Isolation and Characterization of Novel Microsatellite Loci For The Endangered
Orchid Cypripedium japonicum (Orchidaceae)
Author(s): Yumi Yamashita , Ayako Izuno , Yuji Isagi , Takahide Kurosawa , and Shingo Kaneko
Source: Applications in Plant Sciences, 4(2)Published By: Botanical Society of America
DOI: http://dx.doi.org/10.3732/apps.1500097
URL: http://www.bioone.org/doi/full/10.3732/apps.1500097

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Applications in Plant Sciences 2016 4(2): 1500097

Applicati
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in Pl
Plantt Scien
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PRIMER NOTEISOLATION AND CHARACTERIZATION OF NOVEL MICROSATELLITELOCI FOR THE ENDANGERED ORCHID CYPRIPEDIUM JAPONICUM(ORCHIDACEAE)1YUMI YAMASHITA2, AYAKO IZUNO3, YUJI ISAGI3, TAKAHIDE KUROSAWA2, AND SHINGO KANEKO2,4
2Graduate School of Symbiotic Systems Science and Technology, Fukushima University, Kanayagawa 1, Fukushima, Fukushima

960-1296, Japan; and 3Laboratory of Forest Biology, Division of Forest and Biomaterials Science, Graduate School ofAgriculture, Kyoto University, Kitashirakawa, Oiwake-cho, Sakyo-ku, Kyoto 606-8502, Japan• Premise of the study: Twenty-six microsatellite markers were developed for the endangered orchid Cypripedium japonicum(Orchidaceae) to estimate the clonal diversity and genetic structure of the remaining populations in Japan.
• Methods and Results: Microsatellite loci of C. japonicum were isolated using Ion Personal Genome Machine (PGM) sequencing. The primer sets were tested on 55 ramets sampled from two populations in Japan. Sixteen loci showed polymorphism in atleast one population, with two to five alleles per locus. Observed and expected heterozygosities for the two populations ranged
from 0.00 to 0.92 and 0.00 to 0.71, respectively.
• Conclusions: The microsatellite markers developed here provide a useful tool to analyze clonal structure and sexual regeneration status and will help to manage the remaining genetic variation within C. japonicum.
Key words: clonal analysis; conservation genetics; Cypripedium japonicum; Ion PGM sequencing; microsatellites;
Orchidaceae.

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 CriticallyEndangered 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 inJapan.
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 bycareful excavation suggested a high reliability on asexual reproduction by stoloniferous rhizomes (Chiba Prefecture Board ofEducation, 1980). The bias toward asexual reproduction may
have made it difficult to investigate sexual regeneration of thisspecies, and suitable vegetation for safe sites and the fungalsymbionts 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 forC. 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 allozymeanalysis (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). Afteramplification, 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, theresulting 325,984 sequences were screened for potential microsatellite loci

1 Manuscript received 21 August 2015; revision accepted 2 October 2015.

The authors thank Kazuko Iga, Shoue Tohmi, Noriko Hasegawa, andKatsuroh Hasegawa for their help in sampling. We also thank Daisuke
Sugimori for the support in genetic analysis. This study was partly supportedby the Fukushima University research project for the Regeneration ofHarmonies between Human Activity and Nature in Bandai-Asahi NationalPark.
4 Author for correspondence: kaneko.shingo@gmail.com
doi:10.3732/apps.1500097

Applications in Plant Sciences 2016 4(2): 1500097; http://www.bioone.org/loi/apps © 2016 Yamashita et al. Published by the Botanical Society of America.
This work is licensed under a Creative Commons Attribution License (CC-BY-NC-SA).
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Yamashita et al.—Cypripedium japonicum microsatellites

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 rametsfrom a population in the Chiba Prefecture and 31 ramets from a population in
the Hokkaido Prefecture were used (Appendix 1). A tag sequence for fluorescentlabeling 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:
TABLE 1.

10 ng of genomic DNA, 2.5 μL of Multiplex PCR Master Mix, 0.01 μM offorward 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).
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 probabilityof each population calculated by CERVUS 3.0 (Kalinowski et al., 2007)
was 0.000018 in Chiba and 0.00012 in Hokkaido, respectively. Thus, these

Characteristics of 26 microsatellite primers developed for Cypripedium japonicum.aLocus

Primer sequences (5′–3′)

Repeat motif

Fluorescent labelb

Allele size range (bp)

GenBank accession no.

