. The Fournier-Level A, Le Cunff L, Gomez C, Doligez A, Ageorges grapevine transcription factor VvMYBPA1 regulates proanthocyanidin A, Roux C, Bertrand Y, Souquet JM, Cheynier V, This P. 2009. synthesis during fruit development. Plant Physiology 143, 1347–1361. Quantitative genetic bases of anthocyanin variation in grape (Vitis vinifera Braidot E, Petrussa E, Bertolini A, Peresson C, Ermacora P, Loi N, L. ssp. sativa) berry: a quantitative trait locus to quantitative trait nucleotide Terdoslavich M, Passamonti S, Macrì F, Vianello A. 2008. Evidence integrated study. Genetics 183, 1127–1139. for a putative flavonoid translocator similar to mammalian bilitranslocase in Francisco RM, Regalado A, Ageorges A, et al. 2013. ABCC1, an grape berries (Vitis vinifera L.) during ripening. Planta 228, 203–213. ATP binding cassette protein from grape berry, transports anthocyanidin Çakır B, Kılıçkaya O. 2013. Whole-genome survey of the putative ATP- 3-O-glucosides. The Plant Cell 25, 1840–1854. binding cassette transporter family genes in Vitis vinifera. PLoS One 8, Gomez C, Conejero G, Torregrosa L, Cheynier V, Terrier N, e78860. Ageorges A. 2011. In vivo grapevine anthocyanin transport involves Carbonell-Bejerano P, Diago MP, Martínez-Abaigar J, Martínez- vesicle-mediated trafficking and the contribution of anthoMATE Zapater JM, Tardáguila J, Núñez-Olivera E. 2014a. Solar ultraviolet transporters and GST. The Plant Journal 67, 960–970. radiation is necessary to enhance grapevine fruit ripening transcriptional Gomez C, Terrier N, Torregrosa L, et al. 2009. Grapevine MATE-type and phenolic responses. BMC Plant Biology 14, 183. proteins act as vacuolar H+-dependent acylated anthocyanin transporters. Carbonell-Bejerano P, Rodríguez V, Royo1 C, Hernáiz S, Moro- Plant Physiology 150, 402–415. González LC, Torres-Viñals M, Martínez-Zapater JM. 2014b. Grimplet J, Van Hemert J, Carbonell-Bejerano P, Díaz-Riquelme Circadian oscillatory transcriptional programs in grapevine ripening fruits. J, Dickerson J, Fennell A, Pezzotti M, Martínez-Zapater JM. 2012. BMC Plant Biology 14, 78. Comparative analysis of grapevine whole-genome gene predictions, Cardoso S, Lau W, Dias JE, Fevereiro P, Maniatis N. 2012. A functional annotation, categorization and integration of the predicted gene candidate-gene association study for berry colour and anthocyanin sequences. BMC Research Notes 5, 213. content in Vitis vinifera L. PLoS One 7, e46021. He F, Liang NN, Mu L, Pan QH, Wang J, Reeves MJ, Duan CQ. 2012. Carrier G, Huang YF, Le Cunff L, Fournier-Level A, Vialet S, Souquet Anthocyanins and their variation in red wines I. Monomeric anthocyanins JM, Cheynier V, Terrier N, This P. 2013. Selection of candidate genes and their color expression. Molecules 17, 1571–1601. for grape proanthocyanidin pathway by an integrative approach. Plant He F, Mu L, Yan GL, Liang NN, Pan QH, Wang J, Reeves MJ, Duan Physiology and Biochemistry 72, 87–95. CQ. 2010. Biosynthesis of anthocyanins and their regulation in colored Castia T, Franco MA, Mattivi F, Muggiolu G, Sferlazzo G, Versini grapes. Molecules 15, 9057–9091. G. 1992. Characterization of grapes cultivated in Sardinia: chemometric Hichri I, Barrieu F, Bogs J, Kappel C, Delrot S, Lauvergeat V. methods applied to the anthocyanic fraction. Sciences des Aliments 12, 2011. Recent advances in the transcriptional regulation of the flavonoid 239–255. biosynthetic pathway. Journal of Experimental Botany 62, 2465–2483. Candidate genes of the grape anthocyanin pathway | 4439

