Isolation and characterization of microsatellite markers for an endemic tree in East Asia, Quercus variabilis (Fagaceae)1

Ap Applicati tions ons in in Pl Plant t Scien Sciences ces Quercus variabilis Blume (Fagaceae) is a tree native to East Asia found mainly in warm-temperate and subtropical regions (Chen et al., 2012). Previous studies on this species have focused on its physiology and ecology (Lei et al., 2013 ; Du et al., 2014). To date, only a few studies have reported on the genetic diversity and population structure of this species using isozyme, chloro-plast DNA, and microsatellite markers (Zhou et al., 2003 ; Xu et al., 2004 ; Chen et al., 2012). However, the polymorphic iso-zyme loci are limited in number and the chloroplast DNA is maternally inherited, which limits the comprehensive understanding of its genetic diversity and population structure. Xu et al. (2004) developed 16 microsatellite loci for Q. variabilis using primer pairs from three other Quercus L. species; however, these loci do not meet the requirement of high-density markers in landscape genetics (Hall and Beissinger, 2014). Thus, developing a greater number of microsatellite loci for this species is necessary for population genetic and landscape genetic studies of Q. variabilis to progress. Microsatellite markers, or simple sequence repeats (SSRs), are widely used in population genetic and landscape genetic studies, providing useful information to plant natural resources management and promoting future long-term strategies (Ohtsuki et al., 2014 ; Lei et al., 2015). Next-generation sequenc-ing allows for rapid development of a large number of SSR markers (Huang et al., 2014 ; Kumar et al., 2014). Here, we report the development of microsatellite markers for Q. variabilis using Roche 454 pyrosequencing combined with a magnetic bead enrichment protocol. METHODS AND RESULTS Fresh leaves from 24 individuals of Q. variabilis were collected in two wild The samples were stored in silica gel until genomic DNA extraction and geno-typing. Vouchers were deposited at the herbarium of Henan Agricultural University (Herbarium accession no. HHAU-2101-2112 [Song Mountain population] and HHAU-2113-2124 [Meng Mountain population]). Genomic DNA was extracted from approximately 30 mg of dried leaf tissue using a Tiangen Biotech (Beijing, China) DNA extraction kit (Plant no. DP305) following the manufac-turer's protocol. A shotgun library was prepared by shearing 1 μ g of genomic DNA using the DNA Library Preparation Kit (Roche Applied Science, India-napolis, Indiana, USA) following the GS FLX+ library preparation protocol. The shotgun library was further enriched by eight 5 ′-biotinylated probes, namely, and eventually sequenced on a Roche 454 GS FLX …