Isolation and characterization of polymorphic microsatellite markers of Anopheles sinensis,a malaria vector mosquito in the East Asia region

Eleven polymorphic dinucleotide (GA and CA) microsatellites were isolated and characterized in the mosquito Anopheles sinensis; this species is distributed over the East Asia region and is a primary vector of malaria, particularly in Korea. The number of alleles per locus ranged from 4 to 13. The observed heterozygosity (H_O) and expected heterozygosity (H_E) varied from 0.30 to 0.89 and from 0.59 to 0.90, respectively. These microsatellites could be useful in studying the evolution of the widely distributed A. sinensis in diverse environments.     

Nineteen novel microsatellites in Chinese lake gudgeon Sarcocheilichthys sinensis Bleeker, 1971

Nineteen polymorphic microsatellites isolated from a partial genomic DNA library of Sarcocheilichthys sinensis enriched for CA repeats were developed. Variability was tested on 48 unrelated individuals collected from Jiangsu province, China. Observed heterozygosity ranged from 0.200 (Ss236) to 1.00 (Ss223, Ss224), whereas the expected heterozygosity ranged from 0.213 (Ss232) to 0.939 (Ss224). Shannon's information index ranged from 0.405 (Ss236) to 2.988 (Ss224). Sixteen of the 19 microsatellites conformed to Hardy–Weinberg equilibrium. These microsatellites can be used for the evaluation of genetic parameters in populations to supply valuable information for conservation management.     

Isolation and characterization of polymorphic microsatellites from Chinese mitten crab,Eriocheir sinensis

Chinese mitten crab (Eriocheir sinensis) has great commercial significance in Asia, but is an invasive organism in Europe and America. We isolated five novel microsatellites for E. sinensis. All five loci were polymorphic. The average allele number was 16.8 per locus with a range of 13 to 21, while the expected heterozygosity ranged from 0.82 to 0.91 with an average of 0.88. These markers could be applicable to studies of the genetic diversity and population structure of E. sinensis.     

Characterization of microsatellite loci for the Chinese mitten crab,Eriocheir sinensis

The Chinese mitten crab, Eriocheir sinensis is an important human food source in Asia and causes considerable ecological and economical damage as a recent invader of North America and Europe. Here we report the isolation and characterization of 12 highly polymorphic microsatellite loci for E. sinensis. The number of observed alleles per locus ranged from 7 to 40 and the observed heterozygosity from 0.50 to 0.95. These markers should prove a useful tool to investigate the colonization process and its evolutionary implications.     

Characterization of 14 tetranucleotide microsatellite loci derived from Pacific herring

Spawning aggregations of Pacific herring (Clupea pallasi) often exhibit significant interannual variation in allele frequencies of neutral gene markers. We isolated 14 tetranucleotide microsatellites to examine hypothetical processes that may produce this unique genetic signal. We developed and tested primer pairs for each locus and then estimated locus variability in samples (n = 60) from two populations. The number of alleles per locus ranged from five to 49. The expected heterozygosity across loci and populations ranged from 0.20 to 0.96. These microsatellites will be useful for estimating genetic variation in herring on a fine geographical scale.     

Identification and characterization of microsatellite loci in the common fig (Ficus carica L.) and representative species of the genus Ficus

We developed microsatellites in fig (Ficus carica L.). A TC and TG‐enriched genomic library was screened, and after sequencing, primers were designed for 20 microsatellites. Eight primer pairs produced amplification products that were both interpretable and polymorphic in 14 fig cultivars and two French wild‐growing populations of F. carica (n₁ = 9 and n₂ = 10). Number of alleles per locus ranged from three to six. Except for one microsatellite locus, the observed heterozygosity was higher than the expected value. The F. carica microsatellites gave amplification products in 17 other Ficus species in 86% of the cases.     