Hichri I, Heppel SC, Pillet J, Léon C, Czemmel S, Delrot S, Mattivi F, Guzzon R, Vrhovsek U, Stefanini M, Velasco R. 2006. Lauvergeat V, Bogs J. 2010. The basic helix–loop–helix transcription Metabolite profiling of grape: flavonols and anthocyanins. Journal of factor MYC1 is involved in the regulation of the flavonoid biosynthesis Agricultural and Food Chemistry 54, 7692–7702. pathway in grapevine. Molecular Plant 3, 509–523. Matus JT, Poupin MJ, Cañón P, Bordeu E, Alcalde JA, Arce- Huang YF, Bertrand Y, Guiraud JL, Vialet S, Launay A, Cheynier Johnson P. 2010. Isolation of WDR and bHLH genes related to flavonoid V, Terrier N, This P. 2013. Expression QTL mapping in grapevine— synthesis in grapevine (Vitis vinifera L.). Plant Molecular Biology 72, revisiting the genetic determinism of grape skin colour. Plant Science 607–620. 207, 18–24. Mazza G. 1995. Anthocyanins in grapes and grape products. Critical Huang YF, Doligez A, Fournier-Level A, et al. 2012. Dissecting genetic Reviews in Food Science and Nutrition 35, 341–371. architecture of grape proanthocyanidin composition through quantitative Meysman P, Sonego P, Bianco L, et al. 2014. COLOMBOS v2.0: an trait locus mapping. BMC Plant Biology 12, 30. ever expanding collection of bacterial expression compendia. Nucleic Hugueney P, Provenzano S, Verriès C, Ferrandino A, Meudec E, Acids Research 42, D649–D653. Batelli G, Merdinoglu D, Cheynier V, Schubert A, Ageorges A. 2009. Milkowski C, Strack D. 2004. Serine carboxypeptidase-like A novel cation-dependent O-methyltransferase involved in anthocyanin acyltransferases. Phytochemistry 65, 517–524. methylation in grapevine. Plant Physiology 150, 2057–2070. Mizuno H, Hirano K, Okamoto G. 2006. Effect of anthocyanin Ishida T, Hattori S, Sano R, et al. 2007. Arabidopsis TRANSPARENT composition in grape skin on anthocyanic vacuolar inclusion development TESTA GLABRA2 is directly regulated by R2R3 MYB transcription factors and skin coloration. Vitis 45, 173–177. and is involved in regulation of GLABRA2 transcription in epidermal differentiation. The Plant Cell 19, 2531–2543. Oren-Shamir M. 2009. Does anthocyanin degradation play a significant role in determining pigment concentration in plants? Plant Science 177, Downloaded from Jackson RS. 2008. Chemical constituents of grapes and wine. In: Wine 310–316. science: principles and applications . Burlington: Elsevier, 270–331. Passamonti S, Vrhovsek U, Mattivi F. 2002. The interaction of Jeong ST, Goto-Yamamoto N, Hashizume K, Esaka M. 2006. anthocyanins with bilitranslocase. Biochemical and Biophysical Research Expression of the flavonoid ʹ3 -hydroxylase and flavonoid ʹ3 ,5ʹ-hydroxylase Communications 296, 631–636. genes and flavonoid composition in grape (Vitis vinifera). Plant Science 170, 61–69. Pastore C, Zenoni S, Tornielli GB, Allegro G, Dal Santo S, Valentini G, Intrieri C, Pezzotti M, Filippetti I. 2011. Increasing the source/ http://jxb.oxfordjournals.org/ Khater F, Fournand D, Vialet S, Meudec E, Cheynier V, Terrier N. sink ratio in Vitis vinifera (cv ) induces extensive transcriptome 2012. Identification and functional characterization of cDNAs coding for reprogramming and modifies berry ripening. BMC Genomics 12, 631. hydroxybenzoate/hydroxycinnamate glucosyltransferases co-expressed with genes related to proanthocyanidin biosynthesis. Journal of Peng FY, Reid KE, Liao N, et al. 2007. Generation of ESTs in Vitis Experimental Botany 63, 1201–1214. vinifera wine grape (‘Cabernet Sauvignon’) and table grape (‘Muscat Hamburg’) and discovery of new candidate genes with potential roles in Ko JH, Kim JH, Jayanty SS, Howe GA, Han KH. 2006. Loss of function berry development. Gene 402, 40–50. of COBRA, a determinant of oriented cell expansion, invokes cellular defence responses in Arabidopsis thaliana. Journal of Experimental Botany Pindo M, Vezzulli S, Coppola G, Cartwright DA, Zharkikh A, Velasco 57, 2923–2936. R, Troggio M. 2008. SNP high-throughput screening in grapevine using the SNPlex™ genotyping system. BMC Plant Biology 8, 12. Kosambi DD. 1944. The estimation of map distance from recombination at Biblioteca Fondazione Edmund Mach on July 24, 2015 values. Annals of Eugenics 12, 172–175. Pojer E, Mattivi F, Johnson D, Stockley CS. 2013. The case for anthocyanin consumption to promote human health: a review. Koyama K, Numata M, Nakajima