Microsatellite DNA analysis reveals lower than expected genetic diversity in the threatened leopard cat (<Emphasis Type="Italic">Prionailurus bengalensis</Emphasis>) in South Korea

To optimize conservation efforts, it is necessary to determine the risk of extinction by collecting reliable population information for a given species. We developed eight novel, polymorphic microsatellite markers and used these markers in conjunction with twelve existing markers to measure genetic diversity of South Korean populations of leopard cat (Prionailurus bengalensis), a species for which population size and habitat area data are unknown in the country, to assess its conservation status. The average number of alleles and the observed heterozygosity of the species were 3.8 and 0.41, respectively, and microsatellite diversity was lower than the average genetic diversity of 57 populations of 12 other felid species, and lower than that of other mammal populations occurring in South Korea, including the raccoon dog (Nyctereutes procyonoides), water deer (Hydropotes inermis), and endangered long-tailed goral (Naemorhedus caudatus). Furthermore, analysis of genetic structure in the national leopard cat population showed no clear genetic differentiation, suggesting that it is not necessary to divide the South Korean leopard cat population into multiple management units for the purposes of conservation. These results indicate that the genetic diversity of the leopard cat in South Korea is unexpectedly low, and that the risk of local extinction is, as a result, substantial. Thus, it is necessary to begin appropriate conservation efforts at a national level to conserve the leopard cat population in South Korea.     

Isolation and characterization of species-specific microsatellite markers for blue and black wildebeest (<Emphasis Type="BoldItalic">Connochaetes taurinus</Emphasis> and <Emphasis Type="BoldItalic">C. gnou</Emphasis>)

The blue wildebeest (Connochaetes taurinus) is distributed throughout southern and east Africa while the black wildebeest (Connochaetes gnou) is endemic to South Africa and was driven to near extinction in the early 1900s due to hunting pressure and disease outbreaks. Extensive translocation of both species throughout South Africa is threatening the genetic integrity of blue and black wildebeest. To effectively manage these species, genetic tools that can be used to detect hybrid individuals, identify genetically unique subpopulations and determine the levels of genetic diversity are required. In this study, 11 microsatellite markers were developed for wildebeest through next-generation sequencing. The microsatellite loci displayed 2.00–4.14 alleles, unbiased heterozygosity values ranged from 0.32 to 0.60 and observed heterozygosity values ranged from 0.26 to 0.52. The comparatively high level of polymorphism observed in the microsatellite markers indicates that these markers can contribute significantly to our knowledge of population genetic structure, relatedness, genetic diversity and hybridization in these species.     

Isolation and characterization of microsatellite loci from <Emphasis Type="Italic">Spodoptera exigua</Emphasis> (Lepidoptera: Noctuidae)

Ten polymorphic microsatellite loci were isolated and characterized from Spodoptera exigua (Hübner), which is a worldwide economic pest on various crops. The isolated loci were polymorphic, with 3–15 alleles in 40 individuals from four regional populations in Korea. The analyses revealed that 40 individuals had different multilocus genotypes, with the expected heterozygosity ranging from 0.133 to 0.899. Eight of the ten loci did not deviate significantly from Hardy-Weinberg equilibrium. The isolated markers will facilitate population genetic studies of S. exigua.     

Playing hide-and-seek with the tiny dragonfly: DNA barcoding discriminates multiple lineages of <Emphasis Type="Italic">Nannophya pygmaea</Emphasis> in Asia

We examined the utility of DNA barcode data for assessing genetic diversity of the tiny dragonfly Nannophya pygmaea Rambur in Asia. Data analyses inferred from the barcode region of cytochrome oxidase subunit I (COI) were performed with Malaysian N. pygmaea, along with the existing COI haplotypes distributed in Asia. We applied four species delimitation analyses [automatic barcode gap discovery (ABGD), generalised mixed yule coalescent (GMYC), poisson tree processes maximum likelihood (PTP_ML) and poisson tree processes simple heuristic solutions (PTP_sh)] to investigate potential lineages in this geographically widespread species. Based on our dataset, we provisionally recognize four distinct lineages or operational taxonomic units of N. pygmaea, which were represented by the taxa from Japan/Korea, China/Laos/Taiwan, Malaysia and Vietnam, respectively. Phylogenetic analyses showed two well-supported assemblages of N. pygmaea: one restricted to the taxa from Malaysia and Vietnam; and the other covering all populations further north (i.e., China, Japan, Korea, Laos and Taiwan). An extraordinarily high degree of genetic distance (up to >12 %) was detected between these two assemblages—suggesting they represent two separate species.     