Genetic and morphological variation in the venus clam Cyclina?sinensis along the coast of China

Six geographic samples of Cyclina sinensis were collected from the coast along China and analyzed to reveal morphological and genetic variation by using nine allozyme loci and 11 morphological variables. The discriminant function analysis (DFA) of morphology suggested a clear separation between the southern and northern populations. Polymorphism was detected at nine loci across all six populations. The mean allele number ranged from 2.44 to 2.78, and the mean observed heterozygosity ranged from 0.218 to 0.296. High level of genetic differentiation was found between three northern populations and three southern populations. The marked genetic differentiation can be explained by the upwelling of the Zhejiang province and the freshwater outflow of Yangtze River. The results obtained in this study indicate that the northern and southern populations of C. sinensis should be treated as separate units for conservation management.     

Genomewide gene‐associated microsatellite markers for the model invasive ascidian,Ciona intestinalis species complex

The vase tunicate, Ciona intestinalis species complex, has become a good model for ecological and evolutionary studies, especially those focusing on microevolution associated with rapidly changing environments. However, genomewide genetic markers are still lacking. Here, we characterized a large set of genomewide gene‐associated microsatellite markers for C. intestinalis spA (=C. robusta). Bioinformatic analysis identified 4654 microsatellites from expressed sequence tags (ESTs), 2126 of which successfully assigned to chromosomes were selected for further analysis. Based on the distribution evenness on chromosomes, function annotation and suitability for primer design, we chose 545 candidate microsatellites for further characterization. After amplification validation and variation assessment, 218 loci were polymorphic in at least one of the two populations collected from the coast of Arenys de Mar, Spain (N = 24–48), and Cape Town, South Africa (N = 24–33). The number of alleles, observed heterozygosity and expected heterozygosity ranged from 2 to 11, 0 to 0.833 and 0.021 to 0.818, and from 2 to 10, 0 to 0.879 and 0.031 to 0.845 for the Spanish and African populations, respectively. When all microsatellites were tested for cross‐species utility, only 60 loci (25.8%) could be successfully amplified and all loci were polymorphic in C. intestinalis spB. A high level of genomewide polymorphism is likely responsible for the low transferability. The large set of microsatellite markers characterized here is expected to provide a useful genomewide resource for ecological and evolutionary studies using C. intestinalis as a model.     

Isolation of microsatellite loci in the freshwater goby,Rhinogobius sp. (Gobiidae)

The Rhinogobius species complex (Pisces: Gobiidae) includes great variation in colour, morphology and ecological form. In particular, R. sp. OR (Orange type) exhibits some of the greatest colour variation among the species complex, although the genetic relationships among the variations in this fish remain unclear. Thirteen microsatellite loci were identified from R. sp. OR, and all loci were polymorphic with two to 17 alleles per locus. Observed heterozygosity ranged from 0.1 to 0.9, while the expected heterozygosity ranged from 0.19 to 0.96. Multiplex polymerase chain reaction reaction (PCR) were optimized. All loci except Rhi‐5 conform to Hardy–Weinberg equilibrium (P > 0.05).     

Isolation and characterization of dinucleotide microsatellites in greater amberjack, <Emphasis Type="Italic">Seriola dumerili</Emphasis>

Thirteen nuclear-encoded dinucleotide microsatellites were characterized from a genomic DNA library of greater amberjack, Seriola dumerili. The microsatellites include 12 perfect-repeat motifs and one imperfect-repeat motif. The number of alleles at the 13 microsatellites among a sample of 29 fish ranged from 3 to 25; gene diversity (expected heterozygosity) ranged from 0.296 to 0.948, while observed heterozygosity ranged from 0.276 to 0.897. Following Bonferroni correction, genotypes at all 13 microsatellites fit expectations of Hardy-Weinberg equilibrium. One pairwise comparison of microsatellites deviated significantly from expectations of genotypic equilibrium, suggesting that these two microsatellites may be linked. Greater amberjack support commercial and recreational fisheries along both the Atlantic and Gulf coasts of the U.S. and represent a species with potential for worldwide aquaculture. The microsatellites developed will be useful for conservation and population genetic studies of ‘wild’ and domesticated populations of greater amberjack.     

