Comparison between Wild and Hatchery Populations of Korean Pen Shell (Atrina pectinata) Using Microsatellite DNA Markers

Pen shell (Atrina pectinata) is a popular food source with a high commercial value in a number of Asian Pacific areas. The natural A. pectinata population has been declining continuously over the past several decades. Microsatellite DNA markers are a useful DNA-based tool for monitoring the genetic variation of pen shell populations. In this study, 20 polymorphic microsatellite (MS) DNA markers were identified from a partial genomic pen shell DNA library enriched in CA repeats, and used to compare allelic variation between wild and hatchery pen shell populations in Korea. A total of 438 alleles were detected at the 20 MS loci in the two populations. All loci were easily amplified and demonstrated allelic variability, with the number of alleles ranging from 5 to 35 in the wild population and from 5 to 22 in the farmed population. The average observed and expected heterozygosities were 0.69 and 0.82, respectively, in the hatchery samples and 0.69 and 0.83, respectively, in the wild samples. Statistical analysis of fixation index (F(ST)) and analysis of molecular variance (AMOVA) showed minor, but significant, genetic differences between the wild and hatchery populations (F(ST) = 0.0106, CI(95%) = 0.003-0.017). These microsatellite loci may be valuable for future aquaculture and population genetic studies for developing conservation and management plans. Further studies with additional pen shell samples are needed to conclusively determine the genetic diversity between the wild and hatchery populations.     

Wild and hatchery populations of Korean starry flounder (Platichthys stellatus) compared using microsatellite DNA markers

Starry flounder (Platichthys stellatus) is an important sport and food fish found around the margins of the North Pacific. Aquaculture production of this species in Korea has increased because of its commercial value. Microsatellite DNA markers are a useful DNA-based tool for monitoring the genetic variation of starry flounder populations. In this study, 12 polymorphic microsatellite DNA markers were identified from a partial genomic starry flounder DNA library enriched in CA repeats, and used to compare allelic variation between wild and hatchery starry flounder populations in Korea. All loci were readily amplified and demonstrated high allelic diversity, with the number of alleles ranging from 6 to 18 in the wild population and from 2 to 12 in the farmed population. A total of 136 alleles were detected at the 12 microsatellite loci in the two populations. The mean observed and expected heterozygosities were 0.62 and 0.68, respectively, in the hatchery samples and 0.67 and 0.75, respectively, in the wild samples. These results indicate lower genetic variability in the hatchery population as compared to the wild population. Significant shifts in allelic frequencies were detected at eight loci, which resulted in a small but significant genetic differences between the wild and hatchery populations (F(ST) = 0.043, P < 0.05). Further studies with additional starry flounder sample collections are needed for comprehensive determinations of the genetic varieties between the wild and hatchery populations. These microsatellite loci may be valuable for future population genetic studies, monitoring the genetic variation for successful aquaculture management and the preservation of aquatic biodiversity.     

Application of Novel Polymorphic Microsatellite Loci Identified in the Korean Pacific Abalone (Haliotis diversicolor supertexta (Haliotidae)) in the Genetic Characterization of Wild and Released Populations

The small abalone, Haliotis diversicolor supertexta, of the family Haliotidae, is one of the most important species of marine shellfish in eastern Asia. Over the past few decades, this species has drastically declined in Korea. Thus, hatchery-bred seeds have been released into natural coastal areas to compensate for the reduced fishery resources. However, information on the genetic background of the small abalone is scarce. In this study, 20 polymorphic microsatellite DNA markers were identified using next-generation sequencing techniques and used to compare allelic variation between wild and released abalone populations in Korea. Using high-throughput genomic sequencing, a total of 1516 (2.26%; average length of 385 bp) reads containing simple sequence repeats were obtained from 86,011 raw reads. Among the 99 loci screened, 28 amplified successfully, and 20 were polymorphic. When comparing allelic variation between wild and released abalone populations, a total of 243 different alleles were observed, with 18.7 alleles per locus. High genetic diversity (mean heterozygosity = 0.81; mean allelic number = 15.5) was observed in both populations. A statistical analysis of the fixation index (F_ST) and analysis of molecular variance (AMOVA) indicated limited genetic differences between the two populations (F_ST = 0.002, p > 0.05). Although no significant reductions in the genetic diversity were found in the released population compared with the wild population (p > 0.05), the genetic diversity parameters revealed that the seeds released for stock abundance had a different genetic composition. These differences are likely a result of hatchery selection and inbreeding. Additionally, all the primer pair sets were effectively amplified in another congeneric species, H. diversicolor diversicolor, indicating that these primers are useful for both abalone species. These microsatellite loci may be valuable for future aquaculture and population genetic studies aimed at developing conservation and management plans for these two abalone species.     