Development and characterization of twelve microsatellite markers for <Emphasis Type="Italic">Porphyra linearis</Emphasis> Greville

The genus Porphyra (and its sister genus Pyropia) contains important red algal species that are cultivated and/or harvested for human consumption, sustaining a billion-dollar aquaculture industry. A vast amount of research has been focused on species of this genus, including studies on genetics and genomics among other areas. Twelve novel microsatellite markers were developed here for Porphyra linearis. Markers were characterized using 32 individuals collected from four natural populations of P. linearis with total heterozygosity varying from 0.098 to 0.916. The number of alleles per locus ranged from 2 to 18. All markers showed cross amplification with Porphyra umbilicalis and/or Porphyra dioica. These polymorphic microsatellite markers are useful for investigating population genetic diversity and differentiation in P. linearis and may become useful for other genetic research on the reproductive biology of this important species.     

Development of 13 microsatellite markers for <Emphasis Type="Italic">Castanopsis tribuloides</Emphasis> (Fagaceae) using next-generation sequencing

Catanopsis tribuloides is a climax tree species commonly distributed in evergreen forests and has been used to restore degraded areas in northern Thailand. To aid in study of genetic diversity of the species, microsatellite markers, which are specific to C. tribuloides, were developed using whole genome sequencing by next-generation sequencing technology. The primers for microsatellite were developed and screened for optimal annealing temperature by PCR assay. The loci primers specific with C. tribuloides, 13 polymorphic microsatellite primers were successfully developed. The results from genetic information analyzing showed the number of alleles presented were between 2 and 24. Accordingly, the expected and observed heterozygosity obtained were between 0.298 and 0.920 and 0.364 to 1.000, respectively. Null allele frequency was presented 0.000–0.199. Genetic information was generated 10 loci primers significantly deviated from Hardy–Weinberg Equilibrium. All 13 primer pairs of loci were not significant with linkage disequilibrium. A set of microsatellite markers in this study could be applied to gene flow, genetic structure and population genetic studies in the future.     

Development and Characterization of Microsatellite Markers for Genetic Analysis of the Swimming Crab, <Emphasis Type="Italic">Portunus trituberculatus</Emphasis>

This study isolated and characterized 11 novel microsatellite markers for the commercially important swimming crab species, Portunus trituberculatus. Genetic diversity and population structure of two populations of P. trituberculatus in the East China Sea were assessed using these loci. The microsatellite markers produced 242 alleles, varying from 17 to 26 alleles per locus. In all the samples, the range of heterozygosity was 0.6324–0.9403 (observed) and 0.8998–0.9547 (expected). An F-statistic analysis revealed low genetic differentiation between the populations (mean F _ST = 0.0197), with 98% of the variation resulting from the within-population component. In addition, cross-amplification was tested in two other portunid species, and we found that many loci yielded useful information. The high degree of polymorphism exhibited by the 11 microsatellites suggests that these markers will be useful for both aquaculture and studies of natural populations of the genus.     

Development of ten microsatellite markers from the keystone mistletoe <Emphasis Type="Italic">Tristerix corymbosus</Emphasis> (Loranthaceae) using 454 next generation sequencing and their applicability to population genetic structure studies

Tristerix corymbosus (Loranthaceae) is a keystone mistletoe from the South American temperate rainforests. As most mistletoes, T. corymbosus relies on biotic pollination and seed dispersal, which may cause population structure. For a better understanding of its ecology, we isolated and characterized ten polymorphic microsatellite loci for this species. We used 454 Next Generation Sequencing to build a microsatellite library from a high quality DNA sample. We tested 90 sequences from which we obtained ten polymorphic markers. In order to assess their variability, the novel markers were tested in 48 individuals from three locations of the Valdivian Coastal Reserve in Chile. We also estimated genetic differences between pairs of populations using the F_ST statistic. The mean number of alleles per locus in the 48 individuals studied was 7.1 (2–17 alleles per locus). The observed and expected heterozygosity ranged from 0.298 to 0.634 and from 0.310 to 0.881, respectively. There were genetic differences among the three populations assessed, according to the F_ST values (ranging from 0.048 to 0.100, all significant) and, the number of alleles per locus ranged from 3.9 to 5.1. These are the first microsatellite markers developed for T. corymbosus, and they arise as a powerful tool for studying population structure, genetic diversity and gene flow at the landscape scale, along its distribution.     

