Isolation and characterization of microsatellite markers for Viola mirabilis (Violaceae) using a next‐generation sequencing platform

We isolated and characterized microsatellite loci in Viola mirabilis (Violaceae), an endangered species from South Korea. Twenty‐three polymorphic microsatellite loci were developed and tested in Korean, Chinese and Japanese populations. The number of alleles per locus varied from two to eight. The observed and expected heterozygosities within the three populations were 0.000–0.625 and 0.469–0.695, respectively. A total of six loci in the Korean population, one locus in the Chinese population and seven loci in the Japanese population deviated from Hardy–Weinberg equilibrium. We expect that these newly developed microsatellite markers will contribute to understanding the phylogeography and population genetics of V. mirabilis, which will aid in developing conservation strategies for this species.     

Eleven new microsatellite markers in jack mackerel (Trachurus japonicus) derived from an enriched genomic library

Jack mackerel (Trachurus japonicus, Carangidae) are a commercially important fisheries resource in Korea. To understand patterns of genetic variation for conservation and management efforts, we developed microsatellite DNA markers fromT. japonicus. We report the isolation and characterization of eleven microsatellite loci isolated using an enrichment method based on magnetic/biotin capture of microsatellite sequences from a size-selected genomic library. To characterize each locus, 50 individuals from a naturalT. japonicus population in southern Korea were genotyped. All loci except one, KTJ38, were polymorphic with an average of 14 alleles per locus (range 6–23). The mean observed and expected heterozygosities were 0.70 (range 0.46–0.92) and 0.81 (range 0.49–1.00), respectively. Significant deviation from Hardy-Weinberg equilibrium was observed at three loci, KTj3, KTJ20 and KTJ28. Such high variability indicates that these microsatellites are useful markers for high-resolution analysis for population gemetic studies.     

Isolation and characterization of 12 microsatellite loci from Korean water deer (Hydropotes inermis argyropus) for population structure analysis in South Korea

The main goals of this study were to isolate microsatellites markers of Korean water deer (Hydropotes inermis argyropus) and understand the genetic status of the species in South Korea. Twelve new microsatellite loci were isolated and characterized to establish basic population genetic parameters for 45 H. i. argyropus specimens in South Korea. There were no significant regional or genetic structure differences between the mid-eastern and southwestern populations in South Korea according to the population genetic analyses. The number of alleles per locus ranged from two to 13 with an average of 6.08. Mean expected (H _E) and observed heterozygosity (H _O) were 0.622 and 0.533, respectively. Microsatellite variability was also not significant between the two regions (F _ST=0.012). These new markers should facilitate the future population genetics studies of Korean water deer and other closely related species.     

Genetic diversity and genetic structure of the endangered Manchurian trout, <Emphasis Type="Italic">Brachymystax lenok tsinlingensis</Emphasis>, at its southern range margin: conservation implications for future restoration

The Manchurian trout, Brachymystax lenok tsinlingensis (family: Salmonidae), is a cold freshwater fish endemic to Northeast Asia. South Korean populations of this species, which comprise its southern range limit, have recently decreased markedly in size and are now becoming critically endangered. We assessed the current population status of this species in South Korea by estimating the levels of genetic diversity and genetic structure of five natural and four restored populations using mitochondrial DNA (mtDNA) control region sequences and eight nuclear microsatellite loci. Levels of within-population genetic diversity were low, suggesting that past effective population sizes (N _e) have been small. Each population had one or a maximum of two mtDNA haplotypes. Microsatellite allelic richness (AR) was significantly higher for natural populations (mean AR?=?3.51; 95% confidence interval, 3.00–4.03) than for restored populations (mean AR?=?2.61; 2.38–2.98). South Korean populations were significantly genetically isolated from one another, with private mtDNA haplotypes and microsatellite alleles, suggesting that limited gene flow has been occurring among populations. A mtDNA phylogeny revealed that South Korean lineages were more closely related to those of China than to those of North Korea and Russia. Overall, we suggest that future restoration efforts aimed at South Korean populations should consider the genetic characteristics reported here, which should help to fulfil effective conservation strategies for this highly cherished species. Our results will inform other conservation efforts, including assisted migration of freshwater fish populations at the equatorial end of the geographical range limit of the species.     

