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.     

Microsatellite markers for Lycium ruthenicum (Solananeae)

We developed microsatellite markers in Lycium ruthenicum, a desert plant widely distributed in northwestern China. In order to investigate its population genetic structure, genetic diversity, and its evolutionary history, we have isolated 11 novel microsatellite loci primers and characterized them in 24 individuals from 3 populations of L. ruthenicum using the combined biotin capture technique. For these microsatellites, one to seven alleles per locus were identified. The observed heterozygosities ranged from 0 to 0.958, meanwhile the expected heterozygosities ranged from 0 to 0.841. These microsatellite markers could be first useful for population level studies like genetic diversity and structure in this 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.     

Isolation and characterization of eighteen polymorphic microsatellite loci in Schizopygopsis younghusbandi Regan and cross-amplification in three other schizothoracinae species

Eighteen polymorphic microsatellite loci were isolated from Schizopygopsis younghusbandi Regan and the characterization of these loci was assessed in 46 individuals collected from the Yarlung Tsangpo River in Tibet, China. The number of alleles per locus ranged from 2 to 14. The expected heterozygosity and Shannon-Wiener diversity index ranged from 0.022 to 0.879 and from 0.059 to 2.313, respectively. The cross-species amplification and applicability of these loci were tested in three other Schizothoracinae species belonging to Schizothorax and Oxygymnocypris. These loci will be useful for the evaluation of genetic diversity and population genetic structure in S. younghusbandi and other related species.     

Development of twenty-one polymorphic tetranucleotide microsatellite loci for Schizothorax o'connori and their conservation application

Schizothorax o'connori, of the subfamily Schizothoracinae, is an endemic fish in the Yarlung Tsangpo River in Tibet, China. To evaluate the population genetic diversity as a means of devising conservation strategies, a total of 21 polymorphic tetranucleotide microsatellite loci were isolated from S. o'connori using the fast isolation by AFLP of sequences containing repeats (FIASCO) protocol. The characterization of these loci was assessed against a panel of 42 individuals. The number of alleles per locus ranged from 4 to 29 with an average of 17.76. Expected locus-specific heterozygosity and the multilocus Shannon–Wiener diversity index ranged from 0.114 to 0.951 and from 0.280 to 3.131, with an average of 0.851 and 2.338, respectively. The cross-species amplification and applicability of these loci were tested against the two other Schizothorax species from the same river. These microsatellite loci will be valuable for further studies of population genetic diversity and genetic structure, and assessments of the artificial propagation release effect of S. o'connori and other related species.     

Development and characterisation of microsatellite markers for the medicinal plant Scutellaria baicalensis (Lamiaceae)

Scutellaria baicalensis is a popular medicinal plant that is on the verge of extinction due to uncontrolled harvesting, habitat destruction and deterioration of its ecosystem. We isolated and characterised 21 microsatellite loci in this species. Ninety-four individuals from six populations were used to test the polymorphism of the microsatellite loci. The number of alleles per locus ranged from 1 to 13, with a mean of 7.2. Observed and expected heterozygosities varied from 0.000 to 1.000 and 0.000 to 0.938, respectively. Among these new microsatellite markers, only two loci showed significant deviation from Hardy–Weinberg equilibrium. No locus pairs showed significant linkage disequilibrium. The 21 primer pairs were tested in other Scutellaria species. Most of these primer pairs worked successfully, except for Scut18. These new microsatellite markers could be applied to investigate the genetic diversity and population genetic structure of S. baicalensis and its closely related species.     

Isolation and characterization of microsatellite markers in Dendrobium officinale,an endangered herb endemic to China

As an invaluable herb, Dendrobium officinale has been in severe danger since 1950 because of human exploitation and habitat deterioration. In order to investigate the genetic diversity and structure of this species, we isolated and characterized 12 microsatellite loci derived from two microsatellite‐enriched libraries. Twenty‐two individuals from Leye population were analysed. These loci were polymorphic and displayed three to 12 alleles per locus. The observed and expected heterozygosities ranged from 0.150 to 0.624 and from 0.162 to 0.605, respectively. These are the first microsatellite loci characterized from D. officinale that will contribute to research on the conservation, genetic diversity, population structure and individual authentication.     