Genetic Differentiation between Natural and Hatchery Stocks of Japanese Scallop (Mizuhopecten yessoensis) as Revealed by AFLP Analysis

Japanese scallop (Mizuhopecten yessoensis) is a cold-tolerant bivalve that was introduced to China for aquaculture in 1982. In this study, amplified fragment length polymorphism (AFLP) markers were used to investigate levels of genetic diversity within M. yessoensis cultured stocks and compare them with wild populations. Six pairs of primer combinations generated 368 loci among 332 individuals, in four cultured and three wild populations. High polymorphism at AFLP markers was found within both cultured and wild M. yessoensis populations. The percentage of polymorphic loci ranged from 61.04% to 72.08%, while the mean heterozygosity ranged from 0.2116 to 0.2596. Compared with wild populations, the four hatchery populations showed significant genetic changes, such as lower expected heterozygosity and percentage of polymorphic loci, and smaller frequency of private alleles, all indicative of a reduction in genetic diversity. Some genetic structures were associated with the geographical distribution of samples; with all samples from Dalian and Japan being closely related, while the population from Russia fell into a distinct clade in the phylogenetic analysis. The genetic information derived from this study indicated that intentional or accidental release of selected Japanese scallops into natural sea areas might result in disturbance of local gene pools and loss of genetic variability. We recommend monitoring the genetic variability of selected hatchery populations to enhance conservation of natural Japanese scallop resources.     

Isolation and Characterization of Microsatellite Markers in the Domestic Ferret (Mustela putorius furo)

The domestic ferret (Mustela putorius furo) is an important model organism for the study of avian influenza and other diseases of humans and animals, as well as a popular pet animal. In order to evaluate genetic diversity and study disease relationships in ferrets, 22 nuclear microsatellite loci (17 dinucleotide and 5 tetranucleotide) were developed from ferret genomic libraries and organized into seven multiplex sets. Polymorphism was preliminarily assessed in one population in Australia and one in the USA, sampled with 25 individuals each. The loci displayed allelic diversity ranging from 1 to 5 alleles, and expected and observed heterozygosities ranging from 0.04 to 0.65 and 0.04 to 0.76, respectively. Additionally, the loci amplified products in 15 samples from the wild ancestor, European polecat (Mustela putorius) and domestic ferret-polecat hybrids. In polecat/hybrid samples, allelic diversity ranged from 3 to 8 alleles, and expected and observed heterozygosities ranged from 0.13 to 0.81 and 0.13 to 0.80 respectively. These markers will be useful for molecular assessments of genetic diversity and applications to evolution, ecology, and health in domestic ferrets and wild polecats.     

Genetic Diversity and Population Structure: Implications for Conservation of Wild Soybean (Glycine soja Sieb. et Zucc) Based on Nuclear and Chloroplast Microsatellite Variation

Wild soybean (Glycine soja Sieb. et Zucc) is the most important germplasm resource for soybean breeding, and is currently subject to habitat loss, fragmentation and population decline. In order to develop successful conservation strategies, a total of 604 wild soybean accessions from 43 locations sampled across its range in China, Japan and Korea were analyzed using 20 nuclear (nSSRs) and five chloroplast microsatellite markers (cpSSRs) to reveal its genetic diversity and population structure. Relatively high nSSR diversity was found in wild soybean compared with other self-pollinated species, and the region of middle and lower reaches of Yangtze River (MDRY) was revealed to have the highest genetic diversity. However, cpSSRs suggested that Korea is a center of diversity. High genetic differentiation and low gene flow among populations were detected, which is consistent with the predominant self-pollination of wild soybean. Two main clusters were revealed by MCMC structure reconstruction and phylogenetic dendrogram, one formed by a group of populations from northwestern China (NWC) and north China (NC), and the other including northeastern China (NEC), Japan, Korea, MDRY, south China (SC) and southwestern China (SWC). Contrib analyses showed that southwestern China makes the greatest contribution to the total diversity and allelic richness, and is worthy of being given conservation priority.     

