Description of 21 microsatellites for the Chinese bitterling,Rhodeus sinensis Günther, 1868

Chinese bitterling (Rhodeus sinensis) is a freshwater fish endemic to China. During the past several decades, natural populations of this species have become endangered due to habitat fragmentation and water pollution. To help conserve and restore R. sinensis, Illumina Miseq sequencing was used to identify 21 polymorphic microsatellites. The number of alleles varied from 3 to 13, and the observed heterozygosity and expected heterozygosity ranged from 0.3067 to 0.9145 and 0.3845 to 0.9521, respectively. The polymorphism information content ranged from 0.3173 to 0.9105. These novel microsatellites will be useful in both conservation and management of R. sinensis.     

First development and characterization of 27 novel microsatellite markers in the dobsonfly Neoneuromus ignobilis (Megaloptera: Corydalidae) at genome-scale level

The dobsonfly species Neoneuromus ignobilis Navás (Megaloptera: Corydalidae) is endemic to but widely distributed from eastern and southeastern Asia, being an important insect indicator for freshwater biomonitoring. At present, there is no report on the development of microsatellites of Megaloptera. Here, we developed 27 novel microsatellite markers of N. ignobilis from 850,920 candidate microsatellites with the stringent screening criteria considering the amplification success rate, the presence or absence of stutter peaks, the peak intensity, the polymorphism of the loci, the heterozygosity, and the number of alleles. The allele number of 27 microsatellite markers ranges from 3 to 12 with an average value of 6.19 per locus. The observed heterozygosity (H_O) and expected heterozygosity (H_E) revealed a range from 0.000 to 0.947 and 0.000 to 0.842, respectively. We constructed three panels (MP panel, most polymorphic; SS panel, most stringent strategy; ALL panel, total 27 microsatellite markers) and compared the analyses on population genetic diversity and structure. The result showed that the MP panel can significantly improve the analyses of individual assignment and genetic diversity. Accordingly, we advocate selecting the most polymorphic microsatellite marker for analyzing population genetics based on microsatellite data. The present work represents the first study on the microsatellite development of Megaloptera.

     

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.     

Microsatellite loci for studies on population differentiation and connectivity of the red‐bellied yellow tail fusilier,Caesio cuning (Caesionidae)

Ten polymorphic microsatellites were isolated from the red‐bellied yellow tail fusilier Caesio cuning, a reef‐associated fish which occurs widely in the Indo‐Pacific region. The species is exploited by both small‐scale and commercial fisheries. Fifty individuals from six populations were genotyped using primers that reliably amplified 10 polymorphic loci. The number of alleles per locus ranged from three to 11. Observed heterozygosity (H_O) and expected heterozygosity (H_E) ranged between 0.400 to 0.880 and 0.337 to 0.843, respectively. These microsatellite loci may be used to study population structure, genetic diversity and connectivity of C. cuning in the range of its distribution.     

Genetic diversity and gene flow in a Caribbean tree <Emphasis Type="Italic">Pterocarpus officinalis</Emphasis> Jacq.: a study based on chloroplast and nuclear microsatellites

We analysed the molecular diversity of Pterocarpus officinalis, a tree species distributed in Caribbean islands, South and Central America to quantify the genetic variation within island, to assess the pattern of differentiation and infer levels of gene flow; with the overall goal of defining a strategy of conservation. Two hundred two individuals of 9 populations were analysed using three chloroplast and six nuclear microsatellite markers. The observed heterozygosity varied markedly among the populations for nuclear (H _Onuc = 0.20–0.50) and chloroplast microsatellites (H _cp = 0.22–0.68). The continental population from French Guyana showed a higher value of H _Onuc than island populations, and the differences were significant in some cases. The fixation index F _IS ranged from −0.043 to 0.368; a significant heterozygote deficit was detected in 7 populations. The heterozygosity excess method suggested that two populations in Guadeloupe have undergone a recent bottleneck. Global and pairwise F _ST were high for both nuclear (F _STnuc = 0.29) and chloroplast microsatellites (F _STcp = 0.58). The neighbour-joining tree based on both markers, presented a differentiation pattern that can be explained by the seed dispersal by flotation and marine stream. The comparison of Bayesian approach and the method based on allelic frequency demonstrate a very limited number of migrants between populations.     