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 19 polymorphic microsatellite loci from Neosalanx taihuensis,a rapidly invasive and adaptative species

Neosalanx taihuensis can very rapidly adapt to new environmental conditions and quickly becomes the dominant species when introduced to lakes and reservoirs. To better understand the genetic mechanism responsible for this remarkable ability to invade and adapt to diverse ecosystems, 19 polymorphic microsatellite loci were isolated from the genomic DNA of N. taihuensis. These microsatellite loci were identified from 24 individuals from an invasive population sampled in Fuxian Lake, Yunnan Plateau, Southwest China. The number of alleles per locus ranged from 2 to 14. Observed and expected heterozygosities ranged from 0.0417 to 0.8636 and from 0.3112 to 0.9202, respectively. The polymorphism information content (PIC) varied from 0.2583 to 0.8928. These microsatellite loci will be essential in performing molecular analyses of genetic diversity and genetic structure in the future. This will ultimately facilitate better understanding of the genetic mechanisms that allow N. taihuensis to successfully invade and adapt to new ecosystems.     

Genetic diversity of Chinese giant salamander (Andrias davidianus) based on the novel microsatellite markers

Chinese giant salamander (Andrias davidianus) is a rare amphibian species in the world. Microsatellite markers are a promising tool for accurate estimation population structure and genetic diversity. In this paper, we isolated novel microsatellite marker for Chinese giant salamander using fast isolation by AFLP of sequences containing repeats (FIASCO) method. More than 50% sequences in 132 clones had repeat number over ten times with di or trinucleeotide repeat except of (GA)₁₂. Seventy pairs of primers were designed and eleven polymorphic microsatellite loci were characterized for wild and cultivated Chinese giant salamander populations. The allele number was from 3 to 9 in different loci. Polymorphism information content was from 0.544 to 0.702 in cultivated population. The results implied more alleles and PIC were in the wild population than cultivated population. The observed heterozygosities in two populations were higher than 0.553. The data analysis suggested that the cultivated population has lower genetic diversity than wild population, which it??s perhaps owing to inbreeding in artificial breeding. To our knowledge, it??s the first time to isolated microsatellite markers for Chinese giant salamander. The result indicated that the markers were suitable for the population genetic analysis of Chinese giant salamander.     

Development of Microsatellite Markers and Analysis of Genetic Diversity and Population Structure of Colletotrichum gloeosporioides from Ethiopia

Twenty three polymorphic microsatellite markers were developed for citrus plant pathogenic fungus, Colletotrichum gloeosporioides, and were used to analyze genetic diversity and population structure of 163 isolates from four different geographical regions of Ethiopia. These loci produced a total of 118 alleles with an average of 5.13 alleles per microsatellite marker. The polymorphic information content values ranged from 0.104 to 0.597 with an average of 0.371. The average observed heterozygosity across all loci varied from 0.046 to 0.058. The gene diversity among the loci ranged from 0.106 to 0.664. Unweighted Neighbor-joining and population structure analysis grouped these 163 isolates into three major groups. The clusters were not according to the geographic origin of the isolates. Analysis of molecular variance showed 85% of the total variation within populations and only 5% among populations. There was low genetic differentiation in the total populations (F_ST = 0.049) as evidenced by high level of gene flow estimate (N_m = 4.8 per generation) among populations. The results show that Ethiopian C. gloeosporioides populations are generally characterized by a low level of genetic diversity. The newly developed microsatellite markers were useful in analyzing the genetic diversity and population structure of the C. gloeosporioides populations. Information obtained from this study could be useful as a base to design strategies for better management of leaf and fruit spot disease of citrus in Ethiopia.     