Development of Seven Microsatellite Markers Using Next Generation Sequencing for the Conservation on the Korean Population of Dorcus hopei (E. Saunders, 1854) (Coleoptera,Lucanidae)

We developed microsatellite markers for genetic structural analyses of Dorcus hopei, a stag beetle species, using next generation sequencing and polymerase chain reaction (PCR)-based genotyping for regional populations. A total of 407,070,351 base pairs of genomic DNA containing >4000 microsatellite loci except AT repeats were sequenced. From 76 loci selected for primer design, 27 were polymorphic. Of these 27 markers, 10 were tested on three regional populations: two Chinese (Shichuan and Guangxi) and one Korean (Wanju). Three markers were excluded due to inconsistent amplification, genotyping errors, and Hardy-Weinberg equilibrium (HWE). By multi-locus genotyping, the allele number, observed heterozygosity and polymorphism information content of seven microsatellite loci were ranged 2‒10, 0.1333‒1.0000, and 0.1228‒0.8509, respectively. In an analysis on the genetic differentiation among regional populations including one Japanese population and one cross-breeding population, the individual colored bar-plots showed that both Chinese populations were closer to each other than to the Far East Asian populations. In Far East Asian populations, Wanju and Nirasaki populations could not be distinguished from each other because the frequency of genetic contents was very similar in some individuals of two populations. Moreover, the cross-breeding population contained all patterns of genetic contents shown in Chinese, Korean, and Japanese populations, compared with the genetic content frequency of each regional population. As a result, we examined whether the cross-breeding population might be a hybrid population, and might contain a possibility of interbreeding with Chinese populations in parental generations. Therefore, these markers will be useful for analyses of genetic diversity in populations, genetic relationships between regional populations, genetic structure analyses, and origin tests.     

Genetic Characterization of Five Hatchery Populations of the Pacific Abalone (Haliotis discus hannai) Using Microsatellite Markers

The Pacific abalone, Haliotis discus hannai, is a popular food in Eastern Asia. Aquacultural production of this species has increased because of recent resource declines, the growing consumption, and ongoing government-operated stock release programs. Therefore, the genetic characterization of hatchery populations is necessary to maintain the genetic diversity of this species and to develop more effective aquaculture practices. We analyzed the genetic structures of five cultured populations in Korea using six microsatellite markers. The number of alleles per locus ranged from 15 to 64, with an average of 23.5. The mean observed and expected heterozygosities were 0.797 and 0.904, respectively. The inbreeding coefficient F(IS) ranged from 0.054 to 0.184 (mean F(IS) = 0.121 ± 0.056). The genetic differentiation across all populations was low but significant (overall F(ST) = 0.009, P < 0.01). Pairwise multilocus F(ST) tests, estimates of genetic distance, and phylogenetic and principal component analyses did not show a consistent relationship between geographic and genetic distances. These results could reflect extensive aquaculture, the exchange of breeds and eggs between hatcheries and/or genetic drift due to intensive breeding practices. Thus, for optimal resource management, the genetic variation of hatchery stocks should be monitored and inbreeding controlled within the abalone stocks that are being released every year. This genetic information will be useful for the management of both H. discus hannai fisheries and the aquaculture industry.     

Genetic Diversity and Differentiation of the Orange-Spotted Grouper (Epinephelus coioides) Between and Within Cultured Stocks and Wild Populations Inferred from Microsatellite DNA Analysis

In the present study, we employed microsatellite DNA markers to analyze the genetic diversity and differentiation between and within cultured stocks and wild populations of the orange-spotted grouper originating from the South China Sea and Southeast Asia. Compared to wild populations, genetic changes including reduced genetic diversity and significant differentiation have taken place in cultured grouper stocks, as shown by allele richness and heterozygosity studies, pairwise F(st), structure, molecular variance analysis, as well as multidimensional scaling analysis. Although two geographically adjacent orange-spotted grouper populations in China showed negligible genetic divergence, significant population differentiation was observed in wild grouper populations distributed in a wide geographical area from China, through Malaysia to Indonesia. However, the Mantel test rejected the isolation-by-distance model of genetic structure, which indicated the genetic differentiation among the populations could result from the co-effects of various factors, such as historical dispersal, local environment, ocean currents, river flows and island blocks. Our results demonstrated that microsatellite markers could be suitable not only for genetic monitoring cultured stocks but also for revealing the population structuring of wild orange-spotted grouper populations. Meanwhile, our study provided important information for breeding programs, management of cultured stocks and conservation of wild populations of the orange-spotted grouper.     