Isolation and characterization of polymorphic microsatellite markers for the black rockfishSebastes inermis

Black rockfish (Sebastes inermis, Scorpaenidae) is a commercially important fisheries resource in Korea. However, no genetic diversity information is available for wild or released hatchery populations. To study the effects of stock enhancement programs and conservation biology, we developed microsatellite DNA markers fromS. inermis. We report the isolation and characterization of seven microsatellite loci, designated KSi221B, KSi239A, KSi25A, KSi252B, KSi193A, KSi259B, and KSi234, isolated using an enrichment method based on magnetic/biotin capture of microsatellite sequences from a size-selected genomic library. To characterize each locus, 30 individuals from a naturalS. inermis population in Yeosu, Korea, were genotyped. All loci except one, KSi193A, were polymorphic with an average of 9.5 alleles per locus (range 2–20). The mean observed and expected heterozygosities were 0.71 (range 0.43–1.00) and 0.76 (range 0.41–0.90), respectively. Significant deviation from Hardy-Weinberg equilibrium was observed at one locus, KSi221B. Such high variability indicates that these microsatellites should provide useful markers for studies of kinship and population genetics.     

Development and characterization of microsatellite markers for Echinopsis rhodotricha and cross-amplification in other species of Cactaceae

Located in central South America, the Chaco is a large subtropical dry forest characterized by plants with xeromorphic features, including numerous cacti. In the Brazilian Chaco at the northeasternmost part of the Chaquenian region, Echinopsis rhodotricha (Cactaceae, Trichocereeae) is one of the species threatened by intense deforestation caused by expanding pastureland. This study characterizes the microsatellite loci isolated from E. rhodotricha and cross-amplification in thirteen other cactus species. Twelve microsatellite loci were developed from an enriched genomic library. Eight of these were polymorphic and characterized in 48 individuals from three E. rhodotricha populations. The loci showed a mean of 3.2 alleles per locus and overall levels of expected and observed heterozygosities ranging from 0.00 to 0.83 and 0.00 to 0.66, respectively. Five loci showed significant departures from the Hardy–Weinberg equilibrium and also exhibited signs of null alleles. Cross-amplification in other Cactaceae species was successful, ranging from one (Ferocactus latispinus, Cacteae and Harrisia adscendens, Trichocereeae) to twelve loci (Echinopsis calochlora, Trichocereeae). The development of these microsatellite markers will contribute to investigations of population structure, genetic diversity, and gene flow in E. rhodotricha populations, as well as in other cactus species, providing information useful for the creation and delimitation of conservation areas in the Brazilian Chaco region.     

Isolation and characterization of polymorphic microsatellite markers for the endangered Korean freshwater fish Hemibarbus mylodon

Korean barbel, Hemibarbus mylodon, is an endangered freshwater species endemic to Korea. In order to undertake a conservation program for this species in Korea, it is essential to evaluate its genetic diversity and population structure. For this purpose, we developed 10 polymorphic microsatellite markers and examined their allelic variation using a total of 60 wild individuals collected from three different localities. The number of alleles per locus ranged from three to 26. The expected and observed heterozygosities per locus ranged from 0.19 to 0.94 and from 0.20 to 0.98, respectively. Significant departures from Hardy–Weinberg equilibrium expectations occurred in four loci. These markers will be useful for the assessment of genetic diversity for this species.     

Development and validation of microsatellite markers for the tiny dragonfly, <Emphasis Type="Italic">Nannophya pygmaea</Emphasis> (Odonata: Libellulidae), which is endangered in South Korea

The tiny dragonfly, Nannophya pygmaea (Odonata: Libellulidae), is listed as a second-degree endangered wild animal in South Korea. The application of molecular markers to assess genetic diversity and population relationships can provide information necessary to establish an effective conservation strategy. In this study, we developed 12 microsatellite markers specific to N. pygmaea using the NextSeq 500 platform. Forty individuals of N. pygmaea collected from three currently known localities in South Korea were genotyped to validate these markers and to preliminarily assess population genetic characteristics. The observed number of alleles, observed heterozygosity, and expected heterozygosity at a locus ranged from 2 to 9, 0.421–1.0, and 0.508–0.766 in a population with the largest sample size (20 individuals), respectively, thereby validating the suitability of the markers for population analysis. Five of 12 loci showed significant deviation from the Hardy–Weinberg equilibrium in the population. Our preliminary data indicate an absence of inbreeding in all populations and an absence of obvious genetic difference. The microsatellite markers developed in this study will be useful for studying the population genetics of N. pygmaea collected from other regions worldwide, including additional sites in South Korea.     