Development of microsatellite markers using next-generation sequencing for the columnar cactus <Emphasis Type="Italic">Echinopsis chiloensis</Emphasis> (Cactaceae)

The aim of this study was to develop microsatellite markers as a tool to study population structure, genetic diversity and effective population size of Echinopsis chiloensis, an endemic cactus from arid and semiarid regions of Central Chile. We developed 12 polymorphic microsatellite markers for E. chiloensis using next-generation sequencing and tested them in 60 individuals from six sites, covering all the latitudinal range of this species. The number of alleles per locus ranged from 3 to 8, while the observed (Ho) and expected (He) heterozygosity ranged from 0.0 to 0.80 and from 0.10 to 0.76, respectively. We also detected significant differences between sites, with F_ST values ranging from 0.05 to 0.29. Microsatellite markers will enable us to estimate genetic diversity and population structure of E. chiloensis in future ecological and phylogeographic studies.     

Variability of microsatellite loci in <Emphasis Type="Italic">Vincetoxicum</Emphasis> Wolf species in southeastern Ukraine

Genetic diversity of 13 species of the genus Vincetoxicum Wolf found in Ukraine with the use of four of eight nuclear microsatellite markers previously developed for Vincetoxicum atratum from Japan was studied. The number of alleles in studied loci varied in the range from 8 to 25. The expected heterozygosity was 0.690–0.938; the observed heterozygosity varied in the range from 0.205 to 0.806. The total rate of genetic variability of studied species was found to be comparable to the rate of variability of Vincetoxicum atratum from Japan. Microsatellite loci Vinc5, Vinc104, Vinc123, and Vinc124 can be successfully used for estimating the intra- and interspecific polymorphism of the species of genus Vincetoxicum Wolf in Ukraine.     

De novo development and characterization of polymorphic microsatellite markers in a schilbid catfish, <Emphasis Type="Italic">Silonia silondia</Emphasis> (Hamilton, 1822) and their validation for population genetic studies

The stock characterization of wild populations of Silonia silondia is important for its scientific management. At present, the information on genetic parameters of S. silondia is very limited. The species-specific microsatellite markers were developed in current study. The validated markers were used to genotype individuals from four distant rivers. To develop de novo microsatellite loci, an enriched genomic library was constructed for S. silondia using affinity–capture approach. The markers were validated for utility in population genetics. A total number of 76 individuals from four natural riverine populations were used to generate data for population analysis. The screening of isolated repeat sequences yielded eleven novel polymorphic microsatellite loci. The microsatellite loci exhibited high level of polymorphism, with 6–24 alleles per locus and the PIC value ranged from 0.604 to 0.927. The observed (Ho) and expected (He) heterozygosities ranged from 0.081 to 0.84 and 0.66 to 0.938, respectively. The AMOVA analysis indicated significant genetic differentiation among riverine populations (overall F_ST = 0.075; P < 0.0001) with maximum variation (92.5 %) within populations. Cross-priming assessment revealed successful amplification (35–38 %) of heterologous loci in four related species viz. Clupisoma garua, C. taakree, Ailia coila and Eutropiichthys vacha. The results demonstrated that these de novo polymorphic microsatellite loci are promising for population genetic variation and diversity studies in S. silondia. Cross-priming results indicated that these primers can help to get polymorphic microsatellite loci in the related catfish species of family Schilbidae.     