High Genetic Diversity and Low Differentiation of Michelia coriacea (Magnoliaceae), a Critically Endangered Endemic in Southeast Yunnan, China

Michelia coriacea, a critically endangered tree, has a restricted and fragmented distribution in Southeast Yunnan Province, China. The genetic diversity, genetic structure and gene flow in the three extant populations of this species were detected by 10 inter-simple sequence repeat (ISSR) markers and 11 simple sequence repeat (SSR) markers. Examination of genetic diversity revealed that the species maintained a relatively high level of genetic diversity at the species level (percentage of polymorphic bands) PPB = 96.36% from ISSRs; PPL (percentage of polymorphic loci) = 95.56% from SSRs, despite several fragmental populations. Low levels of genetic differentiation among the populations of M. coriacea were detected by Nei's G(st) = 0.187 for ISSR and Wright's F(st) = 0.090 for SSR markers, which is further confirmed by Bayesian model-based STRUCTURE and PCoA analysis that could not reveal a clear separation between populations, although YKP was differentiated to other two populations by ISSR markers. Meanwhile, AMOVA analysis also indicated that 22.84% and 13.90% of genetic variation existed among populations for ISSRs and SSRs, respectively. The high level of genetic diversity, low genetic differentiation, and the population, structure imply that the fragmented habitat and the isolated population of M. coriacea may be due to recent over-exploitation. Conservation and management of M. coriacea should concentrate on maintaining the high level of genetic variability through both in and ex-situ conservation actions.     

Genetic Diversity of Pinus nigra Arn. Populations in Southern Spain and Northern Morocco Revealed By Inter-Simple Sequence Repeat Profiles

Eight Pinus nigra Arn. populations from Southern Spain and Northern Morocco were examined using inter-simple sequence repeat markers to characterize the genetic variability amongst populations. Pair-wise population genetic distance ranged from 0.031 to 0.283, with a mean of 0.150 between populations. The highest inter-population average distance was between PaCU from Cuenca and YeCA from Cazorla, while the lowest distance was between TaMO from Morocco and MA Sierra Mágina populations. Analysis of molecular variance (AMOVA) and Nei's genetic diversity analyses revealed higher genetic variation within the same population than among different populations. Genetic differentiation (Gst) was 0.233. Cuenca showed the highest Nei's genetic diversity followed by the Moroccan region, Sierra Mágina, and Cazorla region. However, clustering of populations was not in accordance with their geographical locations. Principal component analysis showed the presence of two major groups-Group 1 contained all populations from Cuenca while Group 2 contained populations from Cazorla, Sierra Mágina and Morocco-while Bayesian analysis revealed the presence of three clusters. The low genetic diversity observed in PaCU and YeCA is probably a consequence of inappropriate management since no estimation of genetic variability was performed before the silvicultural treatments. Data indicates that the inter-simple sequence repeat (ISSR) method is sufficiently informative and powerful to assess genetic variability among populations of P. nigra.     

Rapid Development of Microsatellite Markers with 454 Pyrosequencing in a Vulnerable Fish, the Mottled Skate, Raja pulchra

The mottled skate, Raja pulchra, is an economically valuable fish. However, due to a severe population decline, it is listed as a vulnerable species by the International Union for Conservation of Nature. To analyze its genetic structure and diversity, microsatellite markers were developed using 454 pyrosequencing. A total of 17,033 reads containing dinucleotide microsatellite repeat units (mean, 487 base pairs) were identified from 453,549 reads. Among 32 loci containing more than nine repeat units, 20 primer sets (62%) produced strong PCR products, of which 14 were polymorphic. In an analysis of 60 individuals from two R. pulchra populations, the number of alleles per locus ranged from 1-10, and the mean allelic richness was 4.7. No linkage disequilibrium was found between any pair of loci, indicating that the markers were independent. The Hardy-Weinberg equilibrium test showed significant deviation in two of the 28 single-loci after sequential Bonferroni's correction. Using 11 primer sets, cross-species amplification was demonstrated in nine related species from four families within two classes. Among the 11 loci amplified from three other Rajidae family species; three loci were polymorphic. A monomorphic locus was amplified in all three Rajidae family species and the Dasyatidae family. Two Rajidae polymorphic loci amplified monomorphic target DNAs in four species belonging to the Carcharhiniformes class, and another was polymorphic in two Carcharhiniformes species.     

