Development and Transferability of EST-SSR Markers for Pinus koraiensis from Cold-Stressed Transcriptome through...

Pinus koraiensis has significant economic and ecological value in Northeast China. However, due to the lack of suitable molecular markers, only a few available microsatellite markers were developed for further population genetics studies. In this study, for the first time we developed expressed sequence tag-simple sequence repeat (EST-SSR) markers from the cold-stressed transcriptome of P. koraiensis using Illumina Sequencing. We identified a total of 7,235 EST-SSRs from 97,376 sequences, and we tested their transferability among seven related Pinus species. The results showed that trinucleotides were the most abundant type of repeat (1287, 18.74%) excluding mononucleotides, followed by dinucleotides (1284, 18.7%) and tetranucleotides (72, 1.05%). The most dominant dinucleotides and trinucleotide repeat motifs were AT/AT (535, 7.79%) and AAT/ATT (103, 1.5%). The observed heterozygosity (Ho) and expected heterozygosity (He) ranged from 0.002 to 0.986 and 0.017 to 0.743, respectively, and the polymorphism information content (PIC) values and number of alleles (Na) varied from 0.029 to 0.794 and 2 to 23, respectively. A total of 8 natural P. koraiensis populations were divided into two main genetic clusters. Furthermore, nine of twenty polymorphic primer pairs were successfully amplified in seven Pinus species, and at least 80% of the successful P. koraiensis EST-SSR primers could be amplified in more than four species (16, 80%). Combined results for the development of EST-SSR markers in P. koraiensis and transferability among related species would contribute to improved studies on the genetic diversity and population structure in P. koraiensis and phylogenetic relationships among Pinus species. They would also provide a significant source for quantitative trait locus analysis.