Generation and characterization of 24 novel EST derived microsatellites from tea plant (Camellia sinensis) and cross-species amplification in its closely related species and varieties

A total of 31 expressed sequence tag (EST)-derived polymorphic microsatellites from tea plant, Camellia sinensis (L.) O. Kuntze, were generated and characterized using the ESTs of the author’s EST sequencing project and other sources. A set of 40 accessions tea germplasms had been used to examine the diversity. Among the 31 microsatellite loci, 24 had two to eight polymorphic alleles. Observed heterozygosity (H _o) were relatively higher (on an average of 0.533), varying from 0.175 (primer 21) to 0.950 (primer 228). Cross-species polymorphic amplification in other four species and two varieties of section Thea (L.) Dyer genus Camellia L. was successful for the 24 loci. Contribution of the 24 novel EST-SSR primers presented here will provide necessary and powerful molecular tools for management and conservation studies on the tea germplasms in the future. Li-Ping Zhao and Zhen Liu contributed equally to this work.     

Isolation and characterization of variable (GT)n repetitive sequences from Atlantic salmon, Salmo salar L.

Microsatellites are islands of long repeats of mono-, di- or trinucleotides evenly distributed in the eukaryotic genome with an average distance of 50–100 kb. They display a high degree of length polymorphism and heterozygosity at individual loci, making them highly useful as markers in the development of genomic maps of eukaryotes. In the present work, we examined the dinucleotide repeat motif (dG-dT)_n in the Atlantic salmon, Salmo salar L., genome. The frequency of (dG-dT)_n microsatellites in salmon correlates well with earlier published estimations. Cloning and sequencing of 45 salmon microsatellites revealed perfect and imperfect repeats, but no compound microsatellites. The distribution of number of repeat units in salmon microsatellites differ significantly from that of higher vertebrates. Salmon tends to have more long repeat stretches and less intermediate length repeats.     

Isolation and characterization of microsatellite loci in Eurycorymbus cavaleriei,a dioecious endemic tree species in China

Eleven dinucleotide microsatellites were developed and characterized for Eurycorymbus cavaleriei, a dioecious, rare and endemic tree species in China. A genomic DNA enrichment protocol was used to isolate microsatellite loci and polymorphism was explored using 30 trees from two natural populations. The observed heterozygosity (H_O) ranged from 0.379 to 0.931, with an average of 0.625. These markers provide powerful tools for the ongoing population and conservation genetics studies of E. cavaleriei.     

Identification of microsatellite loci from Epimedium koreanum and cross‐species amplification in four species of Epimedium (Berberidaceae)

Nineteen polymorphic microsatellite markers were isolated and characterized from a trinucleotide enriched partial genomic library of Epimedium koreanum. The average allele number of these microsatellites was 5.9 per locus, ranging from two to 11. The observed heterozygosity (H_O) and expected heterozygosity (H_E) at the population level were 0.00–0.90 and 0.12–0.90, respectively. In addition, the results of cross‐species amplification of this set of microsatellite markers in four closely related Epimedium species, E. brevicornum, E. sagittatum, E. pubescens and E. wushanense, revealed that these microsatellite markers were useful for population genetic structure evaluation and genotype analysis of major Epimedium species that have been used as traditional Chinese medicines.     

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 loci from Silene tatarica

Five new microsatellite loci were isolated from the perennial plant, Silene tatarica. We characterized S. tatarica individuals originating from two riverbank populations in northern Finland and observed between four and nine alleles per locus. Observed heterozygosity was consistently lower than gene diversity (H_O: 0.0450–0.2385, H_E: 0.1919–0.6187). This deficiency of heterozygous genotypes was observed in most locus/population combinations, and is presumably caused by spatial genetic structuring due to restricted seed flow within subpopulations. The markers presented here are the first microsatellites reported for S. tatarica.     

Characterization of seven genomic and one dbEST-derived microsatellite loci in the river mangrove<Emphasis Type="Italic"> Aegiceras corniculatum</Emphasis> (Myrsinaceae)

We developed seven microsatellite markers from the genome of Aegiceras corniculatum using the FIASCO protocol and one dbEST-derived microsatellite marker. Polymorphism of each locus was assessed in 45 individuals from China, Thailand and Australia. The average allele number of these microsatellites was 4.4 per locus, ranging from 2 to 8. The observed (H _O ) and expected (H _E ) heterozygosity were from 0.178 to 1.000 and from 0.164 to 0.828, respectively. These microsatellite markers would provide a useful tool for genetic and conservation studies of A. corniculatum.     

Genetic diversity of common carp from two largest Chinese lakes and the Yangtze River revealed by microsatellite markers

Six polymorphic microsatellites (eight loci) were used to study the genetic diversity and population structure of common carp from Dongting Lake (DTC), Poyang Lake (PYC), and the Yangtze River (YZC) in China. The gene diversity was high among populations with values close to 1. The number of alleles per locus ranged from 2 to 11, and the average number of alleles among 3 populations ranged from 6.5 to 7.9. The mean observed (H _O) and expected (H _E) heterozygosity ranged from 0.4888 to 0.5162 and from 0.7679 to 0.7708, respectively. Significant deviations from Hardy–Weinberg Equilibrium expectation were found at majority of the loci and in all three populations in which heterozygote deficits were apparent. The analysis of molecular variance (AMOVA) indicated that the percent of variance among populations and within populations were 3.03 and 96.97, respectively. The Fst values between populations indicated that there were significant genetic differentiations for the common carp populations from the Yangtze River and two largest Chinese freshwater lakes. The factors that may result in genetic divergence and significant reduction of the observed heterozygosity were discussed.     