Isolation and Characterization of Microsatellite Markers for <Emphasis Type="Italic">Viola websteri</Emphasis> (Violaceae) and Cross-Species Amplification within the Genus <Emphasis Type="Italic">Viola</Emphasis>

We isolated and characterized microsatellite loci in Viola websteri (Violaceae), an endangered species from Korea and endemic to Northeast Asia. A total of 27 microsatellite loci were developed and tested in Korean and Chinese populations. The number of alleles per locus varied from two to eight. The observed and expected heterozygosities within two populations were 0.000 to 1.000 and 0.080 to 0.816, respectively. Korean and Chinese populations were clearly distinguished by the private alleles from 16 loci. A total of 21 loci out of the 27 developed loci were successfully cross-amplified in 39 other Viola species. We believe that these microsatellite loci will be useful for future studies on genetic diversity and population structure of V. websteri, as well as other Viola species.     

Isolation and characterization of eight microsatellite loci from <Emphasis Type="Italic">Lycorma delicatula</Emphasis> (White) (Hemiptera: Fulgoridae) for population genetic analysis in Korea

Lycorma delicatula (White) is native to China but is becoming an important insect pest in Korea. Polymorphic DNA markers like microsatellites are widely used for characterizing dispersal patterns and capacity of invasive insect pests which can contribute to designing effective management of the species. To facilitate such population genetic studies of L. delicatula in Korea, we isolated and characterized eight microsatellite loci for L. delicatula using a hybridization-biotin enrichment method. We further used these novel microsatellite loci to determine population genetic parameters for 33 L. delicatula specimens collected from Cheonan, South Korea where outbreaks of this species were first reported in Korea. The number of alleles per locus ranged from three to ten, with an average of 6.25. The mean expected (H _E) and observed heterozygosities (H _O) were 0.575 and 0.626, respectively. The eight loci showed no deviation from Hardy–Weinberg equilibrium according to the adjusted significance threshold (P = 0.00625), and there was no linkage disequilibrium between each pair of these eight markers. Bayesian cluster analysis using the program structure revealed no evidence of genetic structuring in L. delicatula samples from Cheonan. These new microsatellite markers will be widely applicable to future ecological genetic studies of L. delicatula, including assessment of the level of gene flow and genetic connectivity among populations that are necessary for effective management and monitoring of the species.     

Isolation and characterization of 19 novel microsatellite loci in Rhododendron aureum and Rhododendron brachycarpum (Ericaceae)

Nineteen polymorphic microsatellite loci are described for Rhododendron aureum Georgi (Ericaceae), an endangered species in Korea, and the closely related Rhododendron brachycarpum. The sequences containing repeat motifs were identified using low-depth next generation sequencing and 148 microsatellite loci were examined for amplification success and the detection of polymorphisms. All 19 loci were polymorphic and the number of alleles for these markers varied from 4 to 25, with an average of 15 alleles per locus. Three R. aureum loci showed departure from Hardy–Weinberg equilibrium (HWE) and one R. brachycarpum locus deviated significantly from HWE. These microsatellite loci will be useful for investigating the genetic diversity and population structure of both species.     

Development and characterization of 24 chloroplast microsatellite markers for two species of <Emphasis Type="Italic">Eranthis</Emphasis> (Ranunculaceae)

Chloroplast microsatellites for two Korean endemic species, Eranthis byunsanensis and E. pungdoensis (Ranunculaceae), were isolated to address the questions of their distributional patterns and evolutionary relationships, using next-generation sequencing. Twenty-four polymorphic chloroplast microsatellite markers for these two species were developed, and then characterized in 65 individuals (55 individuals of E. byunsanensis and 10 individuals of E. pungdoensis). The number of alleles per locus ranged from 2 to 9; the average number of alleles across all the loci scored 4.792. The unbiased diversity per locus ranged from 0.089 to 0.880; the unbiased diversity averaged over all the loci was 0.646. The developed markers were successfully amplified for three congeneric species, E. stellata, E. pinnatifida, and E. longistipitata. The markers developed in this study can provide a valuable and important tool for understanding genetic variations, population structures, evolutionary histories and phylogeography of E. byunsanensis, E. pungdoensis, and related species.     

