DNA Extraction and Application of SSR Markers in Genetic Identification of Grapevine Cultivars
Chapter from the book: Vucelić-Radović, B et al. 2019. Application of Molecular Methods and Raman Microscopy/Spectroscopy in Agricultural Sciences and Food Technology.
Microsatellite markers (SSR markers) are widely used in grapevine genetic research for identification of cultivars, parentage analysis, and genetic characterization of germplasm. Aim of this work was extraction of total DNA, primer selection and design, PCR protocols and analysis of DNA sequences with special emphasize on variability between collected samples of different grapevine cultivars. The material used in this study were samples of grapevine leaves of different autochthonous and introduced cultivars from grapevine collection on Experimental field “Radmilovac” at the Faculty of Agriculture, University in Belgrade and from the National fruit collection “Brogdale” from UK. Standard set of nine primers for grapevine was used. Analyses were performed in Molecular Genetics Laboratory, School of Agriculture, Policy and Development, University of Reading, Reading, UK. Extraction and purification of total DNA from fresh and frozen plant material (grapevine leaves) was performed using a DNeasy ® Plant Mini (Qiagen Inc.) kit. The concentration of extracted DNA was measured by NanoDrop spectrophotometer and stored on -20°C until use. In the study, we utilized the protocol for Type-it Microsatellite.