Nine microsatellite DNA primers for <Emphasis Type="Italic">Hippophae rhamnoides</Emphasis> ssp. <Emphasis Type="Italic">sinensis</Emphasis> (Elaeagnaceae)

Hippophae rhamnoides ssp. sinensis occurs mainly in the arid regions of northwest China. The wood stands of this subspecies play an important role in maintaining the local ecosystems in these regions. In addition, the genetic characteristics are essential to understand the historical range changes of this subspecies and its morphological differentiation with other subspecies. In this study, we developed nine microsatellite loci for this subspecies for the first time. We used the combining biotin capture method to enrich AG/CT/AC/GT/CG/GTG/CCA microsatellites. Twenty-six microsatellites were isolated from the enriching library and nine of them were found to be polymorphic through screening 12 distantly distributed individuals. The number of alleles per locus ranged from three to twelve and expected heterozygosity from 0.2659 to 0.4767, respectively. We further performed cross-priming tests in another subspecies and two congeneric species. These firstly isolated loci will provide a useful tool to investigate the genetic structure of this subspecies and its morphological differentiation from the other subspecies.     

Polymorphic microsatellites identified by cross-species amplifications in the European Coot Fulica atra

We tested the cross-amplification of 26 microsatellites developed for passerines and an additional three developed for Gallinula species in eight European Coots from two populations. Sixteen microsatellite markers successfully amplified, of which nine were polymorphic with 2–6 alleles (mean 3.7 alleles) and an expected heterozygosity (H _e) ranging from 0.375 to 0.805 (mean H _e = 0.589). On average, we found 2.22 alleles/locus and a mean H _e of 0.440 in one nest, and 2.56 alleles/locus and a mean H _e of 0.494 in the other one. These nine polymorphic markers could be of potential use in studies of genetic variability, population structure and reproductive strategy of European Coots.     

Isolation and characterization of 11 microsatellite loci from Camellia sinensis in Taiwan using PCR-based isolation of microsatellite arrays (PIMA)

We report 11 novel microsatellite primer pairs for the wild tea, Camellia sinensis (L.) O. Kuntze forma formosensis Kitamura. These simple sequence repeat markers were tested in 24 samples collected from wild tea populations, and in cultivars and C. japonica. The number of alleles ranged from 4 to18. The expected (H _E) and observed (H _O) heterozygosity were 0.687–0.946 and 0.042–0.792, respectively. All loci were significantly deviated from Hardy-Weinberg expectations due to the heterozygote deficiency, indicating a dramatic loss of genetic polymorphisms in the rare species. Significant LD was discovered in most loci. These primers may provide a tool for understanding demography and population structure in wild tea.     

Eight polymorphic microsatellite loci for the Chinese medicinal plant Artemisia annua L. (Asteraceae)

Artemisia annua is an important medicinal plant from which Artemisinin was extracted to cure malaria effectively. We developed eight microsatellite markers from the genome of A. annua using the FIASCO protocol. Polymorphism of each locus was assessed in 54 individuals from two Chinese populations. The average allele number of these microsatellites was 3.1 per locus, ranging from 2 to 6. The observed (H _O) and expected (H _E) heterozygosity were from 0.019 to 0.907 and from 0.055 to 0.793, respectively. These microsatellite markers would provide a useful tool for genetic studies of A. annua. H.-R. Huang and G. Zhou have contributed equally to this work.     

Eleven polymorphic simple sequence repeat markers from expressed sequence tags of Pacific oyster Crassostrea gigas EST database

Oyster (Crassostrea gigas) is widely distributed in coastal areas of world. We developed and evaluated simple sequence repeat (SSR) markers from expressed sequence tags (ESTs) of C. gigas and to amplify EST-SSR in C. gigas. Characteristics of 11 EST-SSR loci were investigated using 45 C. gigas individuals. The number of alleles per locus ranged from two to thirteen. The observed heterozygosity (H _o) ranged from 0.0889 to 0.7333 and the expected heterozygosity (H _e) ranged from 0.0859 to 0.8981. Because of their high level of polymorphism, our 11 single-locus EST-SSR markers will be valuable tools for research on mating system, population genetics and systemic evolution of oyster in the future.