Isolation and characterization of 28 microsatellite loci for a Korean endemic, Lespedeza maritima (Fabaceae)1

Ap Applicati tions ons in in Pl Plant t Scien Sciences ces Plants of species within Lespedeza Michx. sect. Macrolespedeza Maxim. (Fabaceae) are deciduous shrubs mainly distributed in the temperate forests of East Asian countries such as Korea,, hybridization is frequent among these species. Therefore, their taxonomic entities are often unclear. One representative, L. maritima Nakai, is a Korean endemic that primarily occurs at low elevations on mountains along the southern coast of the Korean Peninsula. This species is distinguished from others within the genus by its unique morphological traits, e.g., leafl ets with leathery surfaces and revolute margins, as well as abundant sprouting from the basal portion of the plant. Despite these distinctive characteristics, the taxo-nomic entity of L. maritima has been controversial. First described by Nakai (1923) , this species has since then been regarded either as a hybrid between L. cyrtobotrya Miq. and L. maximowiczii C. K. Schneid. (Lee, 1965) or as a synonym of L. thunbergii (DC.) Nakai subsp. thunbergii (= L. formosa (Vogel) Koehne subsp. velutina (Nakai) S. Akiyama & H. Ohba) (Akiyama, 1988), thus making its boundary of taxonomic delimi-tation more ambiguous. Previous phylogenetic analysis (Xu et al., 2012) indicated low resolution for chloroplast DNA (cpDNA) and nuclear ribosomal internal transcribed spacer (nrITS) sequences among species. In plants such as those of Quercus L., codominant polymorphic microsatellite markers have been used to confi rm that hybridization is frequent among those species (see, e.g., Lee et al., 2014). Advances in sequencing techniques, i.e., next-generation sequencing (NGS), have made it possible for researchers to obtain microsatellite libraries rapidly. Thus, microsatellite markers could be applied to resolve the above-mentioned debate about L. maritima and other Lespedeza species if they were available for that genus. Here, we used NGS to develop 28 microsatellite markers to clarify the taxonomic entity of L. maritima. To avoid repeated collections from the same plant, we selected individuals that grew at least 5 m apart. The leaves were preserved with silica gel, and voucher specimens were deposited in the Herbarium of Inha University (IUI), Incheon, Korea (Appendix 1). Genomic DNA, used to acquire the microsatellite library, was extracted from the silica gel–dried leaves of one individual (voucher no. DP149122) with a DNeasy Plant Mini Kit (QIAGEN, Seoul, Korea). The extracted DNA (300 ng) was fragmented to 500 bp using a Covaris S220 were generated. Through SSR_pipeline version 0.951 (Miller et al., 2013), we …