Using of amplicon melting curves to assess the suitability of Norway spruce nSSR loci for multiplexing

Microsatellite, or SSR markers (Simple Sequence Repeats) are one of the most powerful tools for assessing genetic variation. They are widely used to study the genetic diversity and population genetic structure of coniferous species. The most promising is the multiplexing of SSR loci. It allows to automate microsatellite analysis, significantly increase the accuracy of studies and reduce their cost. At the moment, there are no universally accepted multiplexes of Norway spruce nuclear microsatellites and their creation is very important. The aim of the study is the selection from number of published nSSR the loci suitable for multiplexing and compilation from them the original multiplexes convenient for analyzing the genetic diversity and identification of valuable genotypes of Picea abies (L.) Karst. For the screening the loci with specific amplification in a wide range of annealing temperature, Real-Time PCR in a temperature gradient with the analysis of melting curves of amplicons for each locus separately was used. Initially, multiplexes were created using the Multiplex Manager 1.0 program. After their modification, 7 multiplex panels were proposed (3 of two, and 1 of three, and 3 of four loci). Multiplexes were tested on 30 trees, originating from four geographically different populations of the southern taiga region of the European part of the Russian Federation. Multiplex PCR was performed with direct primers labeled FAM, HEX, ROX fluorescent dyes and reverse unlabeled primers. PCR results were detected using the Applied Biosystems 3500 genetic analyzer. To determine the length of DNA fragments, the GeneScan 600 LIZ v2.0 size standard was used.