Development of microsatellite markers to genetically differentiate populations of Octopus minor from Korea and China

Of the more than 300 octopus species, Octopus minor is one of the most popular and economically important species in Eastern Asia, including Korea, along with O. vulgaris, O. ocellatus, and O. aegina. We developed 19 microsatellite markers from Octopus minor and eight polymorphic markers were developed to analyze the genetic diversity and relationships among four octopus populations from Korea and three from China. The number of alleles per locus varied from 10 to 49, and allelic richness per locus ranged from 2 to 16.4 across all populations. The average allele number among the populations was 11.1, with a minimum of 8.3 and a maximum of 13.6. The mean allelic richness was 8.7 in all populations. The Hardy-Weinberg equilibrium (HWE) test revealed significant deviation in 19 of the 56 single-locus sites, and null alleles were presumed in five of eight loci. The pairwise F ( ST ) values between populations from Korea and China differed significantly in all pairwise comparisons. The genetic distances between the China and Korea samples ranged from 0.161 to 0.454. The genetic distances among the populations from Korea ranged from 0.033 to 0.090, with an average of 0.062; those among populations from China ranged from 0.191 to 0.316, with an average of 0.254. The populations from Korea and China formed clearly separated into clusters via an unweighted pair group method with arithmetic mean dendrogram. Furthermore, a population from muddy flats on the western coast of the Korean Peninsula and one from a rocky area on Jeju Island formed clearly separated subclusters. An assignment test based on the allele distribution discriminated between the Korean and Chinese origins with 96.9 % accuracy.     

Isolation and characterization of 18 polymorphic microsatellite markers for the “Female Ginseng” Angelica sinensis (Apiaceae) and cross-species amplification

Angelica sinensis (Apiaceae) is an endangered alpine herb that is widely used as a medicinal plant in traditional Chinese medicine (TCM). Wild populations of A. sinensis have become quite rare in China. Thus, population genetics studies of this species are urgently needed for its effective conservation and sustainable use. However, to date, no microsatellite loci have been isolated in A. sinensis. To address this issue, we isolated 18 polymorphic loci and genotyped 120 individuals collected from 6 populations. The number of alleles per locus ranged from 1.2 to 5.5, and the average was 2.4. The observed and expected heterozygosity per locus for a population varied, respectively, from 0.000 to 0.983 (averaged at 0.198) and from 0.066 to 0.661 (averaged at 0.333). Deviation from the Hardy–Weinberg equilibrium (p < 0.01) was observed for 4 to 14 loci in various populations. These microsatellite markers were cross-amplified in 10 species affinis, and 7 loci were successfully amplified in all species. These microsatellite markers are useful for genetic studies, the conservation management of A. sinensis, and identification of A. sinensis.     

Polymorphic microsatellite loci for population studies of the Japanese scallop,Patinopecten yessoensis

Fifteen microsatellite loci were developed to examine the population structure of Japanese scallop, Patinopecten yessoensis. These markers were tested in samples from two geographically distant populations: the Sakhalin Island (Russia) and Dalian City (China). The mean numbers of alleles, observed and expected heterozygosities between the two populations were summarized by locus, but none of them show significant divergence between the two populations. Most loci conformed to Hardy–Weinberg expectation with the exception of loci HLJX‐06, HLJX‐12, HLJX‐13 and HLJX‐28, which had heterozygote deficits.     

