A microsatellite-based system for the identification and legal protection of grapevine varieties

Abstract

More than 6,000 plants have been analyzed to evaluate a system based on 9 microsatellites that could be useful for most of the issues related to the genotyping of grapevine varieties: variety identification, DUS testing, and identification of Essentially Derived Varieties (EDVs). The 9 microsatellite markers selected are: VVS2, VVMD5, VVMD27, VVMD28, ssrVrZAG29, ssrVrZAG62, ssrVrZAG67, ssrVrZAG83 and ssrVrZAG112. The selection of these markers was based on different criteria: availability (public), map position (genetically independent), polymorphism (high), allele size range (that allows multiplexing), and 'quality' (ease of amplification, absence of known null alleles, absence of alleles differing only in 1 bp). A genotyping system was optimized, including the design of a multiplex PCR with the 9 markers, and capillary electrophoresis and fluorescence analysis in an automatic sequencer. More than 1,300 accessions (2,600 plants) of Vitis vinifera L. from the collection of grapevine varieties at the "El Encín" state have been analyzed with this system. Until now, the conclusion reached for all those accessions studied that presented the same genotype at the 9 microsatellite loci is that they arise from the same embryo. Once the genotype table has been built up, any plant belonging to any of these varieties can be easily and unequivocally identified, with the exception of the sports and EDVs, which require a morphological description. Regarding the technical exams for legal protection of new varieties (DUS test), the lower number of different alleles between different varieties, and the higher number of different alleles within a variety (mutations) have been used to determine a minimum distance that allows the establishing of Distinction. Uniformity and Stability have been evaluated by studying about 4,000 plants of 19 different varieties.