Pursuit for transcriptome microsatellites towards estimation of genetic variation, species discrimination and detection of interspecific hybrids within the genus Centaurium

Abstract

Over the last few decades microsatellites have been the markers of choice for applications in both plant population and conservation genetics. Unassembled 114 bp long transcriptome sequences of Centaurium erythraea Rafn, obtained by Illumina GAIIx System, were complementary paired to make short contigs of 170 -200 bp which were used as a starting material for microsatellite mining. Identification and bioinformatics filtering of microsatellite-containing sequences were carried out employing open source and free software components operational on an average performance computer. The designing of primers was performed for 16 selected sequences. Transferability of microsatellite loci was assayed on 22 individual plants originating in the Balkan Peninsula which belong to 7 different taxa of the genus Centaurium including three species, two subspecies and five specimens with assumed hybrid constitution. Out of 16 primer pairs tested, 9 were successfully amplified showing high-quality and reproducible bands on 2.5% agarose gels. Seven out of nine functional loci amplified in all taxa accessions, while two loci had the ability to clearly discriminate between the taxa. Further validation of developed microsatellite markers was performed by Lab-on-a-Chip technology using Agilent 2100 Bioanalyzer in order to detect polymorphism of microsatellite loci and to assess their range. Eight loci displayed clearly polymorphic patterns. The number of observed alleles across loci was 3 to 11. Owing to considerable transferability and variability, obtained microsatellite loci are standing for powerful tool for future studies on discrimination of species within the genus Centaurium, estimation of their genetic variation, and for tracking interspecific hybridization among them.