Complete genome sequence of a novel grapevine-infecting member of the genus Polerovirus, grapevine polerovirus 1.

Double-stranded RNAs and total RNAs purified from grapevine (Vitis vinifera) phloem scrapings of two varieties held in the INRAE (France) grapevine germplasm collection were analyzed by high-throughput sequencing. BLAST annotation revealed contigs with homology to Polerovirus genus members. The full genome sequence of one isolate (KT) was determined (5651 nucleotides [nt]), and a partial sequence representing about half of the genome was assembled for a second isolate (KS) that was found to share 95% nt sequence identity with the KT isolate. The genome has a typical polerovirus organization, containing six open reading frames (ORFs) as well as a putative additional ORF3a. Based on genome organization and phylogenetic relationships, the new virus belongs to the genus Polerovirus but, similar to the recently described persimmon polerovirus 1, is characterized by a highly divergent coat-protein/readthrough domain. Considering the species demarcation criteria for the family Luteoviridae, these two isolates, together with a closely related sequence recently deposited in the GenBank database (LC507098), represent a new Polerovirus species for which the name "Grapevine polerovirus 1" is proposed.

Complete Nucleotide Sequence of an Isolate of Grapevine Satellite Virus and Evidence for the Presence of Multimeric Forms in an Infected Grapevine.

The complete nucleotide sequence of an isolate of grapevine satellite virus (GV-Sat) was determined by next-generation sequencing (NGS) and compared with the single available complete sequence. The NGS data unexpectedly provided evidence for the existence of multimeric forms of GV-Sat, which were experimentally confirmed, allowing the redefinition of GV-Sat genomic ends.

High throughput analysis of grape genetic diversity as a tool for germplasm collection management.

Using 20 SSR markers well scattered across the 19 grape chromosomes, we analyzed 4,370 accessions of the INRA grape repository at Vassal, mostly cultivars of Vitis vinifera subsp. sativa (3,727), but also accessions of V. vinifera subsp. sylvestris (80), interspecific hybrids (364), and rootstocks (199). The analysis revealed 2,836 SSR single profiles: 2,323 sativa cultivars, 72 wild individuals (sylvestris), 306 interspecific hybrids, and 135 rootstocks, corresponding to 2,739 different cultivars in all. A total of 524 alleles were detected, with a mean of 26.20 alleles per locus. For the 2,323 cultivars of V. vinifera, 338 alleles were detected with a mean of 16.9 alleles per locus. The mean genetic diversity (GDI) was 0.797 and the level of heterozygosity was 0.76, with broad variation from 0.20 to 1. Interspecific hybrids and rootstocks were more heterozygous and more diverse (GDI = 0.839 and 0.865, respectively) than V. vinifera cultivars (GDI = 0.769), Vitis vinifera subsp. sylvestris being the least divergent with GDI = 0.708. Principal coordinates analysis distinguished the four groups. Slight clonal polymorphism was detected. The limit between clonal variation and cultivar polymorphism was set at four allelic differences out of 40. SSR markers were useful as a complementary tool to traditional ampelography for cultivar identification. Finally, a set of nine SSR markers was defined that was sufficient to distinguish 99.8% of the analyzed accessions. This set is suitable for routine characterization and will be valuable for germplasm management.

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

Polymorphisms in the grape transcription factor family VvMybA are responsible for variation in anthocyanin content in the berries of cultivated grapevine (Vitis vinifera L. subsp. sativa). Previous study has shown that white grapes arose through the mutation of two adjacent genes: a retroelement insertion in VvMybA1 and a single-nucleotide polymorphism mutation in VvMybA2. The purpose of this study was to understand how these mutations emerged and affected genetic diversity at neighbouring sites and how they structured the genetic diversity of cultivated grapevines. We sequenced a total of 3225 bp of these genes in a core collection of genetic resources, and carried out empirical selection tests, phylogenetic- and coalescence-based demographic analyses. The insertion in the VvMybA1 promoter was shown to have occurred recently, after the mutation of VvMybA2, both mutations followed by a selective sweep. The mutational pattern for these colour genes is consistent with progressively relaxed selection from constrained ancestral coloured haplotypes to light coloured and finally white haplotypes. Dynamics of population size in the VvMybA genes showed an initial exponential growth, followed by population size stabilization. Most ancestral haplotypes are found in cultivars from western region, whereas recent haplotypes are essentially present in table cultivars from eastern regions where intense breeding practices may have replaced the original diversity. Finally, the emergence of the white allele was followed by a recent strong exponential growth, showing a very fast diffusion of the initial white allele.

Toward the authentication of varietal wines by the analysis of grape (Vitis vinifera L.) residual DNA in must and wine using microsatellite markers.

In an attempt to develop a technique for the identification of grape cultivars in commercial wines, a method for the extraction of DNA from must and experimental wines was adapted and optimal PCR conditions for the amplification of this DNA were established. DNA was analyzed during the fermentation process for six cultivars (Chardonnay, Clairette blanche, Grenache noir, Merlot, Muscat blanc à petits grains, and Syrah). The extractions were performed on solid parts in suspension as well as on the aqueous fraction. Expected profiles for these cultivars were obtained with DNA extracted from the solid parts during all of the fermentation process and for the wine. The analysis of DNA extracted from aqueous fractions was less reproducible, and microsatellite amplifications were obtained only in the case of Clairette blanche, Merlot, and Syrah wines. Results demonstrate that the purification process is adequate for the analysis but that the DNA concentration represents the main limiting factor. Technical improvements of the method are discussed.