Genetic variability comparison of cultured Israeli carp (<Emphasis Type="Italic">Cyprinus carpio</Emphasis>) from Korea using microsatellites

In aquaculture, cultured fish often undergo continuous cross-fertilization without any inflow of new broodstock. This lowers genetic diversity, leading to increased disease rates and decreased survival rates. To improve the mass production and easy culture of Israeli carp, it is essential to investigate the population structure and genetic diversity of these fish. However, such a survey has not yet been performed on Korean Israeli carp. In this study, we used seven microsatellite markers to analyze the genetic diversity and association of cultured Israeli carp from Korea and China. The average numbers of alleles per locus (N _A ) for two Korean (KorA and KorB) and two Chinese (ChA and ChB) populations were as follows: KorA (10.42), KorB (14.43), ChA (20.57) and ChB (20.71). The expected heterozygosity (H _e ) ranged from 0.672 to 0.897 and from 0.827 to 0.938 in the Korean sample and Chinese sample respectively. The genetic diversity of the Korean Israeli carp was about half that of the Chinese carp. The diversity of the Korean Israeli carp was very low, suggesting that the immunity of this population could be weak, and that diversity–recovery studies are urgently needed. Therefore, our results may therefore form the foundation for future research efforts towards genetic monitoring and selective breeding, continuous research needs to be conducted in order to recover the genetic diversity of the Korean Israeli carp.     

Population structure and variability of Pacific herring (<Emphasis Type="Italic">Clupea pallasii</Emphasis>) in the White Sea,Barents and Kara Seas revealed by microsatellite DNA analyses

Pacific herring, Clupea pallasii, have recently colonised the northeast Atlantic and Arctic Oceans in the early Holocene. In a relatively short evolutionary time, the herring formed a community with a complex population structure. Previous genetic studies based on morphological, allozyme and mitochondrial DNA data have supported the existence of two herring subspecies from the White Sea and eastern Barents and Kara Seas (C. p. marisalbi and C. p. suworowi, respectively). However, the population structure of the White Sea herring has long been debated and remains controversial. The analyses of morphological and allozyme data have previously identified local spawning groups of herring in the White Sea, whereas mtDNA markers have not revealed any differentiation. We conducted one of the first studies of microsatellite variation for the purpose of investigating the genetic structure and relationship of Pacific herring among ten localities in the White Sea, the Barents Sea and the Kara Sea. Using classical genetic variance-based methods (hierarchical AMOVA, overall and pairwise F _ST comparisons), as well as the Bayesian clustering, we infer considerable genetic diversity and population structure in herring at ten microsatellite loci. Genetic differentiation was the most pronounced between the White Sea (C. p. marisalbi) versus the Barents and Kara seas (Chesha–Pechora herring, C. p. suworowi). While microsatellite variation in all C. pallasii was considerable, genetic diversity was significantly lower in C. p. suworowi, than in C. p. marisalbi. Also, tests of genetic differentiation were indicating significant differentiation within the White Sea herring between sympatric summer- and spring-spawning groups, in comparison with genetic homogeneity of the Chesha–Pechora herring.     

Development and characterization of 12 polymorphic microsatellite loci in the sea sandwort, <Emphasis Type="Italic">Honckenya peploides</Emphasis>

Codominant marker systems are better suited to analyze population structure and assess the source of an individual in admixture analyses. Currently, there is no codominant marker system using microsatellites developed for the sea sandwort, Honckenya peploides (L.) Ehrh., an early colonizer in island systems. We developed and characterized novel microsatellite loci from H. peploides, using reads collected from whole genome shotgun sequencing on a 454 platform. The combined output from two shotgun runs yielded a total of 62,669 reads, from which 58 loci were screened. We identified 12 polymorphic loci that amplified reliably and exhibited disomic inheritance. Microsatellite data were collected and characterized for the 12 polymorphic loci in two Alaskan populations of H. peploides: Fossil Beach, Kodiak Island (n?=?32) and Egg Bay, Atka Island (n?=?29). The Atka population exhibited a slightly higher average number of alleles (3.9) and observed heterozygosity (0.483) than the Kodiak population (3.3 and 0.347, respectively). The overall probability of identity values for both populations was PID?=?2.892e^?6 and PID_sib?=?3.361e^?3. We also screened the 12 polymorphic loci in Wilhelmsia physodes (Fisch. ex Ser.) McNeill, the most closely related species to H. peploides, and only one locus was polymorphic. These microsatellite markers will allow future investigations into population genetic and colonization patterns of the beach dune ruderal H. peploides on new and recently disturbed islands.     