Isolation and characterization of polymorphic microsatellite loci in a traditional Chinese medicinal plant, Schisandra sphenanthera

Schisandra sphenanthera (Schisandraceae) is a traditional Chinese medicinal plant. In this study, nine polymorphic microsatellite loci were developed and characterized for the species. A genomic DNA enrichment protocol was used to isolate microsatellite loci and polymorphism was explored using 36 individuals sampled from a natural population. The observed number of alleles ranged from 2 to 10 with an average of 5.2. Expected and observed heterozygosity ranged from 0.30 to 0.82 and 0.33 to 0.97, respectively. These microsatellites have been directly applied to both population and conservation genetic studies of S. sphenanthera.     

Isolation and characterization of seven tetranucleotide microsatellite loci in mungbean,Vigna radiata

A simple and rapid method for isolating tetranucleotide microsatellites in mungbean, Vigna radiata, based on the 5′‐anchored polymerase chain reaction technique, revealed 15 microsatellite sequences. We report on the characterization of seven polymorphic microsatellite loci in V. radiata. The number of alleles per locus ranged from 2 to 6, whereas the observed heterozygosity ranged from 0 to 0.561. Tetranucleotide markers are useful because they amplify fewer stutter bands thus making scoring easier. These markers will be useful for detecting genetic variation in mungbean varieties for germplasm management and development of the crop.     

Development and characterization of eleven microsatellite markers for an endangered cavefish (Triplophysa rosa Chen and Yang, 2005) using 454 sequencing

Triplophysa rosa is an endangered cave‐dwelling and endemic fish species found only in Chongqing, southwestern China. The genetic data available for this species is very limited. Polymorphic microsatellites were identified in the genome of T. rosa using 454 sequencing. Of the 145 loci screened, 106 were amplified successfully and 11 showed polymorphic patterns. The number of alleles ranged from 2 to 6, and the observed and expected heterozygosity varied from 0.061 to 0.543 (mean = 0.349) and from 0.248 to 0.789 (mean = 0.551), respectively. The polymorphic information content (PIC) ranged from 0.215 to 0.744 (mean = 0.486), indicating moderate levels of polymorphism. In addition, cross‐species amplification was tested for the 106 loci in Triplophysa moquensis, which showed a high level of transferability (76.4%), implying that the microsatellite markers developed here could be used effectively for other closely related species.     

Population genetic inferences using immune gene SNPs mirror patterns inferred by microsatellites

Single nucleotide polymorphisms (SNPs) are replacing microsatellites for population genetic analyses, but it is not apparent how many SNPs are needed or how well SNPs correlate with microsatellites. We used data from the gopher tortoise, Gopherus polyphemus—a species with small populations, to compare SNPs and microsatellites to estimate population genetic parameters. Specifically, we compared one SNP data set (16 tortoises from four populations sequenced at 17 901 SNPs) to two microsatellite data sets, a full data set of 101 tortoises and a partial data set of 16 tortoises previously genotyped at 10 microsatellites. For the full microsatellite data set, observed heterozygosity, expected heterozygosity and F_ST were correlated between SNPs and microsatellites; however, allelic richness was not. The same was true for the partial microsatellite data set, except that allelic richness, but not observed heterozygosity, was correlated. The number of clusters estimated by structure differed for each data set (SNPs = 2; partial microsatellite = 3; full microsatellite = 4). Principle component analyses (PCA) showed four clusters for all data sets. More than 800 SNPs were needed to correlate with allelic richness, observed heterozygosity and expected heterozygosity, but only 100 were needed for F_ST. The number of SNPs typically obtained from next‐generation sequencing (NGS) far exceeds the number needed to correlate with microsatellite parameter estimates. Our study illustrates that diversity, F_ST and PCA results from microsatellites can mirror those obtained with SNPs. These results may be generally applicable to small populations, a defining feature of endangered and threatened species, because theory predicts that genetic drift will tend to outweigh selection in small populations.     

