Real-Time PCR and analysis of amplicon melting curves to assess the suitability of SSR loci of Scots pine for multiplexing

Abstract

Nuclear microsatellite markers are considered to be among the most powerful tools for assessing genetic resources. To date, a great variety of primers for SSR loci of pines has been developed. As regards Scots pine, selection of neutral steadily amplifiable loci not containing null alleles and their multiplexing are high-priority tasks, as there are no generally accepted multiplex panels of loci for this species. Authors of published multiplexes tend not to use loci due to their unstable amplification or the presence of single nucleotide repeats. The aim of the paper was to create the new multiplexes of reliable previously published pine nSSR loci in view of preliminary data based on the analysis of melting curves of products after Real-Time polymerase chain reaction (PCR) for each locus separately. Initially, microsatellite multiplexes were created using Multiplex Manager 1.0. DNA was extracted by the CTAB method from samples of pine needles. Real-Time PCR was performed in CFX96 thermal cyclers (BIO-RAD, USA). Fragment analysis of PCR products labeled with FAM, HEX and ROX dyes was performed on a 3500 genetic analyzer (Applied Biosystems). GeneScanTM 500 LIZ size standard was used. As a result of the study, 3 multiplexes have been proposed.