Quantitative Genetic Bases of Anthocyanin Variation in Grape (Vitis viniferaL. ssp.sativa) Berry: A Quantitative Trait Locus to Quantitative Trait Nucleotide Integrated Study

Abstract

The combination of QTL mapping studies of synthetic lines and association mapping studies of natural diversity represents an opportunity to throw light on the genetically based variation of quantitative traits. With the positional information provided through quantitative trait locus (QTL) mapping, which often leads to wide intervals encompassing numerous genes, it is now feasible to directly target candidate genes that are likely to be responsible for the observed variation in completely sequenced genomes and to test their effects through association genetics. This approach was performed in grape, a newly sequenced genome, to decipher the genetic architecture of anthocyanin content. Grapes may be either white or colored, ranging from the lightest pink to the darkest purple tones according to the amount of anthocyanin accumulated in the berry skin, which is a crucial trait for both wine quality and human nutrition. Although the determinism of the white phenotype has been fully identified, the genetic bases of the quantitative variation of anthocyanin content in berry skin remain unclear. A single QTL responsible for up to 62% of the variation in the anthocyanin content was mapped on a Syrah 3 Grenache F 1 pseudo-testcross. Among the 68 unigenes identified in the grape genome within the QTL interval, a cluster of four Myb-type genes was selected on the basis of physiological evidence (VvMybA1, VvMybA2, VvMybA3, and VvMybA4). From a core collection of natural resources (141 individuals), 32 polymorphisms revealed significant association, and extended linkage disequilibrium was observed. Using a multivariate regression method, we demonstrated that five polymorphisms in VvMybA genes except VvMybA4 (one retrotransposon, three single nucleotide polymorphisms and one 2-bp insertion/deletion) accounted for 84% of the observed variation. All these polymorphisms led to either structural changes in the MYB proteins or differences in the VvMybAs promoters. We concluded that the continuous variation in anthocyanin content in grape was explained mainly by a single gene cluster of three VvMybA genes. The use of natural diversity helped to reduce one QTL to a set of five quantitative trait nucleotides and gave a clear picture of how isogenes combined their effects to shape grape color. Such analysis also illustrates how isogenes combine their effect to shape a complex quantitative trait and enables the definition of markers directly targeted for upcoming breeding programs.

Evolution of the VvMybA gene family, the major determinant of berry colour in cultivated grapevine (Vitis vinifera L.)

“…Amplification primers were designed using Primer 3 software (Lincoln S., MIT, Boston, MA, USA). Half of these were anchored in the coding sequence of the genes and the other half in the flanking non-coding and nonconserved regions; the primers are listed in Fournier-Level et al (2009). PCR fragments were amplified, sequenced and analysed as described by Le Cunff et al (2008).…”

“…Network analysis was carried out using the median-joining method as described in Bandelt et al (1999) and implemented in Network v4.5.02 (Fluxus Technology, Sudbury, UK). Supplementary analysis was carried out using only the polymorphisms shown to be associated with berry colour and with frequency superior to 0.1 in Fournier-Level et al (2009). We reconstructed another haplotype set with 18 SNPs.…”

“…The presence of two polymorphisms with a major influence on grape colour (Fournier-Level et al, 2009) helped us to define four haplogroups, among which one was discarded from the analyses because of its recombined origin. These haplogroups were treated separately in the analyses.…”

“…The small number of generations and vegetative propagation imply limited recombination and have helped to maintain extensive linkage disequilibrium (LD; Barnaud et al, 2006), especially in the context of loci linked within a 43-kb region (Fournier-Level et al, 2009). Nonetheless, Schierup and Hein (2000) showed that recombination led to overestimation of the length of internal branches, regardless of its intensity, conflicting with the hypothesis of constant evolutionary rate along the coalescent phylogeny (Hudson, 1983).…”

“…This locus is constituted of a cluster of three MYB-type transcription factor genes, among which VvMybA1 and VvMybA2 were shown to be functionally involved in berry pigmentation (Kobayashi et al, 2002;Walker et al, 2007). A third expressed gene, VvMybA3, is statistically associated with berry colour determinism but not functionally validated (Fournier-Level et al, 2009). The rise of the white berry genotype was recently demonstrated to be the result of the silencing of the first two functional genes (Kobayashi et al, 2005;Walker et al, 2007).…”