To acquire an NGS library for H. capitata, we used the DNeasy Plant Kit (Qiagen, Seoul, Korea) to extract genomic DNA from a fresh leaf of a single plant sample collected from Baegunsan Mt., Gwangyang-si, Jeollanam-do, Korea (Table 1). We evaluated the quality of the DNA using a NanoDrop ND-1000 (NanoDrop Technologies, Wilmington, DE, USA). The DNA passed the quality check (concentration 317.95 ng/μL; A260/280 = 1.83; A260/230 = 2.37) was sequenced on the Illumina MiSeq platform (LAS Inc., Seoul, Korea). To identify microsatellites, sequence reads were screened using SSR-pipeline version 0.951 (Miller et al., 2013). The parameter settings for the detection of di-, tri-, or tetranucleotide motifs were flanking regions larger than 100 bp and a minimum of 10, 6, and 4 repeats, respectively. In addition, AT and TA repeats, G + C contents under 30%, and G + C contents above 80%, as well as reads with additional repeat were removed. The resulting sequences were grouped by the repeat motives and mapped to the reference using Geneious R 10.1.3. For each assembled contig, sites with identities < 80% and qualities < 50% were removed. We selected fragments with unique patterns that had less than 20 reads, two separate alleles, little variation at the site to which the primer was attached, and no additional single nucleotide polymorphisms in the flanking region. The final selected reads were deduplicated through de novo assembly and used to develop microsatellite markers. Primer pairs were designed using Primer 3 (Rozen and Skaletsky, 2000), and three sets of M13 tag sequences were added (5′-CACGACGTTGTAAAACGAC-3′, 5′-TGTGGAAT TGTGAGCGG-3′, and 5′-CTATAGGGCACGCGTGGT-3′) to the forward primer with 6FAM, VIC, and NED fluorescent dyes, respectively. For multiplex PCR (nine primer sets), each marker was 100–300 bp. To select microsatellite markers with infraspecific polymorphism for H. capitata, 90 microsatellite markers were verified using 32 individuals collected from Baegunsan Mt. The PCR protocol consisted of initial denaturation at 95°C for 10 min, followed by 35 cycles of denaturation at 95°C for 30 s, annealing at 56°C for 1 min 30 s, and extension for 1 min at 72°C, and a final extension at 4°C for 10 min. The PCR products were analyzed using an ABI 3730XL sequencer with the GeneScan 500LIZ Size Standard (Thermo Fisher Scientific, Waltham, MAs, USA). Allele sizes and peaks for each sample were determined using Peak Scanner version 2.0 (Thermo Fisher Scientific). The number of alleles and the values for H_E (expected heterozygosity) and H_O (observed heterozygosity) were analyzed using GeneAlEx 6.5 (Peakall and Smouse, 2005). Deviations from Hardy–Weinberg equilibrium (HWE) and linkage disequilibrium were estimated using GENEPOP 4.0 (Rousset, 2008). In addition, Micro-checker version 2.2.3 (Van Oosterhout et al., 2004) was used to determine the null allele frequencies and scoring errors.

Using the 90 newly designed primer pairs, we developed 28 polymorphic microsatellite loci from H. capitata (Table 2). The number of alleles per locus ranged from 1 to 13 (average, 4.7). H_E and H_O values ranged from 0.000 to 0.832 and from 0.000 to 0.844, respectively. After Bonferroni correction, three markers (Hca069, Hca70, and Hca72) showed a significant deviation from HWE (p < 0.0018), but the deviation was not consistent across the three populations of H. capitata (Table 3). We tested the 28 loci for cross-amplification in its two closely related species of H. minor and H. venusta (Table 4). From the results, 13 loci were successfully amplified and were polymorphic in both H. minor and H. venusta, with 2–10 alleles per locus. The newly developed microsatellite markers will provide a powerful genetic tool for elucidating the phylogeographic patterns of Hosta capitata populations in Korea and Japan. Furthermore, these results will be beneficial for studying the genetic delimitation of H. capitata and its related species in sect. Lamellatae.