CHARACTERIZATION OF MICROSATELLITE FLANKING SEQUENCES IN VITIS SPECIES

Abstract

Simple sequence repeats (SSRs) or microsatellites have become an important source of highly polymorphic genetic markers. They are widely used in the genetic analysis of populations, the construction of linkage maps, in cultivar fingerprinting and pedigree construction. The SSR unique flanking sequences were used in several eukaryotic taxa for transferring microsatellites isolated from a source species to a related species. Cross-species transportability of microsatellite markers is very attractive in Vitis, as non-V. vinifera species and inter-specific hybrids are commonly used as rootstocks and raw material in breeding programs. This work provides new insights into (1) the transportability of microsatellites isolated from Vitis vinifera to different Vitis species and (2) the possibility of using microsatellite flanking sequences for taxonomic and phylogenetic inference. Eleven microsatellites isolated from grapevine (Vitis vinifera) were used to assay the conservation of priming sites in 14 non-V. vinifera species, including relevant taxa for breeding and Parthenocissus quinquefolia as a representative of a related genus. Cross-species amplification was obtained in 94% of the 176 genotype x locus tested combinations. Sequencing of PCR products was performed for three microsatellite loci amplified in 10 different species. A microsatellite repeat was found to be present in all the cases. The repeat region was often longer in V. vinifera than in non-source species whose repetitive sequences showed frequent interruptions of the core repeat. In spite of these constraints, which could reduce the polymorphism of microsatellites in non-source species, the results demonstrate the possibility of extending the use of microsatellite markers to wild germplasm and inter-specific hybrids. Point mutations have been found in microsatellite flanking regions and these variations have been used to investigate the genetic relationship among taxa. The Neighbour-joining tree obtained on the base of 10 nucleotide variations, showed that there is no clear dividing line between American, Asian and European species and that the actual taxonomy which reflects the geographical distribution of species should be revised.