Development of microsatellite markers for the carnivorous plant <Emphasis Type="Italic">Genlisea aurea</Emphasis> (Lentibulariaceae) using genomics data of NGS

Genlisea aurea A.St.-Hil. is a carnivorous plant endemic species to Brazil in the Lentibulariaceae family. Very few studies have addressed the genetic structure and conservation status of G. aurea and the Lentibulariaceae. Microsatellites markers are advantageous tools that can be employed to predict the vulnerability of Lentibulariaceae species. Therefore, the development of molecular markers focusing the population analyses of Genlisea for future genetic studies and conservation actions are essential. Thus, we developed simple sequence repeats (SSRs) based on in silico analyses of G. aurea draft genome assembly. We characterized 40 individuals from several populations and identified 12 loci that were polymorphic, with heterozygosity between 0.123 and 0.650. We demonstrated that the G. aurea SSR markers work cross-species in Genlisea filiformis, G. repens, G. tuberosa and G. violacea. These markers will be important for future population, phylogeographic and conservation studies in G. aurea and other Genlisea species.     

Genome-wide single nucleotide polymorphisms (SNPs) for a model invasive ascidian <Emphasis Type="Italic">Botryllus schlosseri</Emphasis>

Invasive species cause huge damages to ecology, environment and economy globally. The comprehensive understanding of invasion mechanisms, particularly genetic bases of micro-evolutionary processes responsible for invasion success, is essential for reducing potential damages caused by invasive species. The golden star tunicate, Botryllus schlosseri, has become a model species in invasion biology, mainly owing to its high invasiveness nature and small well-sequenced genome. However, the genome-wide genetic markers have not been well developed in this highly invasive species, thus limiting the comprehensive understanding of genetic mechanisms of invasion success. Using restriction site-associated DNA (RAD) tag sequencing, here we developed a high-quality resource of 14,119 out of 158,821 SNPs for B. schlosseri. These SNPs were relatively evenly distributed at each chromosome. SNP annotations showed that the majority of SNPs (63.20%) were located at intergenic regions, and 21.51% and 14.58% were located at introns and exons, respectively. In addition, the potential use of the developed SNPs for population genomics studies was primarily assessed, such as the estimate of observed heterozygosity (H _O ), expected heterozygosity (H _E ), nucleotide diversity (π), Wright’s inbreeding coefficient (F _IS ) and effective population size (Ne). Our developed SNP resource would provide future studies the genome-wide genetic markers for genetic and genomic investigations, such as genetic bases of micro-evolutionary processes responsible for invasion success.     

Potential of cross-species microsatellite markers to assess population genetics of the endemic,endangered Nilgiri tahr (<Emphasis Type="Italic">Nilgiritragus hylocrius</Emphasis>)

Nilgiri tahr, the only wild representative of the Caprinae subfamily in Southern India, is endangered due to population decline, decreasing range size and limited geographical distribution, which together with habitat loss and fragmentation, further reduce its long-term viability. Planning conservation and management strategies to rehabilitate the species will require information on its population status and genetics. With an objective to assess the population genetics of Nilgiri tahr, we identified a panel of polymorphic microsatellite markers that amplify across the Caprinae species. We screened 50 pellet samples collected from four herds belonging to the largest remnant population of Nilgiri tahr in Eravikulam National Park, with 19 microsatellite markers, of which 17 polymorphic markers were selected for further tests. We observed varied levels of polymorphism (2–8 alleles) and heterozygosity (0.0476–0.8421). Probability of identity for individuals was 0.0018 at 10 loci and for siblings was 0.0062 at 13 loci, signifying the usefulness of these markers to study wild herds. Overall, observed and expected heterozygosities were H _o = 0.4280 ± 0.2376 and H _e = 0.4464 ± 0.2265, respectively, and the F _IS value was 0.0138 (p = 0.63). Our results validate the use of cross-species markers in wild populations of Nilgiri tahr to identify individuals and determine genetic diversity, which can be further used to understand population dynamics of this species.