Vitis californica and Vitis californica × Vitis vinifera Hybrids are Hosts for Grapevine leafroll-associated virus-2 and -3 and Grapevine virus A and B.

Vitis and non-Vitis spp. surrounding nine Napa Valley vineyards were surveyed for Grapevine leafroll-associated virus (GLRaV)-1 to -5 and -9, Grapevine virus A (GVA), Grapevine virus B (GVB), and Grapevine virus D (GVD). Vitis spp. from three riparian areas not adjacent to vineyards were also included. DNA fingerprinting and probability analyses indicated that the Vitis samples consisted primarily of Vitis californica followed by V. californica × V. vinifera hybrids. Single and mixed infections of GLRaV-2, -3, GVA, or GVB were detected by conventional or quantitative reverse-transcription polymerase chain reaction in 6 of the 66 V. californica and 11 of the 19 V. californica × V. vinifera hybrids. GLRaV-1, -4, -5, -9, and GVD were not detected. Phylogenetic analysis of GLRaV-2 and -3 partial coat protein gene nucleotide sequences indicated that the isolates from V. californica and V. californica × V. vinifera hybrids were closely related to isolates from V. vinifera.

Development of a standard set of microsatellite reference alleles for identification of grape cultivars.

In order to investigate the comparability of microsatellite profiles obtained in different laboratories, ten partners in seven countries analyzed 46 grape cultivars at six loci (VVMD5, VVMD7, VVMD27, VVS2, VrZAG62, and VrZAG79). No effort was made to standardize equipment or protocols. Although some partners obtained very similar results, in other cases different absolute allele sizes and, sometimes, different relative allele sizes were obtained. A strategy for data comparison by means of reference to the alleles detected in well-known cultivars was proposed. For each marker, each allele was designated by a code based on the name of the reference cultivar carrying that allele. Thirty-three cultivars, representing from 13 to 23 alleles per marker, were chosen as references. After the raw data obtained by the different partners were coded, more than 97% of the data were in agreement. Minor discrepancies were attributed to errors, suboptimal amplification and visualization, and misscoring of heterozygous versus homozygous allele pairs. We have shown that coded microsatellite data produced in different laboratories with different protocols and conditions can be compared, and that it is suitable for the identification and SSR allele characterization of cultivars. It is proposed that the six markers employed here, already widely used, be adopted as a minimal standard marker set for future grapevine cultivar analyses, and that additional cultivars be characterized by means of the coded reference alleles presented here. The complete database is available at http://www.genres.de/eccdb/vitis/ Cuttings of the 33 reference cultivars are available on request from the Institut National de la Recherche Agronomique Vassal collection (didier.vares@ensam.inra.fr).

A microsatellite marker based framework linkage map of Vitis vinifera L.

We have constructed a framework linkage map based on microsatellite markers for Vitis vinifera L., the European wine grape. The mapping population consisted of 153 progeny plants from a cross of Vitis vinifera cvs. Riesling x Cabernet Sauvignon. One hundred fifty-two microsatellite markers and one polymorphic EST marker have been mapped to 20 linkage groups (2 n=38). The map covers 1,728 cM with an average distance between markers of 11.0 cM. Estimates of genome size, expected genome coverage, and observed genome coverage were determined with 135-140 markers. Genome length estimates differed between paternal and maternal data sets. Observed approximate genome coverage was 65% versus an expected coverage of 90%. Meiotic recombination rates were not significantly different between maternal and paternal parents. This map has been adopted as a reference map for the International Grape Genome Program.

Isolation and characterization of new polymorphic simple sequence repeat loci in grape (Vitis vinifera L.).

Four new simple sequence repeat (SSR) loci (designated VVMD5, VVMD6, VVMD7, and VVMD8) were characterized in grape and analyzed by silver staining in 77 cultivars of Vitis vinifera. Amplification products ranged in size from 141 to 263 base pairs (bp). The number of alleles observed per locus ranged from 5 to 11 and the number of diploid genotypes per locus ranged from 13 to 27. At each locus at least 75% of the cultivars were heterozygous. Alleles differing in length by only 1 bp could be distinguished by silver staining, and size estimates were within 1 or 2 bp, depending on the locus, of those obtained by fluorescence detection at previously reported loci. Allele frequencies were generally similar in wine grapes and table grapes, with some exceptions. Some alleles were found only in one of the two groups of cultivars. All 77 cultivars were distinguished by the four loci with the exception of four wine grapes considered to be somatic variants of the same cultivar, 'Pinot noir', 'Pinot gris', 'Pinot blanc', and 'Meunier'; two table grapes that are known to be synonymous, 'Keshmesh' and 'Thompson Seedless'; and three table grapes, 'Dattier', 'Rhazaki Arhanon', and 'Markandi', the first two of which have been suggested to be synonymous. Although the high polymorphism at grape SSR loci suggests that very few loci would theoretically be needed to separate all cultivars, the economic and legal significance of grape variety identification requires the increased resolution that can be provided by a larger number of loci. The ease with which SSR markers and data can be shared internationally should encourage their broad use, which will in turn increase the power of these markers for both identification and genetic analysis of grape. Key words : grape, Vitis, microsatellite, simple sequence repeat, DNA typing, identification.