Combining microsatellite markers and capillary gel electrophoresis with laser-induced fluorescence to identify the grape (Vitis vinifera) variety of musts

摘要

In this work, a new method that combines the use of microsatellite markers (VVMD5 and ZAG79) together with capillary gel electrophoresis with laser-induced fluorescence (CGE–LIF) is developed and applied to the identification of Albariño and Moscatel Grano Menudo musts. The GCE-LIF method uses commercially available products including polymers, DNA-intercalating dyes and bare fused silica capillaries to provide reproducible and sensitive separations of DNA fragments for grapevine characterization. The CGE–LIF procedure offers highly resolved separations of DNA fragments from 48 to 1031 bp in ca. 30 min with efficiencies up to 1.8×106 plates/m allowing the separation of fragments that differ in 4 bp. The use of different DNA standards (i.e., 100 bp ladder, Ф×174 and pBR322) and their effect on size assignment of the amplified DNA is also investigated. It is demonstrated that the microsatellite markers (VVMD5 and ZAG79) provide DNA amplification patterns specific for Albariño and Moscatel Grano Menudo grapes that can be adequately differentiated by using CGE–LIF. Moreover, the DNA sizes determined by this CGE–LIF method are corroborated using a more standard procedure (i.e., an automatic genetic analyzer with a commercial kit) demonstrating the usefulness of this new methodology.