Microsatellite markers for Saussurea polylepis (Asteraceae), a vulnerable continental island species endemic to Korea

Premise Nuclear microsatellite markers were developed for Saussurea polylepis (Asteraceae), a vulnerable species with very limited distribution in a few southwestern continental islands of the Korean peninsula, in order to facilitate future population genetic studies. Methods and Results Based on the Illumina sequence data, a total of 21 microsatellite primer pairs were designed and tested for their suitability. Nineteen of these primers, with two to 11 alleles per locus, were polymorphic in three natural populations of S. polylepis. The levels of expected and observed heterozygosity ranged from 0.000 to 0.842 and 0.000 to 0.933, respectively. Sixteen of these simple sequence repeat (SSR) markers were successfully cross‐amplified in five congeneric species, namely S. gracilis, S. grandifolia, and S. tanakae for all 21 loci, and S. maximowiczii and S. pulchella for 18 loci. Conclusions The SSR markers developed here will be useful for future population genetic studies on S. polylepis and related species.


METHODS AND RESULTS
Total genomic DNA was extracted from a fresh leaf sample collected in Hong-do, Korea (Appendix 1), and deposited in the National Center for Biotechnology Information (NCBI) BioSample database (BioSample accession number SAMN10362162) using a DNeasy Plant Mini Kit (QIAGEN, Carlsbad, California, USA), following the manufacturer instructions. An Illumina paired-end genomic library was constructed using a TruSeq DNA LT Sample Prep Kit (Illumina, San Diego, California, USA), and the library sequencing was con ducted with the Illumina MiSeq platform at the Macrogen Company (Seoul, Korea). A total of 15,210,742 paired-end reads (4.5 Gbp, NCBI Sequence Read Archive BioProject ID PRJNA503488) were obtained. The raw reads were trimmed to remove low-quality bases using Sickle (Joshi and Fass, 2011) and adapters using Scythe (v0.994 BETA) (Buffalo, 2014). The paired-end reads were then assembled us ing SPAdes version 2.4 (Bankevich et al., 2012), and a total of 814,626 contigs were obtained. MIcroSAtellite identification software (MISA) with default settings (Thiel et al., 2003) was used to detect simple se quence repeat (SSR) motifs with a repeat unit ranging from two to six nucleotides. The minimum number of nucleotide repeats was set to six for dinucleotide repeats and five for trinucleotide, tetranucleotide, pentanucleotide, and hexanucleotide repeats, and a total of 40,773 regions were found. Primer3 version 2.3.6 software (Koressaar and Remm, 2007;Untergasser et al., 2012) was used to design SSR prim ers with the following settings: optimal conditions of length of 20 bp (18-27 bp), annealing temperature of 60°C (57-63°C), and product size range of 100-300 bp. Of the 40,773 regions, 35 candidate primer pairs were randomly selected and tested for amplification efficiency using eight individuals of S. polylepis sampled from three island pop ulations of Hueksan-do, Hong-do, and Ui-do (Appendix 1). The PCR amplifications were performed in a total volume of 20 μL containing 1 μL of genomic template DNA, 0.5 μL of 10 mM forward primer with fluorescent dye (Table 1), 0.5 μL of 10 mM reverse primer, 2.5 μL (10×, with 2.5 mM MgCl 2 ) of PCR buffer, 0.5 μL (each 10 mM) of dNTPs, and 0. showed polymorphisms across all eight individuals. These 19 primer pairs were subsequently scored using 48 individuals of S. polylepis sampled from the same three island populations and five individu als from each of the other species, namely S. gracilis, S. grandifolia, S. maximowiczii, S. pulchella, and S. tanakae (Appendix 1), for con generic cross-transferability of the markers. The PCR thermocycling conditions and genotyping method were the same as described above. Population genetic diversity parameters, including number of alleles, expected heterozygosity, and observed heterozygosity, were calculated using GenAlEx v6.502 (Peakall and Smouse, 2006). Deviations from Hardy-Weinberg equilibrium were calculated for each locus with GENEPOP 4.2 (Rousset, 2008). Of the 21 SSRs de veloped in this study, 19 were polymorphic. The expected and ob served heterozygosity values ranged from 0.000 to 0.842 and from 0.000 to 0.933, respectively (Table 2). Of the 19 polymorphic loci, five in the Hong-do population, five in the Heuksan-do population, and three in the Ui-do population deviated from Hardy-Weinberg equilibrium (Table 2).    Cross-amplification of the 21 primer pairs was conducted in five widely distributed congeneric species of Saussurea on the Korean peninsula. Three species (S. gracilis, S. grandiflora, and S. tanakae) were all successfully amplified and polymorphic for the 21 loci, while two species (S. maximowiczii and S. pulchella) were success fully amplified and polymorphic for 18 loci.

CONCLUSIONS
The results of the present study represent the second set of SSR markers developed for the genus Saussurea. The 21 SSR markers developed in this study will be useful for studying genetic diver sity, gene flow, and population structure in S. polylepis. In addi tion, the resulting population genetic studies will help to develop appropriate conservation strategies and management plans of the endangered S. polylepis on continental islands in Korea. Lastly, the successful cross-amplification of these markers among com monly found Saussurea species in Korea will provide a novel pop ulation genetic tool in one of the most speciose groups in Korea.

ACKNOWLEDGMENTS
The authors thank the Korean National Park Service for special per mission to collect samples of Saussurea polylepis from the islands of Hong-do, Hueksan-do, and Ui-do. This study represents a part of the first author's Ph.D. dissertation submitted to Sungkyunkwan University.

AUTHOR CONTRIBUTIONS
S.A.Y. and S.-C.K. designed the project. S.A.Y. collected samples in the field, conducted the experiments, analyzed the data, and wrote the draft of the manuscript. S.-C.K. supervised the study and re vised the manuscript. All authors approved the final version of the manuscript.

DATA ACCESSIBILITY
All sequence information was deposited in the National Center for Biotechnology Information (NCBI) Sequence Read Archive (BioProject ID PRJNA503488), and genomic DNA was deposited in the NCBI BioSample database (accession number SAMN10362162).
Primer sequences were uploaded to GenBank and accession num bers are provided in Table 1.

LITERATURE CITED
APPENDIX 1. Locality and voucher information of three Saussurea polylepis populations and five congeneric species used in this study.

Species
Collection locality Geographic coordinates a N Voucher specimen b