Isolation and Characterization of Microsatellite Loci in Pistacia weinmannifolia (Anacardiaceae)

Fourteen polymorphic microsatellite loci were isolated from the genomic DNA of Pistacia weinmannifolia, using the Fast Isolation by AFLP of Sequences Containing repeats (FIASCO) method, and screened on 12 individuals from each of two wild populations. The 14 polymorphic loci had an average of 4.1 alleles per locus varying from 1 to 9. The observed (H(o)) and expected (H(e)) heterozygosities across the two populations ranged from 0.000 to 0.933 and from 0.000 to 0.906, respectively. Tests for departure from Hardy-Weinberg equilibrium (HWE) and genotypic linkage disequilibrium (LD) were conducted for each of the two populations separately. It was found that no locus significantly deviated from HWE proportions and no significant LD was detected between loci (p < 0.001). In the test of cross-species utility, we successfully amplified nine (64.2%) of 14 loci in P. chinensis and four (28.6%) in P. mexicana. The relatively high level of polymorphism for these markers will facilitate further studies of gene flow, population structure and evolutionary history of P. weinmannifolia and its congeners.     

Isolation and Characterisation of 11 Polymorphic Microsatellite Markers in Papaver rhoeas L. (Corn Poppy), a Major Annual Plant Species from Cultivated Areas

Papaver rhoeas, an annual plant species in the Papaveraceae family, is part of the biodiversity of agricultural ecosystems and also a noxious agronomic weed. We developed microsatellite markers to study the genetic diversity of P. rhoeas, using an enriched microsatellite library coupled with 454 next-generation sequencing. A total of 13,825 sequences were obtained that yielded 1795 microsatellite loci. After discarding loci with less than six repeats of the microsatellite motif, automated primer design was successful for 598 loci. We tested 74 of these loci for amplification with a total of 97 primer pairs. Thirty loci passed our tests and were subsequently tested for polymorphism using 384 P. rhoeas plants originating from 12 populations from France. Of the 30 loci, 11 showed reliable polymorphism not affected by the presence of null alleles. The number of alleles and the expected heterozygosity ranged from 3 to 7.4 and from 0.27 to 0.73, respectively. A low but significant genetic differentiation among populations was observed (F_ST = 0.04; p < 0.001). The 11 validated polymorphic microsatellite markers developed in this work will be useful in studies of genetic diversity and population structure of P. rhoeas, assisting in designing management strategies for the control or the conservation of this species.     

Eleven Novel Polymorphic Microsatellite Loci for Oval Squid Sepioteuthis Lessoniana (Shiro-Ika Type)

The oval squid Sepioteuthis lessoniana is one of the most economically important squid species in Japan; however, its population structure is poorly understood due to the lack of hypervariable markers. Such information is critical for managing sustainable fisheries, as well as for ensuring the existence of wild S. lessoniana stocks. Eleven candidate microsatellite loci were isolated from a small insert genomic DNA library. Polymorphisms in these 11 loci were screened in 24 wild individuals. The number of alleles per locus was found to range from 5 to 19 alleles, and the observed heterozygosity ranged from 0.292 to 0.958. No evidence for linkage disequilibrium was detected among all the loci. The genotypic proportions conformed to Hardy-Weinberg equilibrium, except at one locus. In conclusion, these polymorphic microsatellite loci may be used to develop a genetic framework to manage S. lessoniana in the future.     

Isolation and Characterization of Microsatellite Loci in the Chinese Cobra Naja atra (Elapidae)

We characterize thirteen polymorphic microsatellite loci isolated from Naja atra genomic libraries, which were enriched for AC-motif microsatellites. The thirteen loci were screened on a group of 48 individuals from two populations, one in Yong'an and the other in Ganzhou. These markers revealed a relatively high degree of genetic diversity (4-12 alleles per locus) and heterozygosity (Ho ranged from 0.213-0.854 and He ranged from 0.301-0.838). Tests for departure from Hardy-Weinberg equilibrium and for linkage disequilibrium were conducted for each of the two populations separately. After sequential Bonferroni correction, none of the 13 loci showed significant departures from Hardy-Weinberg equilibrium. Hierarchical analysis of molecular variance indicated that a small but significant (P < 0.001) proportion (16.0%) of the total variation in the microsatellite DNA data were attributable to differences among populations, indicating geographical structuring and restricted gene flow. It could be attributable to the Wuyi mountains in the area having a sufficiently isolating effect to significantly reduce gene flow. Our microsatellite data also showed a low N(m) (1.31) value in the two populations from mainland China. Thus, the Yong'an and Ganzhou populations could be treated as distinct evolutionarily significant units (ESUs). The high level of polymorphism revealed by these microsatellite markers will be useful for the study of gene flow, population structure and evolutionary history of N. atra.     

