Certification of Russian red clover (Тrifolium pratense L.) varieties based on SSR and SRAP markers

Molecular-genetic certification is a powerful strategies and efficient addition to the traditional methods of variety testing and agricultural crops identification. Russia, as well as a world in a whole, introduces the current DNA technologies in the breeding programs, in a variety registration process and in a system of seed production. However, the traditional approaches, based on observation and recording the morphological characters, are the prevalent now for the forage crops. It influences negatively on efficiency of selection, increases the terms and coasts of the new varieties development, registration and breeders rights protection. In this paper, the results of creation a system for identification and genetic certification of Russian red clover cultivars on the base of SSR and SRAP-markers are submitted for the first time. The seeds of 15 domestic varieties from gene pool collection of Federal Williams Research Center of Forage Production and Agroecology and 6 accessions of foreign breeding from Vavilov All-Russian Institute of Plant Genetic Resources were used for investigations. The genome DNA was extracted from 7-day seedlings’ tissue. Bulk DNA samples were formed from 30 individual genotypes per each variety. We used basic SDS-method in own modifications. Quantity and quality of extracted DNA was analyzed by agarose gel electrophoresis and measurement of concentration and purity. The final concentration of DNA samples was 30 ng/ml. PCR amplification was performed using 35 SSR from the Red Clover Marker Database (http://marker.kazusa.or.jp/Red_clover), and 40 SRAP markers. A total of 476 PCR products were generated with SSR markers for 12 red clover varieties. A set of eight microsatellite loci was selected for identification the tested samples. With application of 40 SRAP markers, we selected 18 informative combinations for analysis of the red clover collection of 16 varieties. Total 812 PCR products were revealed and 85 (10.5 %) among them were determined as polymorphic. The set of 7 informative markers were identified for samples differentiation on the base of SRAP analysis. Unique varieties-specific DNA fragments were sequenced (Evrogen Lab company, Russia) for validation the results of analysis. Nucleotide sequences, identifying Russian red clover varieties Trifon, Mars, Topas, Atlant, Tetraploidniy VIK, Meteor, VIK 77, were included in the GenBank NCBI (https://www.ncbi.nlm.nih.gov/). The data of DNA fingerprinting we used for development the molecular-genetic formulas representing microsatellite loci allele composition and polymorphism in exon and intron regions of genome. As a result of this study, 10 etalon genetic certificates were designed for Russian red clover varieties.