Optimization of PCR Conditions to Amplify Microsatellite Loci in Grapevine (Vitis vinifera L.) Genomic DNA

Abstract

A total of three different primer pairs were optimized for polymerase chain reaction (PCR) to amplify microsatellite loci in total genomic DNA of grapevine (Vitis vinifera L.). Different concentrations of MgCl2, DNA and different regimes of annealing temperature were optimized. For all the primer pairs, 2.5 mM MgCl2 concentration was found optimum. For DNA concentration, 100 ng in the final reaction volume was suitable for good amplification. Annealing temperatures 56°C, 61°C and 58°C were found optimum to amplify with primer pairs VVMD5, scu04vv and VMC8E6, respectively. The other reagents used in PCR and temperature regimes (denaturation and extension temperature) were kept constant. The protocol has been successfully applied producing scorable and clear amplicons in all cultivars studied. These loci can be used to evaluate the genetic variability and cultivar relatedness in autochthonous Vitis vinifera cultivars from Tunisia