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.

PCR assays that identify the grapevine dieback fungus Eutypa lata.

Eutypa lata is the causal fungal agent of Eutypa dieback, a serious grapevine necrotic disease. The erratic and delayed (1 to 2 months) appearance of characteristic conidia on culture media and the presence of numerous microorganisms in decaying wood make it difficult either to identify or to detect E. lata in grapevine wood samples. We designed six pairs of PCR primers for diagnosis of E. lata. Three primer pairs were derived from ribosomal DNA internal transcribed spacer sequences, and three pairs were derived from randomly amplified polymorphic DNA fragments. The six primer pairs could be used to amplify DNAs extracted from all of the E. lata isolates tested. They did not amplify DNAs from fungi and bacteria representing more than 50 different species of microorganisms associated with grapevine. We developed a simple protocol, leading to a rapid release of DNA, that enabled us to identify E. lata from pure or mixed cultures as well as from grapevine wood samples. Identification of E. lata in wood was achieved within a few hours, instead of the several weeks required for classical cultures on agar medium. We believe that the procedure described here can be adapted to detect other microorganisms involved in woody plant diseases.

Variation in Pathogenicity and Genetic Structure in the Eutypa lata Population of a Single Vineyard.

ABSTRACT Fifty-five isolates of Eutypa lata were collected in 1994 from a single vineyard, each from a different vine that showed either shoot and foliar symptoms of Eutypa dieback or only abnormalities during the period of 1990 to 1994. These isolates showed a large variation in pathogenicity after inoculation on cuttings in the greenhouse. Variability also was observed for cultural traits and radial growth rate on potato dextrose agar (PDA) medium, but no relation was found between these characteristics and pathogenicity. The isolates were paired on PDA medium, and a barrage reaction was observed for all pairings, indicating the isolates were vegetatively incompatible. Thirty-two random amplified polymorphic DNA (RAPD) markers were used to study the genetic relatedness of the isolates and the genetic structure of the population. Fifty-five different RAPD patterns were observed, confirming the genetic uniqueness of each isolate. Gametic disequilibrium values were calculated among all pairs of the 32 putative RAPD loci, and only one value was significant. Unweighted pair-group method with arithmetic averages analysis of distance data, as well as a heterogeneity statistic (Fst), did not indicate any population substructure. The results strongly suggest that isolates originated from a random-mating population and that the disease was propagated in this vineyard by ascospores produced from diverse outside sources. Southern hybridization performed for five markers indicated that the two-allele assumption made to interpret RAPD data may be violated for markers that are similar in size. However, the exclusion of such markers did not change the conclusions of the study.