Cypj025

F: TTCGAGATGCTTCCGACCC
R: TTGGCCGAGTTCGTTCGAG
F: TGTCAGTGTCGCTGCCTTC
R: AGTTCACGACCCGATTGTCF: TCACTGAGAGGTGTGATTCC
R: CATTGCATGCTTGTGTTGT
F: TTTTGGATCAAATCATCACCT
R: CTTCTTTAGAGGAAGATCCAAGA
F: TGAGGCTACCAGTTAATGTCTG
R: ATCTTCCTCTCCACCAATCA
F: ACAAGAACCTGCCAGAAAAC
R: GACAAGATTTTCAATTCATCACTC
F: GCATCATTCAAGGTGTCAAA
R: CTTCCTCCCTCTCTCTTTCC
F: ATTCATAAAACCAGGGCTGA
R: TCAAAGGATGGTGGAGAAGT
F: TCGATGACATTGATATGGAAG
R: AGGGATGATCTTTTCCTTCA
F: CCTCAATAGGGACACACACA
R: AGTTCAATGGAACCCTCAAA
F: GGTGAATTATATGATGGAAGCA
R: TTGCTGTTATTACTCCCACCTF: TTAAGGGACTTTCTCTGATTCAAC
R: CCAATCACTTCCTAGCTGGC
F: CCATCAGGCCACCATTCTG
R: TGGTGTCTCCTTATTGTGATTGC
F: AGTTGGGTATCGAGGTGGC
R: AGACTAAGCTATGGTAACTACATTCC
F: CCAGGACCTTAGCCCTGAC
R: CCCTCTCAGATCTCCTACAAAGG
F: AGTTGGCAAGGATCTTATTGGC
R: GCCCAGGCCCTTATTCAAAG
F: ACACCCATATTTGAGGATGGC
R: AGCAGTTCCTAATGGCAAGG
F: AGCTCTCATACTGAGGGTTG
R: TATGCACTTGGCACATTCG
F: ACCGATGAAATTTGGCAGAGG
R: CACTCCCGCCATTAGAACC
F: TGCTAACATTTGCAACAAAGC
R: TGCTTGGTGATGGAGGAAAC
F: TCCTCCAGCACTTTGTCGG
R: TCCTACAAGCCTCCACTGC
F: ACTAGCATCGCTGAAAGTGC
R: TGAGGAGAGACTCCATGAACG
F: AATCAATTCCCATTTAAAACTCTC
R: ATTTAGGCCAAAACAGAGGA
F: ACCGGTGATGAAGGAAAATA
R: TGATTTGAAGCCTAATATATATF: AAGAGGTTGGCTTTTGGATT
R: CAACGATGAGTTCGTAAAGG
F: AAGCCAAAAGAGAAGCTTGA
R: GAACTTGAACCCGAGAGAGA(TTG)9VIC

236–239

LC73788(GCG)10

FAM

191–199

LC73789(AC)12FAM

160

LC73790(AC)10

FAM154–156

LC73791(AG)12

FAM

131–135

LC73792(AG)10FAM

122–124

LC73793(GA)10

VIC

104

LC73794(GA)11

FAM

158–162

LC73795(GA)23FAM

125–131

LC73796(AG)11

VIC

128–157

LC73797(AC)11

VIC

173–177

LC73798

(CT)9

FAM

240

LC73799

(GA)7

FAM

221–223

LC73800(GA)13FAM

174–176

LC73801

(GA)6

VIC

375

LC73802

(TA)6

VIC

247–249

LC73803

(TG)9

FAM

311

LC73804(CT)10VIC

217–219

LC73805

(CT)8

FAM

258

LC73806(AG)10FAM

174–176

LC73807(AG)10

VIC

180

LC73808(GA)10

VIC

277

LC73809(CT)10VIC

234LC73810(TA)10VIC

220–226

LC73811(TC)11

VIC

168–170

LC73812(CT)10FAM

214

LC73813

Cypj047
Cypj060
Cypj061
Cypj062
Cypj065
Cypj069
Cypj082
Cypj091
Cypj094
Cypj100
Cypj114
Cypj122
Cypj140
Cypj147
Cypj179
Cypj180
Cypj196
Cypj197
Cypj202
Cypj204
Cypj205
Cypj216
Cypj218
Cypj224
Cypj233

a Annealing temperature for all reactions was 57°C.

b Sequence of the fluorescent labels: FAM = 5′-CACGACGTTGTAAAACGAC-3′, VIC = 5′-TGTGGAATTGTGAGCGG-3′.

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Applications in Plant Sciences 2016 4(2): 1500097
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TABLE 2.

Yamashita et al.—Cypripedium japonicum microsatellites

Genetic variation of the 16 polymorphic microsatellite loci for two populations of Cypripedium japonicum in Japan.
Chiba (N = 24, G = 24)

Hokkaido (N = 31, G = 19)

Locus

A

Ho

He

PID

A

Ho

He

PID

Cypj025
Cypj047
Cypj061
Cypj062
Cypj065
Cypj082
Cypj091
Cypj094
Cypj100
Cypj122
Cypj140
Cypj179
Cypj196
Cypj202
Cypj218
Cypj22424222345322222410.04
0.17
0.38
0.08
0.46
0.17
0.92
0.54
0.58
0.38
0.38
0.29
0.04
0.29
0.46
0.00

