Cypripedium L. (Orchidaceae) is a genus rich in horticulturally important species, including many endangered taxa. Cypripedium japonicum Thunb. is an attractive terrestrial orchid distributed widely throughout temperate forests in Japan, Korea, and China (Cribb, 1997). However, because of recent habitat destruction and extraction for horticultural purposes, remaining populations of this species have declined and become fragmented, and C. japonicum is now classified as Critically Endangered in Korea (Lee, 2009) and Vulnerable in Japan (Ministry of the Environment, 2015). In this critical situation, the necessity for the in situ and ex situ conservation of genetic resources has been highlighted in Korea (Lee, 2009), and in situ recovery programs are underway for several populations in Japan.
Although immediate establishment of appropriate conservation programs is needed for C. japonicum, ecological studies and knowledge of sexual regeneration for this species are limited. Field observations reported a low fruiting rate in Japan (Hasegawa et al., 1987; Yamashita, personal observation), and observations of connections of underground organs by careful excavation suggested a high reliability on asexual reproduction by stoloniferous rhizomes (Chiba Prefecture Board of Education, 1980). The bias toward asexual reproduction may have made it difficult to investigate sexual regeneration of this species, and suitable vegetation for safe sites and the fungal symbionts for its germination are not yet known. Therefore, clarifying the current status of sexual reproduction in remaining populations can provide basic information about in situ conservation programs of this endangered species.
In this study, we developed microsatellite markers for C. japonicum using the Ion Personal Genome Machine (PGM; Life Technologies, Waltham, Massachusetts, USA) sequencing to investigate the current status of sexual and asexual regeneration. This genetic analysis aims to reveal clonal structures accumulated as a result of asexual reproduction as well as sexual regeneration status. The data will also provide information about genetic variation and differences among remaining populations, which have not been possible to elucidate by either allozyme analysis (Chung et al., 2009) or intersimple sequence repeat (ISSR) analysis (Qian et al., 2014).
METHODS AND RESULTS
A fresh leaf sample was taken from a ramet growing in a native population in Soma, Fukushima Prefecture, Japan (Appendix 1). Genomic DNA was extracted using the DNeasy Plant Mini Kit (QIAGEN, Germantown, Maryland, USA) following the manufacturer's instructions. The DNA fragment library was constructed using an Ion Xpress Plus Fragment Library Kit (Life Technologies). Emulsion PCR was performed for the fragment library with capture beads using an Ion PGM Template OT2 400 Kit (Life Technologies). After amplification, the desired beads were enriched and the amplified DNA fragments were sequenced using an Ion PGM Sequencing 400 Kit (Life Technologies) and an Ion 318 Chip v2 (Life Technologies). A total of 326,901 sequences (mean read length 220 bp) were obtained. After filtering for identical reads, the resulting 325,984 sequences were screened for potential microsatellite loci using MSATCOMMANDER (Faircloth, 2008) using default settings. Primers were designed for all sequences containing more than six di- or trinucleotide repeats using Primer3 software (Rozen and Skaletsky, 1999) with the default settings, resulting in a total of 238 primer pairs for screening. Twenty-six primer pairs showing clear peak patterns were selected after an amplification trial using eight ramets from populations in the Chiba and Fukushima prefectures, Japan (Appendix 1).
To test the genetic variation of the 26 selected microsatellite loci, 24 ramets from a population in the Chiba Prefecture and 31 ramets from a population in the Hokkaido Prefecture were used (Appendix 1). A tag sequence for fluorescent labeling was added to each of the forward primers (Boutin-Ganache et al., 2001). PCR amplification was done in 5-µL reactions using the QIAGEN Multiplex PCR Kit (QIAGEN). Each reaction contained the following components: 10 ng of genomic DNA, 2.5 µL of Multiplex PCR Master Mix, 0.01 µM of forward primer, 0.2 µM of reverse primer, and 0.1 µM of fluorescently labeled tag primer. Amplifications used the following program: 95°C for 15 min; 33 cycles at 94°C for 30 s, 57°C for 1.5 min, and 72°C for 1 min; and an extension at 60°C for 30 min. Product sizes were determined using an ABI PRISM 3130 Genetic Analyzer and GeneMapper software (Applied Biosystems, Foster City, California, USA).
Characteristics of 26 microsatellite primers developed for Cypripedium japonicum.a
Of the 26 loci tested, 16 were polymorphic and 10 were monomorphic (Table 1). In the Chiba population, all 24 ramets showed distinct multilocus genotypes, whereas in the Hokkaido population, 19 multilocus genotypes were detected across 31 ramets. The combined nonexclusion probability of each population calculated by CERVUS 3.0 (Kalinowski et al., 2007) was 0.000018 in Chiba and 0.00012 in Hokkaido, respectively. Thus, these microsatellite markers have sufficient resolution in clonal analysis, and ramets that showed identical genotypes in the Hokkaido population were from the same genets, probably produced by asexual propagation via rhizome elongation.
Genetic variation of the 16 polymorphic microsatellite loci for two populations of Cypripedium japonicum in Japan.
Genetic variation was evaluated for 24 genets from the Chiba population and 19 genets from the Hokkaido population using GenAlEx version 6.2 (Peakall and Smouse, 2006) and CERVUS 3.0 (Kalinowski et al., 2007). The observed and expected heterozygosities (Ho and He) were 0.00–0.92 (mean 0.20) and 0.00–0.71 (mean 0.19), respectively (Table 2). Deviations from Hardy–Weinberg equilibrium (HWE) and linkage disequilibrium (LD) between loci were tested using FSTAT version 2.9.3 (Goudet, 1995). Significance levels were adjusted using the Bonferroni correction for multiple testing. None of the loci exhibited a significant deviation from HWE (P < 0.05) in either of the populations, and there was no evidence of LD for any locus pairs.
We have developed 26 microsatellite markers for C. japonicum that will be useful for assessing the clonal structure and sexual regeneration status of remaining populations of C. japonicum. The results presented here indicate that sexual regeneration may be contributing more to maintaining the number of ramets than previously expected. These markers also have enough resolution to investigate genetic variation and differences among remaining populations, which are essential for handling the priority of genets and populations for the in situ and ex situ conservation of this species.
 The authors thank Kazuko Iga, Shoue Tohmi, Noriko Hasegawa, and Katsuroh Hasegawa for their help in sampling. We also thank Daisuke Sugimori for the support in genetic analysis. This study was partly supported by the Fukushima University research project for the Regeneration of Harmonies between Human Activity and Nature in Bandai-Asahi National Park.