Quantitative genetic bases of anthocyanin variation in grape (Vitis vinifera L. 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 x 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.

Figure 1.—

Distribution of the anthocyanin content in berry skin for the year 2005, expressed in logarithm of milligrams of anthocyanin/gram of fresh berry skin. The quantity of anthocyanin displays a continuous variation from 0 to 33.2 mg of anthocyanin per gram of fresh berry skin, with an overrepresented sample of white-berried cultivars displaying no anthocyanin.

Figure 2.—

(a) Presentation of VvMybA gene cluster and SSR markers, (b) position of the QTNs, and (c) level of association between markers and total anthocyanin content of berry skin along scaffold 97 of the grape genome browser. Along the x-axes, the dashed lines correspond to nonlinear scales. In the association tests, the microsatellite markers are presented in red, and the genic polymorphisms in blue; for the genic polymorphisms, dots correspond to QTNs and diamonds to the other polymorphisms. The Bonferonni threshold is equal to 6.25E-4.

Figure 3.—

LD plot based on R² values for the SNPs and indels associated with the total anthocyanin content of berry skin on VvMybA1, -A2, and -A3 genes in the lower diagonal and overall level of LD for the full genes in the upper diagonal. QTNs selected through stepwise cofactor selection (far left column) are framed in black. Presented R² values are estimated according to Remington et al. (2001).