Characterization of polymorphic microsatellite markers and genetic diversity in wild bronze featherback, Notopterus notopterus (Pallas, 1769)

Six polymorphic microsatellite DNA loci were identified in the primitive fish, bronze featherback, Notopterus notopterus for the first time and demonstrated significant population genetic structure. Out of the six primers, one primer (NN90) was specific to N. notopterus (microsatellite sequence within the RAG1 gene) and five primers were product of successful cross-species amplification. Sixty-four primers available from 3 fish species of order Osteoglossiformes and families Notopteridae and Osteoglossidae were tested to amplify homologous microsatellite loci in N. notopterus. Fifteen primer pairs exhibited successful cross-priming PCR product. However, polymorphism was detected only at five loci. To assess the significance of these six loci (including NN90) in population genetic study, 215 samples of N. notopterus from five rivers, viz Satluj, Gomti, Yamuna, Brahmaputra and Mahanadi were analyzed. The five sample sets displayed different diversity levels and observed heterozygosity ranged from 0.6036 to 0.7373. Significant genotype heterogeneity (P < 0.0001) and high F_ST (0.2205) over all loci indicated that the samples are not drawn from the same genepool. The identified microsatellite loci are promising for use in fine-scale population structure analysis of N. notopterus.     

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.     

Patterns of geographic distribution have a considerable influence on population genetic structure in one common and two rare species of Rhododendron (Ericaceae)

Genetic diversity is essential for species to sustain their populations and evolutionary potential. In order to develop effective conservation strategies for rare species, it is necessary to understand differences in patterns of genetic diversity between common and rare species. Data about population genetic structure is important to design effective conservation strategies for rare species. In this study, we compared the genetic diversity and population genetic structure of a common species, Rhododendron weyrichii, to those of two rare species, Rhododendron sanctum and Rhododendron amagianum, with different geographic distributions. We analyzed five microsatellite loci in 16 populations of R. weyrichii, 9 populations of R. sanctum, and 6 populations of R. amagianum. As expected, the level of genetic diversity indicated by allelic richness and gene diversity was lower for the rare species R. sanctum than for the common species R. weyrichii. However, there was no statistically significant difference in genetic diversity between R. weyrichii and the other rare species, R. amagianum. Analyses of the isolation-by-distance pattern, neighbor-joining trees, and Bayesian clustering indicated that R. sanctum had a strong population genetic structure whereas R. amagianum exhibited very weak genetic structure among populations and that there was moderate population genetic structure for R. weyrichii. Therefore, the degree and pattern of population genetic structure in each species was unrelated to its rarity and instead merely reflected its geographic distribution.     

Isolation and characterization of microsatellite markers for Achyranthes bidentata (Amaranthaceae) using next-generation sequencing platform

Achyranthes bidentata Blume (Amaranthaceae) is a perennial herb that is widely distributed in India, Java, China, and Japan. The natural resources of A. bidentata in its geo-authentic product area have rapidly declined in recent years because of the over-collection of its roots. To devise adequate conservation and management strategies for this species, its genetic diversity and population structure should be characterized. Roche 454 pyrosequencing combined with magnetic bead enrichment was used to develop microsatellite markers for A. bidentata. A total of 903 microsatellite loci were identified from 42,004 individual sequence reads. One hundred microsatellite loci were selected to test the primer amplification efficiency across 16 individuals from two A. bidentata populations. Of these tested markers, 8 yielded polymorphic amplification products, 29 yielded single alleles. For polymorphic primer pairs, the number of alleles per locus ranged from 2 to 4, with an average of 2.75. The observed and expected heterozygosities ranged from 0.353 to 0.671 and 0.250 to 0.938, respectively. The inbreeding coefficient varied from −0.692 to 0.627. This set of markers will provide useful tools for examining genetic diversity and population structure, and aid in better understanding of the conservation of A. bidentata.     