Isolation and characterization of microsatellite DNA primers for <Emphasis Type="Italic">Sinadoxa corydalifolia</Emphasis> (Adoxaceae)

Sinadoxa corydalifolia is the only species of Sinadoxa (Adoxaceae) with the aberrant morphology. This species has become extremely endangered in the Qinghai-Tibetan Plateau. To provide a population-level genetic profile for investigation and conservation of genetic diversity of this species, we developed 10 new microsatellite loci for this species by the combining biotin capture method. About 31 microsatellites were screened from the library, 10 of the screened microsatellites are polymorphic. The number of alleles per locus in 18 individuals ranged from 3 to 11, expected heterozygosity and observed heterozygosity ranged from 0.3071 to 0.6243 and from 0.1675 to 0.4357, respectively. We further performed cross-priming tests of these primers in another species of the Adoxaceae: Adoxa moschatellina and found 9 of 10 successfully amplified the targeted sequences. These newly developed loci provide a useful tool to investigate the genetic diversity and design the conversation measures of S. corydalifolia and study the genetic divergence and the initial speciation pattern between it and the related species in the Adoxaceae.     

Isolation of seven polymorphic microsatellites in Ophioblennius atlanticus atlanticus (Perciformes,Blenniidae)

We isolated and characterized seven polymorphic microsatellite loci of Ophioblennius atlanticus atlanticus (Valenciennes, 1836) using an optimized protocol to construct and screen a microsatellite‐enriched genomic library. The analysis of variability was performed in 16 specimens from Faial Island (Azores, Portugal). The mean number of alleles was 8.71 ± 2.43 and the level of expected heterozygosity ranged from 0.764 to 0.903. The total exclusionary probabilities using these loci for the first and the second parent were 0.985 and 0.998, respectively, suggesting that these microsatellites are a useful tool for large‐scale parentage analysis.     

Isolation and characterization of 14 polymorphic microsatellite loci in the ark shell Scapharca subcrenata (Bivalvia: Arcidae)

The ark shell Scapharca subcrenata is an important fishery resource, but has been suffering from severe population decline due to the deterioration of the coastal environment and over-exploitation. To investigate the genetic variation and structure among populations of S. subcrenata, 14 polymorphic microsatellite loci were developed. The number of alleles per polymorphic locus ranged from 11 to 29 and the observed and expected heterozygosity varied from 0.400 to 0.923 and from 0.705 to 0.965, respectively. These microsatellites will help advance the investigation of the genetic population structure and genetic diversity in this species.     

Development of microsatellite markers for the Manila clam Ruditapes philippinarum

We isolated nine polymorphic microsatellites from the Manila clam Ruditapes philippinarum. These loci provide one class of highly variable genetic marker as the number of alleles ranged from six to 22, and the observed and expected heterozygosity ranged from 0.136 to 0.909 and from 0.553 to 0.954, respectively. We consider that these loci are potentially useful for detailing the genetic structure and gene flow among R. philippinarum populations.     

PCR primers for trinucleotide and tetranucleotide microsatellites in greater amberjack,Seriola dumerili

Eighteen nuclear‐encoded microsatellites from a genomic DNA library of greater amberjack, Seriola dumerili, were isolated and characterized. The microsatellites include 13 perfect (five tetranucleotide and eight trinucleotide) and five imperfect (three tetranucleotide, one trinucleotide and one combination dinucleotide/trinucleotide) repeat motifs. The number of alleles at the 18 microsatellites among a sample of 29 fish ranged from two to 20; gene diversity (expected heterozygosity) ranged from 0.068 to 0.950, whereas observed heterozygosity ranged from 0.069 to 0.966. Following Bonferroni correction, genotypes at all 18 microsatellites fit expectations of Hardy–Weinberg equilibrium, and all pairwise comparisons of microsatellites did not deviate significantly from genotypic equilibrium. Greater amberjack support commercial and recreational fisheries along both the Atlantic and the Gulf coasts of the USA and represent a species with potential for worldwide aquaculture. The microsatellites developed will be useful for population genetic studies of ‘wild’ populations and breeding studies of domesticated populations.