Isolation and Characterization of Microsatellite Markers for Passiflora contracta

Passiflora contracta Vitta (Passifloraceae) is an endemic species of the Atlantic Rainforest, one of the most species-rich ecoregions in the world, although extremely endangered. We have developed an enriched microsatellite library in order to fine-scale studies of the genetic structure of P. contracta. Twelve pairs of microsatellite primers were designed, and seven loci were successfully amplified and characterized by genotyping two wild populations of P. contracta. All seven loci were polymorphic, with an average number of alleles found being 4.8 and 5 per population. The cross-species transferability was tested using sister species Passiflora ovalis Vell. Ex Roemer. The development of these markers will contribute to the studies of population genetics in P. contracta as well as future studies concerning diversity patterns in the Atlantic Rainforest, and may also help to establish strategies for the conservation of this species.     

Loss of Genetic Diversity in the Cultured Stocks of the Large Yellow Croaker, Larimichthys crocea, Revealed by Microsatellites

The large yellow croaker (Larimichthys crocea) is the most important mariculture fish species in China and the wild stocks of this croaker have collapsed in the past decades due to high fishing pressure and habitat degradation. Due to a lack of wild croaker samples, however, studies concerning the genetic changes of the cultured croaker stocks compared to their wild counterparts were never conducted. Here, we collected three wild populations in the northern and central East China Sea during fisheries survey and investigated the differences in terms of genetic diversity and differentiation between and within cultured stocks and wild populations. Our results demonstrated that the cultured croaker had significantly reduced genetic diversity in contrast to the wild populations, and also presented statistically significant differentiation from the wild, indicating that enhancement of the current wild stock should be conducted with caution. These changes may be caused by founder effects, artificial selection and random genetic drift. With a relatively high level of genetic diversity, the wild populations showed important value for improving the ongoing breeding program of this croaker. Further, we detected no differentiation among the wild populations, suggesting that the wild croaker in the northern and central East China Sea should be considered as one unit for management and conservation.     

Genetic Diversity and Population Structure of Siberian apricot (Prunus sibirica L.) in China

The genetic diversity and population genetic structure of 252 accessions from 21 Prunus sibirica L. populations were investigated using 10 ISSR, SSR, and SRAP markers. The results suggest that the entire population has a relatively high level of genetic diversity, with populations HR and MY showing very high diversity. A low level of inter-population genetic differentiation and a high level of intra-population genetic differentiation was found, which is supported by a moderate level of gene flow, and largely attributable to the cross-pollination and self-incompatibility reproductive system. A STRUCTURE (model-based program) analysis revealed that the 21 populations can be divided into two main groups, mainly based on geographic differences and genetic exchanges. The entire wild Siberia apricot population in China could be divided into two subgroups, including 107 accessions in subgroup (SG) 1 and 147 accessions in SG 2. A Mantel test revealed a significant positive correlation between genetic and geographic distance matrices, and there was a very significant positive correlation among three marker datasets. Overall, we recommend a combination of conservation measures, with ex situ and in situ conservation that includes the construction of a core germplasm repository and the implement of in situ conservation for populations HR, MY, and ZY.     

Conservation Genetics of an Endangered Lady’s Slipper Orchid: Cypripedium japonicum in China

Knowledge about the population genetic variation of the endangered orchid, Cypripedium japonicum, is conducive to the development of conservation strategies. Here, we examined the levels and partitioning of inter-simple sequence repeat (ISSR) diversity (109 loci) in five populations of this orchid to gain insight into its genetic variation and population structure in Eastern and Central China. It harbored considerably lower levels of genetic diversity both at the population (percentage of polymorphic loci (PPL) = 11.19%, Nei’s gene diversity (H) = 0.0416 and Shannon’s information index (I) = 0.0613) and species level (PPL = 38.53%, H = 0.1273 and I = 0.1928) and a significantly higher degree of differentiation among populations (the proportion of the total variance among populations (Φ_pt) = 0.698) than those typical of ISSR-based studies in other orchid species. Furthermore, the Nei’s genetic distances between populations were independent of the corresponding geographical distances. Two main clusters are shown in an arithmetic average (UPGMA) dendrogram, which is in agreement with the results of principal coordinate analysis (PCoA) analysis and the STRUCTURE program. In addition, individuals within a population were more similar to each other than to those in other populations. Based on the genetic data and our field survey, the development of conservation management for this threatened orchid should include habitat protection, artificial gene flow and ex situ measures.