Identification of Microsatellite Markers for Plasmopara viticola and Establishment of High throughput Method for SSR Analysis

The Oomycete Plasmopara viticola is the causal organism of downy mildew on grapevine (Vitis spp.). In order to set up the techniques for investigating downy mildew disease dynamics and genetic structure, co-dominant, neutral, highly reproducible and polymorphic microsatellite markers for P. viticola were developed. Five markers, two with a (TC)_n repeat (loci BER and ISA), two with a (TC)_n(AC)_n repeat (loci CES and REX) and one with a (CT)_n(CTAT)_n repeat (locus GOB), were selected. Simple sequence repeat (SSR) markers revealed different degrees of polymorphism within 190 oil spots (disease symptoms) collected from an infected Italian vineyard. The most polymorphic SSR marker GOB showed 43 alleles (Nei's expected gene diversity H_e = 0.89) while CES, ISA, BER and REX showed 14 (H_e = 0.71), 4 (H_e = 0.57), 3 (H_e = 0.24) and 1 allele (H_e = 0), respectively. A high throughput DNA extraction method, that allowed molecular analysis of this obligate pathogen directly in the host without any isolation procedure, was developed. The quality and quantity of oil spots did not influence the SSR analysis. Amplified SSR loci were separated by electrophoresis on a Beckman–Coulter 2000XL sequencer and automatically analysed. The objective of this study was to develop molecular biological tools and methods that allow high throughput analysis of the downy mildew populations.