Vitis Vinifera L.)
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Heredity (2010) 104, 351–362 & 2010 Macmillan Publishers Limited All rights reserved 0018-067X/10 $32.00 www.nature.com/hdy ORIGINAL ARTICLE Evolution of the VvMybA gene family, the major determinant of berry colour in cultivated grapevine (Vitis vinifera L.) A Fournier-Level1, T Lacombe1, L Le Cunff1,2, J-M Boursiquot1,2 and P This1 1INRA UMR 1097 Diversite´ et Adaptation des Plantes Cultive´es, Montpellier, France and 2UMT Ge´no-Vigne, INRA-IFV, Montpellier, France Polymorphisms in the grape transcription factor family VvMybA2, both mutations followed by a selective sweep. VvMybA are responsible for variation in anthocyanin content The mutational pattern for these colour genes is consistent in the berries of cultivated grapevine (Vitis vinifera L. subsp. with progressively relaxed selection from constrained ances- sativa). Previous study has shown that white grapes arose tral coloured haplotypes to light coloured and finally white through the mutation of two adjacent genes: a retroelement haplotypes. Dynamics of population size in the VvMybA insertion in VvMybA1 and a single-nucleotide polymorphism genes showed an initial exponential growth, followed by mutation in VvMybA2. The purpose of this study was to population size stabilization. Most ancestral haplotypes are understand how these mutations emerged and affected found in cultivars from western region, whereas recent genetic diversity at neighbouring sites and how they haplotypes are essentially present in table cultivars from structured the genetic diversity of cultivated grapevines. eastern regions where intense breeding practices may have We sequenced a total of 3225 bp of these genes in a core replaced the original diversity. Finally, the emergence of the collection of genetic resources, and carried out empirical white allele was followed by a recent strong exponential selection tests, phylogenetic- and coalescence-based demo- growth, showing a very fast diffusion of the initial white allele. graphic analyses. The insertion in the VvMybA1 promoter Heredity (2010) 104, 351–362; doi:10.1038/hdy.2009.148; was shown to have occurred recently, after the mutation of published online 18 November 2009 Keywords: anthocyanin; white grape; grape domestication; selection Introduction in the late Neolithic, with the establishment of viticulture from 5000 BC in Syria (Goor, 1966). Owing to its easy Major domestication events of today’s most widespread vegetative propagation, grapevine has been widely crops started between 12 000 and 10 000 years ago, at the distributed, exchanged and spread across the Mediterra- end of the Epipalaeolithic, from their wild relatives. This nean basin for millennia (Unwin, 1991). Distinct uses was illustrated by the first domestic rye grains found in of grapes for wine making in the West and table con- Abu Hureyra in Syria (Zohary and Hopf, 2000). These sumption on the Eastern shores led to strong disruptive events led to continuous morphological change during selection, with smaller and juicy berried Western the transition from wild plants in natural conditions to cultivars and bigger and fleshy berried Eastern cultivars. domesticated crops in controlled conditions (Brown et al., The diffusion of grapevines from Transcaucasus natu- 2009). Evidence for these domestication events at the rally led to an East-to-West pattern of colonization, molecular level has been widely emphasized in plants, as following human migrations (Mc Govern, 2003). Further- for example with the Y1 locus in maize (Palaisa et al., more, propagation by cuttings, generation overlap and 2004) and the qSW5 locus in rice (Shomura et al., 2008). possible secondary domestication events, as well as This artificial selection process has favoured the fixation hybridization with sympatric wild relatives (Levadoux, of alleles that were considered beneficial in cultural 1956), led to a very complex pattern of admixture conditions. Consequently, the crop genetic diversity is (Aradhya et al., 2003; Arroyo-Garcia et al., 2006). believed to have been shaped by selection and adapta- Although nuclear and chloroplastic microsatellite mar- tion, either ecological or anthropic in changing environ- kers helped to resolve questions about the grape genetic mental conditions (Ehrenreich and Purugganan, 2006; structure and cultivar evolution (Arroyo-Garcia et al., Brown et al., 2009). 