Microsatellite population genetics of the emerald ash borer (Agrilus planipennis Fairmaire): comparisons between Asian and North American populations

The emerald ash borer (EAB) (Agrilus planipennis Fairmaire) (Coleoptera; Buprestidae), is an invasive wood-boring beetle native to northeast Asia. This species was first detected in Michigan USA in 2002, and is a significant threat to native and ornamental ash tree species (Fraxinus spp.) throughout North America. We characterized seven polymorphic microsatellite markers for EAB and used these to investigate EAB population structure in the early invasive populations within North America and in comparison with Asia. We found 2–9 alleles per microsatellite locus, no evidence of linkage disequilibrium, and no association with known coding sequences, suggesting that these markers are suitable for population genetic analysis. Microsatellite population genetic structure was examined in 48 EAB populations sampled between 2003 and 2008 from five regions, three in the introduced range, Michigan (US) and Ontario and Quebec (Canada) and two Asian regions, China and South Korea, where EAB is native. We found significant genetic variation geographically but not temporally in EAB populations. Bayesian clustering analyses of individual microsatellite genotypes showed strong clustering among multiple North American populations and populations in both China and South Korea. Finally, allelic richness and expected heterozygosity were higher in the native range of EAB, but there was no difference in observed heterozygosity, suggesting a significant loss of alleles upon introduction but no significant change in the distribution of alleles within and among individuals.     

Microsatellite DNA Analysis Revealed a Drastic Genetic Change of Plasmodium vivax Population in the Republic of Korea During 2002 and 2003

Background

Vivax malaria was successfully eliminated in the Republic of Korea (South Korea) in the late 1970s, but it was found to have re-emerged from 1993. In order to control malaria and evaluate the effectiveness of malaria controls, it is important to develop a spatiotemporal understanding of the genetic structure of the parasite population. Here, we estimated the population structure and temporal dynamics of the transmission of Plasmodium vivax in South Korea by analyzing microsatellite DNA markers of the parasite.

Methodology/Principal Findings

We analyzed 14 microsatellite DNA loci of the P. vivax genome from 163 South Korean isolates collected from 1994 to 2008. Allelic data were used to analyze linkage disequilibrium (LD), genetic differentiation and population structure, in order to make a detailed estimate of temporal change in the parasite population. The LD analysis showed a gradual decrease in LD levels, while the levels of genetic differentiation between successive years and analysis of the population structure based on the Bayesian approach suggested that a drastic genetic change occurred in the South Korean population during 2002 and 2003.

Conclusions/Significance

Although relapse and asymptomatic parasite carriage might influence the population structure to some extent, our results suggested the continual introduction of P. vivax into South Korea through other parasite population sources. One possible source, particularly during 2002 and 2003, is North Korea. Molecular epidemiology using microsatellite DNA of the P. vivax population is effective for assessing the population structure and temporal dynamics of parasite transmission; information that can assist in the elimination of vivax malaria in endemic areas.     

Characterization of 11 novel polymorphic microsatellite loci in the threatened Korean loach,Iksookimia koreensis,isolated using a next-generation sequencing method

To investigate the genetic diversity and population structure of Iksookimia koreensis, we characterized 11 novel polymorphic microsatellite markers developed using next-generation sequencing. The number of alleles per locus ranged from 4 to 10 (average = 6.26). Observed and expected heterozygosities ranged from 0.333 to 0.866 and from 0.375 to 0.866, respectively. No loci showed significant deviation from Hardy–Weinberg equilibrium (HWE). These loci were also used successfully to study the genetic diversity of a closely related species, Iksookimia longicorpa. Four of the 11 loci amplified in the two species showed different allele frequency and distribution, indicating deep genetic divergence between I. koreensis and I. longicorpa. The newly developed microsatellite markers reported here will provide a useful tool for examining gene flow, population genetic structure, and genetic diversity of these species.     

Ten polymorphic microsatellites from freshwater pearl mussel,Hyriopsis cumingii

Freshwater pearl mussel, Hyriopsis cumingii, is the most economically important mussel species in China. Due to overexploitation and changes of water quality, H. cumingii is facing serious population decline and local extinction. We have isolated and characterized 10 polymorphic microsatellite loci from a genomic DNA library enriched for CA and AG repeats with the aim of developing a set of codominant DNA markers for analysing genetic diversity and population structure of this species. The average allele number of the 10 markers was 7.8 per locus, ranging from two to 16 in 24 unrelated individuals. Nine of 10 markers conformed to Hardy–Weinberg equilibrium and inherited independently, suggesting these microsatellites could be useful for studying population genetics, designing conservation strategies and developing breeding programs.