Development of Polymorphic Genic-SSR Markers by cDNA Library Sequencing in Boxwood, Buxus spp. (Buxaceae)

Premise of the study: Genic microsatellites or simple sequence repeat (genic-SSR) markers were developed in boxwood (Buxus taxa) for genetic diversity analysis, identification of taxa, and to facilitate breeding. Methods and Results: cDNA libraries were developed from mRNA extracted from leaves of Buxus sempervirens ‘Vardar Valley’ and sequenced using the Illumina MiSeq system. Approximately 11.9 million base pairs of sequence data were examined and 845 genic-SSRs were identified, including 469 dinucleotide, 360 trinucleotide, seven tetranucleotide, one pentanucleotide, and eight hexanucleotide repeats. Primer pairs were designed for 71 selectively chosen genic-SSRs containing trinucleotide repeat motifs and were used to amplify the corresponding loci in 18 diverse boxwood accessions. Twenty-three primer pairs amplified polymorphic loci, with two to 10 alleles per locus. Conclusions: These novel polymorphic genic-SSR markers will aid in evaluating genetic diversity of boxwood germplasm and allow verification of hybrids and cultivars for breeding programs.