2006; This et al., 2006), the molecular bases of trait Grape (Vitis vinifera L. subsp. sativa) was among the adaptation are still poorly investigated in non-model first perennial domesticated crops. Its cultivation started organisms. The primary domestication syndromes in grape are hermaphroditism and the size of berries and Correspondence: Dr P This, UMR Diversite´ et adaptation des plantes bunches (Levadoux, 1956). Colour variation is neither cultive´es, INRA, 2 place Viala, Montpellier 34060, France. E-mail: [email protected] considered as a primary target for selection nor Received 3 June 2009; revised 24 August 2009; accepted 17 correlated with a particular geographic origin (Sefc September 2009; published online 18 November 2009 et al., 2000; Lijavetzky et al., 2006). Molecular evolution of grape colour A Fournier-Level et al 352 Grape berry colour is due to the presence of a single netic analyses. Finally, we investigated how the demo- pigment family, the anthocyanins, which vary highly in graphic history of the colour locus has been affected by concentration among grape cultivars (Mazza, 1995). these successive mutational events, using Bayesian recon- While in the ancestral wild vines population, no colour struction of effective population size dynamics. variation was reported, neither at the interspecific level (Cadle-Davidson and Owens, 2008) nor at the intraspe- Materials and methods cific level (Olmo, 1976). The diversification of the grape colour locus led to the emergence of white genotypes, a Plant material derived state (Zohary and Hopf, 2000) that arose from ancestral coloured vine populations. Between these two The reference plant material consisted of 137 individuals from a core collection of Vitis vinifera L. subsp. sativa, pools, we observe a continuous range of pale-coloured genotypes, from pink to red. Thus, in contrast to the maximizing agro-morphological diversity for 50 qualita- tive and quantitative traits (Barnaud et al., 2006). Names, primary domestication traits, growers within each grape- vine growing region have selected distinct colour origins and colour profile of the cultivars are shown in Supplementary Table 1. The outgroup consisted of four variants, leading to a diversification process. Vitis sp Vitis balanseana Vitis Recent investigations on the genetic bases of the trait Asian . genotypes: (Vba), amurensis (Vam), Vitis pentagona (Vpe) and Vitis coignetiae provided evidence that a single gene cluster, located on chromosome 2, is responsible for most of this variation in (Vco). The material is conserved at INRA Domaine de Vassal (http://bioweb.ensam.inra.fr/collections_vigne/). colour, and that colour phenotype is due to the combined additive effect of the VvMybA gene alleles (Fournier- Level et al., 2009). This locus is constituted of a cluster of Molecular analysis three MYB-type transcription factor genes, among which A square inch (80–100 mg) of fresh young leaf was VvMybA1 and VvMybA2 were shown to be functionally harvested from each genotype. DNA was extracted using involved in berry pigmentation (Kobayashi et al., 2002; Qiagen DNA Plant Mini Kit (Qiagen SA, Courtaboeuf, Walker et al., 2007). A third expressed gene, VvMybA3, is France) with minor modifications, as described by statistically associated with berry colour determinism Adam-Blondon et al. (2004). but not functionally validated (Fournier-Level et al., We aimed to sequence all three VvMybA genes that have 2009). The rise of the white berry genotype was recently been shown to have a quantitative effect on colour demonstrated to be the result of the silencing of the first phenotype. Amplification primers were designed using two functional genes (Kobayashi et al., 2005; Walker et al., Primer 3 software (Lincoln S., MIT, Boston, MA, USA). Half 2007). VvMybA1 gene silencing is due to the insertion of these were anchored in the coding sequence of the genes of Gret1, a gipsy-type retrotransposon in its promoter and the other half in the flanking non-coding and non- (Lijavetzky et al., 2006; This et al., 2007). For VvMybA2, conserved regions; the primers are listed in Fournier-Level the single-nucleotide polymorphism (SNP) K980 in the et al. (2009). PCR fragments were amplified, sequenced and coding sequence, which modifies a putative a-helix of analysed as described by Le Cunff et al. (2008). the R2R3 recognition domain, also led to a non- Presence of the Gret1 retroelement in the MybA1 locus functional gene (Walker et al., 2007). promoter was genotyped using two forward primers, Since the publication of the genome sequence of Vitis one specific for the wild allele placed before the insert vinifera (Jaillon et al., 2007), the resequencing of genes of and the other specific for the white allele inside the interest in large genetic resource collections has become insert, and a common reverse primer placed in the first feasible, helping to answer hypotheses about the evolu- gene exon (Kobayashi et al., 2005; This et al., 2007). PCR tion of grape traits. We analysed the sequence poly- conditions were identical to those described by Kobaya- morphism of these three genes in a core collection of shi et al. (2005). Amplified fragments described