Isolation and characterization of 10 new compound microsatellite markers for a mangrove tree species,Avicennia marina (Forsk.) Vierh. (Avicenniaceae)

Avicennia marina is an ecologically important mangrove tree species. We isolated 10 polymorphic microsatellite loci from this species using an improved technique. Our isolated loci provided compound microsatellite markers with polymorphism of two to six alleles per locus. The observed and expected heterozygosities ranged from 0.025 to 0.625 and from 0.096 to 0.767, respectively. These markers would be the useful tools for researching on the genetic diversity and population genetic structure of A. marina.     

Genetic diversity of the Chestnut blight fungus Cryphonectria parasitica in four French populations assessed by microsatellite markers

Microsatellites are powerful markers to infer population genetic parameters. Here, 13 microsatellite loci isolated from a genomic and a cDNA library of Cryphonectria parasitica were used to characterize the genetic diversity and structure of four French populations. Twelve of these loci were polymorphic within populations, and average gene diversity (H_e) was estimated to be 0.35. There was a lower genetic diversity in a south-eastern population relative to three south-western populations. In these three populations, microsatellite genotypic diversity was higher than vegetative compatibility type diversity. A high genetic differentiation (G_ST = 0.27) suggested a low gene flow and/or founder effects of French populations which are in agreement with low dispersal of spores and different introductions of this species in southern France. This study demonstrates the significance of these microsatellite loci to assess gene flow and reproductive system in this important pathogen.     

Microsatellites reveal substantial among-population genetic differentiation and strong inbreeding in the relict fern Dryopteris aemula

Background and Aims

A previous study detected no allozyme diversity in Iberian populations of the buckler-fern Dryopteris aemula. The use of a more sensitive marker, such as microsatellites, was thus needed to reveal the genetic diversity, breeding system and spatial genetic structure of this species in natural populations.

Methods

Eight microsatellite loci for D. aemula were developed and their cross-amplification with other ferns was tested. Five polymorphic loci were used to characterize the amount and distribution of genetic diversity of D. aemula in three populations from the Iberian Peninsula and one population from the Azores.

Key Results

Most microsatellite markers developed were transferable to taxa close to D. aemula. Overall genetic variation was low (H_T = 0·447), but was higher in the Azorean population than in the Iberian populations of this species. Among-population genetic differentiation was high (F_ST = 0·520). All loci strongly departed from Hardy–Weinberg equilibrium. In the population where genetic structure was studied, no spatial autocorrelation was found in any distance class.

Conclusions

The higher genetic diversity observed in the Azorean population studied suggested a possible refugium in this region from which mainland Europe has been recolonized after the Pleistocene glaciations. High among-population genetic differentiation indicated restricted gene flow (i.e. lack of spore exchange) across the highly fragmented area occupied by D. aemula. The deviations from Hardy–Weinberg equilibrium reflected strong inbreeding in D. aemula, a trait rarely observed in homosporous ferns. The absence of spatial genetic structure indicated effective spore dispersal over short distances. Additionally, the cross-amplification of some D. aemula microsatellites makes them suitable for use in other Dryopteris taxa.     

Variability of allozyme and cpSSR markers in the populations of Siberian spruce

The variability of 21 allozyme and three microsatellite loci of chloroplast DNA (cpDNA) was studied in the populations of Siberian spruce (Picea obovata Ledeb.) from Irkutsk oblast, Magadan oblast, Buryatia, and Mongolia. It was demonstrated that the highest level of genetic diversity among the examined populations at both allozyme and microsatellite loci was observed in the Tulyushka population from Irkutsk oblast. The lowest level of genetic diversity was observed in marginal isolated populations of Bogd Uul and Magadan. In the relict spruce population from Olkhon Island, differing from the other populations in the lowest allelic diversity of both types of markers, no expected decline of expected heterozygosity and haplotype diversity was observed. In this population, the variability parameters mentioned were close to the population mean. The obtained intrapopulation and intraspecific variability parameters of allozyme and microsatellite loci of chloroplast DNA and the data on the population differentiation at these loci indicate that the given markers can be used for the analysis of the population structure of Siberian spruce.     

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.