0.04
0.16
0.40
0.08
0.51
0.16
0.71
0.64
0.58
0.36
0.31
0.25
0.04
0.25
0.38
0.00

0.92
0.71
0.44
0.85
0.38
0.72
0.14
0.21
0.26
0.48
0.53
0.59
0.92
0.59
0.43
1.00

1
2
2
1
1
1
3
3
2
1
1
2
2
1
2
2

0.00
0.05
0.63
0.00
0.00
0.00
0.42
0.58
0.32
0.00
0.00
0.26
0.42
0.00
0.16
0.26

0.00
0.15
0.51
0.00
0.00
0.00
0.35
0.65
0.40
0.00
0.00
0.23
0.40
0.00
0.15
0.23

1.00
0.74
0.38
1.00
1.00
1.00
0.48
0.21
0.45
1.00
1.00
0.62
0.45
1.00
0.74
0.62

Note: A = number of alleles; G = number of genets; He = expected heterozygosity; Ho = observed heterozygosity; N = number of analyzed ramets;
PID = probability of identity.

microsatellite markers have sufficient resolution in clonal analysis, and ramets that showed identical genotypes in the Hokkaido population werefrom the same genets, probably produced by asexual propagation via rhizome
elongation.
Genetic variation was evaluated for 24 genets from the Chiba populationand 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 (mean0.20) and 0.00–0.71 (mean 0.19), respectively (Table 2). Deviations fromHardy–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.

CONCLUSIONS
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.
LITERATURE CITED
BOUTIN-GANACHE, I., M. RAPOSO, M. RAYMOND, AND C. F. DESCHEPPER.
2001. M13-tailed primers improve the readability and usability ofmicrosatellite analyses performed with two different allele-sizing
methods. BioTechniques 31: 24–26.

http://www.bioone.org/loi/apps

CHIBA PREFECTURE BOARD OF EDUCATION. 1980. Report on Cypripedium
japonicum at Noruto-machi, the natural monument of Chiba Prefecture.
Chiba Prefecture Board of Education, Chiba, Japan [in Japanese].
CHUNG, J. M., K. W. PARK, C.-S. PARK, S.-H. LEE, M. G. CHUNG, AND
M. Y. CHUNG. 2009. Contrasting levels of genetic diversity between
the historically rare orchid Cypripedium japonicum and the historically common orchid Cypripedium macranthos in South Korea.
Botanical Journal of the Linnean Society 160: 119–129.
CRIBB, P. 1997. The genus Cypripedium. Timber Press, Portland, Oregon,USA.
FAIRCLOTH, B. C. 2008. MSATCOMMANDER: Detection of microsatellite repeat arrays and automated, locus-specific primer design.
Molecular Ecology Resources 8: 92–94.
GOUDET, J. 1995. FSTAT: A computer program to calculate F-statistics,version 1.2. Journal of Heredity 86: 485–486.
HASEGAWA, A., M. NAKASUGI, AND M. GOI. 1987. A seed harvesting
method of Cypripedium japonicum Thunberg. Technical Bulletin ofFaculty of Agriculture, Kagawa University 38: 63–70 [in Japanesewith English abstract].
KALINOWSKI, S. T., M. L. TAPER, AND T. C. MARSHALL. 2007. Revising how thecomputer program CERVUS accommodates genotyping error increases
success in paternity assignment. Molecular Ecology 16: 1099–1106.
LEE, B. C. 2009. Rare plants: Data book of Korea. Korea National
Arboretum, Pocheon, Korea.
MINISTRY OF THE ENVIRONMENT. 2015. Red Data Book 2014–Threatened wildlife of Japan, Vol. 8, Vascular Plants. Gyosei, Tokyo, Japan [in Japanese].
PEAKALL, R., AND P. E. SMOUSE. 2006. GenAlEx6: Genetic analysis
in Excel. Population genetic software for teaching and research.
Molecular Ecology Notes 6: 288–295.
QIAN, X., Q.-J. LI, F. LIU, M.-J. GONG, C.-X. WANG, AND M. TIAN. 2014.
Conservation genetics of an endangered Lady’s Slipper Orchid:
Cypripedium japonicum in China. International Journal of Molecular
Sciences 15: 11578–11596.
ROZEN, S., AND H. SKALETSKY. 1999. Primer3 on the WWW for general users and for biologist programmers. In S. Misener and S. A. Krawetz[eds.], Methods in molecular biology, vol. 132: Bioinformatics methods
and protocols, 365–386. Humana Press, Totowa, New Jersey, USA.

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APPENDIX 1.

Voucher and location information for the Cypripedium japonicum populations used in this study. One voucher was collected from eachpopulation sampled.
Collection locality

GPS coordinatesa

Soma, Fukushima Prefecture, Japan

37°46′N, 140°42′E

FKSE 22462 (Kazuko Iga 977)

1

Yotsukaido, Chiba Prefecture, Japan
Nikappu, Hokkaido Prefecture, Japan

35°39′N, 140°12′E42°21′N, 142°18′E

FKSE 87328 (Yumi Yamashita 735)
FKSE 86822 (Yumi Yamashita 636)

2431CollectorKazuko Iga and Yumi
Yamashita
Yumi Yamashita
Yumi Yamashita

Voucher specimen accession no.b

No. of ramets

a Precise GPS coordinates were not included for conservation purposes.
b All vouchers were deposited in the Herbarium of the Faculty of Symbiotic Systems Science (FKSE), Fukushima University, Fukushima, Japan.

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