Multiplex analysis of microsatellite markers in DNA certification of sweet cherry varieties

Abstract. Microsatellite DNA markers are widely used in genetic studies of many fruit crops, including sweet cherry. They provide data that improve the efficiency of breeding and genetic research. Improving the methodological aspects of SSR genotyping is an important issue in order to reduce the costs of time, reagents and consumables while maintaining the accuracy of the analysis. One of these methods is the use of multiplex PCR, when several SSR loci are analyzed in one reaction. The aim of these studies was to improve the elements of the SSR fingerprinting methodology for sweet cherry varieties by developing multiplex sets of SSR markers. As a result of testing microsatellite DNA markers in the formation of multiplex sets, optimization of such parameters as the ratio of primer concentrations of DNA markers included in one multiplex set; the number of reaction cycles, the annealing temperature of primers during PCR; the degree of dilution of PCR products during their denaturation during fragment analysis on the ABIprism automatic genetic analyzer and Nanofor 05. Eight multiplex kits were developed, including 21 microsatellite DNA markers. Data on the polymorphism of DNA markers and on the length ranges of amplified fragments are presented. The obtained multiplex kits were successfully used for DNA certification of sweet cherry varieties representing the gene pool of the South of Russia. Part of the data on the DNA profiles of sweet cherry varieties was registered as an intellectual property object in the form of a database of SSR fingerprints of sweet cherry varieties. The obtained data can also be used to assess the genetic structure and diversity of collections of genetic resources of this crop. Keywords: sweet cherry, genotyping, SSR-markers, SSR-fingerprinting, multiplex analysis