I, Goto-Yamamoto N, Matsumura Comprehensive Reviews in Food Science and Food Safety 12, 483–508. H, Tanaka N. 2014. Functional characterization of a new grapevine MYB transcription factor and regulation of proanthocyanidins biosynthesis in Qi T, Song S, Ren Q, Wu D, Huang H, Chen Y, Fan M, Peng W, grapes. Journal of Experimental Botany 65, 4433–4449. Ren C, Xie D. 2011. The jasmonate-ZIM-domain proteins interact with the WD-repeat/bHLH/MYB complexes to regulate jasmonate-mediated Laitinen RAE, Ainasoja M, Broholm SK, Teeri TH, Elomaa P. 2008. anthocyanin accumulation and trichome initiation in Arabidopsis thaliana. Identification of target genes for a MYB-type anthocyanin regulator in The Plant Cell 23, 1795–1814. Gerbera hybrida. Journal of Experimental Botany 59, 3691–3703. Rossetto M, Vanzani P, Mattivi F, Lunelli M, Scarpa M, Rigo A. 2002. Lecourieux F, Kappel C, Lecourieux D, Serrano A, Torres E, Arce- Synergistic antioxidant effect of catechin and malvidin 3-glucoside on Johnson P, Delrot S. 2014. An update on sugar transport and signalling free radical-initiated peroxidation of linoleic acid in micelles. Archives of in grapevine. Journal of Experimental Botany 65, 821–832. Biochemistry and Biophysics 408, 239–245. Lijavetzky D, Carbonell-Bejerano P, Grimplet J, Bravo G, Flores Soubeyrand E, Basteau C, Hilbert G, van Leeuwen C, Delrot S, P, Fenoll J, Hellín P, Oliveros JC, Martínez-Zapater JM. 2012. Gomès E. 2014. Nitrogen supply affects anthocyanin biosynthetic Berry flesh and skin ripening features in Vitis vinifera as assessed by and regulatory genes in grapevine cv. Cabernet-Sauvignon berries. transcriptional profiling. PLoS One 7, e39547. Phytochemistry 103, 38–49. Malacarne G, Costantini L, Coller E, Battilana J, Velasco R, Stange M, Utz HF, Schrag TA, Melchinger AE, Würschum T. 2013. Vrhovsek U, Grando MS, Moser C. 2015. Regulation of flavonol content High-density genotyping: an overkill for QTL mapping? Lessons learned and composition in (Syrah×Pinot Noir) mature grapes: integration of from a case study in maize and simulations. Theoretical and Applied transcriptional profiling and metabolic quantitative trait locus analyses. Genetics 126, 2563–2574. Journal of Experimental Botany 66, 4441–4453. St-Pierre B, De Luca V. 2000. Evolution of acyltransferase genes: origin Manning K, Tör M, Poole M, Hong Y, Thompson AJ, King GJ, and diversification of the BAHD superfamily of acyltransferases involved in Giovannoni JJ, Seymour GB. 2006. A naturally occurring epigenetic secondary metabolism. In: Romeo JT, Ibrahim R, Varin L, De Luca V, eds. mutation in a gene encoding an SBP-box transcription factor inhibits Evolution of metabolic pathways . Oxford: Elsevier Science, 285–315. tomato fruit ripening. Nature Genetics 38, 948–952. Sudha G, Ravishankar GA. 2003. The role of calcium channels in Marinova K, Kleinschmidt K, Weissenböck G, Klein M. 2007. anthocyanin production in callus cultures of Daucus carota. Plant Growth Flavonoid biosynthesis in barley primary leaves requires the presence of Regulation 40, 163–169. the vacuole and controls the activity of vacuolar flavonoid transport. Plant Physiology 144, 432–444. Sweetman C, Wong DCJ, Ford CM, Drew DP. 2012. Transcriptome analysis at four developmental stages of grape berry (Vitis vinifera cv. Marrs KA, Alfenito MR, Lloyd AM, Walbot V. 1995. A glutathione Shiraz) provides insights into regulated and coordinated gene expression. S-transferase involved in vacuolar transfer encoded by the maize gene BMC Genomics 13, 691. Bronze-2. Nature 375, 397–400. Teixeira A, Eiras-Dias J, Castellarin SD, Gerós H. 2013. Berry Mateos A, Herrero J, Tamames J, Dopazo J. 2002. Supervised neural phenolics of grapevine under challenging environments. International networks for clustering conditions in DNA array data after reducing noise Journal of Molecular Sciences 14, 18711–18739. by clustering gene expression profiles. In: Lin S, Johnson K, eds. Methods of microarray data analysis II . Boston: Kluwer Academic Publishers, Terrier N, Torregrosa L, Ageorges A, Vialet S, Verriès C, Cheynier 91–103. V, Romieu C. 2009. Ectopic expression of VvMybPA2 promotes 4440 | Costantini et al. proanthocyanidin biosynthesis in grapevine and suggests additional Wong DCJ, Sweetman C, Drew DP, Ford CM. 2013. VTCdb: a gene targets in the pathway. Plant Physiology 149, 1028–1041. co-expression database for the crop species Vitis vinifera (grapevine). BMC Genomics 14, 882. Tohge T, Nishiyama Y, Hirai MY, et al. 2005. Functional genomics by integrated analysis of metabolome and transcriptome of Arabidopsis Yonekura-Sakakibara K, Nakayama T, Yamazaki M, Saito K. 2009. plants over-expressing an MYB transcription factor. The Plant Journal 42, Modification and stabilization of anthocyanins. In: Gould K, Davies K, 218–235. Winefield C, eds. Anthocyanins: biosynthesis, functions, and applications . New York: Springer Science+Business Media, LLC, 169–190. Troggio M, Malacarne G, Coppola G, et al. 2007. A dense single- nucleotide polymorphism-based genetic linkage map of grapevine (Vitis Yonekura-Sakakibara K, Tohge T, Matsuda F, Nakabayashi R, vinifera L.) anchoring Pinot Noir bacterial artificial chromosome contigs. Takayama H, Niida R, Watanabe-Takahashi A, Inoue E, Saito K. 2008. Comprehensive flavonol profiling and transcriptome coexpression Genetics 176, 2637–2650. analysis leading to decoding gene–metabolite correlations in Arabidopsis. Vanderauwera S, Zimmermann P, Rombauts S, Vandenabeele The Plant Cell 20, 2160–2176. S, Langebartels C, Gruissem W, Inze D, Van Breusegem F. 2005. Yoo J, Shin DH, Cho MH, Kim TL, Bhoo SH, Hahn TR. 2011. Genome-wide analysis of hydrogen peroxide-regulated gene expression in An ankyrin repeat protein is involved in anthocyanin biosynthesis in Arabidopsis reveals a high light-induced transcriptional cluster involved in Arabidopsis. Physiologia Plantarum 142, 314–325. anthocyanin biosynthesis. Plant Physiology 139, 806–821. Zamboni A, Di Carli M, Guzzo F, et al. 2010. Identification of putative Van Ooijen JW. 2006. JoinMap 4, Software for the calculation of genetic stage-specific grapevine berry biomarkers and omics data integration into linkage maps in experimental populations . Wageningen, The Netherlands: networks. Plant Physiology 154, 1439–1459. Kyazma BV.

Zenoni S, Ferrarini A, Giacomelli E, Xumerle L, Fasoli M, Malerba Downloaded from Van Ooijen JW. 2009. MapQTL 6, Software for the mapping of G, Bellin D, Pezzotti M, Delledonne M. 2010. Characterization of quantitative trait loci in experimental populations of diploid species . transcriptional complexity during berry development in Vitis vinifera using Wageningen, The Netherlands: Kyazma BV. RNA-Seq. Plant Physiology 152, 1787–1795. Velasco R, Zharkikh A, Troggio M, et al. 2007. A high quality draft Zhang Y, Zheng S, Liu Z, Wang L, Bi Y. 2011. Both HY5 and HYH are consensus sequence of the genome of a heterozygous grapevine variety. necessary regulators for low temperature-induced anthocyanin accumulation in Arabidopsis seedlings. Journal of Plant Physiology 168, 367–374.

PLoS One 12, e1326. http://jxb.oxfordjournals.org/ Zhao J, Dixon RA. 2009. The ‘ins’ and ‘outs’ of flavonoid transport.

Vitrac X, Larronde F, Krisa S, Decendit A, Deffieux G, Mérillon JM. Trends in Plant Science 15, 72–80. 2000. Sugar sensing and Ca2+ calmodulin requirement in Vitis vinifera cells producing anthocyanins Phytochemistry 53, 659–665. Ziliotto F, Corso M, Rizzini FM, Rasori A, Botton A, Bonghi C. 2012. Grape berry ripening delay induced by a pre-véraison NAA treatment Voorrips RE. 2002. MapChart: software for the graphical presentation of is paralleled by a shift in the expression pattern of auxin- and ethylene- linkage maps and QTLs. Journal of Heredity 93, 77–78. related genes. BMC Plant Biology 12, 185. at Biblioteca Fondazione Edmund Mach on July 24, 2015