Development of 12 novel microsatellite loci in a traditional Chinese medicinal plant, Epimedium brevicornum and cross-amplification in other related taxa

A total of 12 polymorphic microsatellite loci were developed and characterized for a Chinese medicinal plant, Epimedium brevicornum (Berberidaceae). A genomic DNA enrichment protocol was used to isolate microsatellite loci and polymorphism was explored using 38 individuals from one natural population. The observed number of alleles ranged from 2–14. The ranges of observed and expected heterozygosity were 0.00–0.83 and 0.15–0.88, respectively. In addition, successful cross-species amplification of this set of microsatellite markers in other four medicinal Epimedium species suggested that they would provide a useful tool for the genetic and conservation studies of Epimedium species.     

Isolation and characterization of 23 microsatellite loci in Astragalus camptodontus (Leguminosae), a traditional medicinal plant in Yunnan province

Astragalus camptodontus is a traditional medicinal plant in Yunnan province, and it is used as one of substitutes for the Chinese medicine Astragali radix, namely “Huangqi”. Twenty-three microsatellite loci were developed from its nuclear genome using a fast isolation protocol by amplified fragment length polymorphism of sequences containing repeats (FIASCO). Two wild populations collected from Northwest Yunnan were used to assess polymorphism of each locus. The number of alleles per locus ranged from 2 to 5 with a mean of 4.2. Values for H_O and H_E ranged from 0.042 to 0.875 and from 0.254 to 0.793, with average of 0.403 and 0.616, respectively. These polymorphic loci will facilitate further studies on genetic diversity, population structure and gene flow of A. camptodontus and its allied species.     

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.     

Isolation and characterization of 12 microsatellite loci from cutlassfish (Trichiurus haumela)

The cutlassfish (Trichiurus haumela) is an important commercial fish species. In the present study, we report 12 polymorphic microsatellite DNA markers for cutlassfish. The number of alleles per locus ranged from 2 to 9 in a sample of 26 individuals. Observed and expected heterozygosities per locus varied from 0.2727 to 0.9583 and from 0.4059 to 0.7926, respectively. Two loci (Trha25 and Trha27) showed significant departure from Hardy–Weinberg equilibrium after sequential Bonferroni correction (P < 0.0042). No significant linkage disequilibrium between pairs of loci was found. These microsatellite markers provide powerful tools for investigating genetic population structure, population history and conservation management of cutlassfish. Jin-Zhen Bi and Chang-Wei Shao contributed equally to this work.     

Characterization of polymorphic microsatellite loci in a traditional Chinese medicinal plant,Gastrodia elata

Genetic characterization of germplasm resource is essential for the conservation and efficient utilization of a traditional Chinese medicinal plant, Gastrodia elata. Thirty‐two primer pairs flanking microsatellite repeats were designed and tested using 32 individuals from eight wild populations. A total of 13 microsatellite loci were found highly polymorphic, with three to 10 alleles per locus and gene diversity ranging from 0.400 to 0.841. These microsatellites have been directly applied to the ongoing population and conservation genetics studies.     

Isolation and characterization of polymorphic microsatellite loci in <Emphasis Type="Italic">Achnatherum sibiricum</Emphasis> (Poaceae)

Achnatherum sibiricum is a threaten and toxic perennial bunchgrass mainly in the north of China. We developed 11 polymorphic microsatellite loci from A. sibiricum by combining biotin capture method. After validating and scoring, these loci were polymorphic in a test population with the range of alleles from four to 13 per locus. The observed and expected heterozygosities ranged from 0.1649 to 0.5624 and from 0.3071 to 0.8826, respectively. All 11 microsatellite markers were in Hardy–Weinberg equilibrium. These polymorphic microsatellites will be useful for genetic diversity analysis and population structure construction for A. sibiricum.     

Isolation and characterization of microsatellite loci in Acer opalus (Aceraceae), a sexually-polymorphic tree, through an enriched genomic library

An enrichment protocol was used to isolate and characterize microsatellite loci in Acer opalus, a Mediterranean tree species. Highly polymorphic microsatellite loci were required for paternity analyses in a population of this species. Eight microsatellite loci were amplified and a total of 87 alleles were detected in a sample of 142 individuals from one population, allowing the identification of each individual with a unique multilocus genotype. The paternity exclusion probabilities varied from 0.261 for locus Aop820 to 0.806 for locus Aop450, and the parent-pair exclusion probabilities varied from 0.433 for Aop820 to 0.940 for Aop450. The cumulative probabilities of exclusion for paternity and parentage of the eight loci were both higher than 0.999, supporting the usefulness of these microsatellite loci for future paternity and parentage analyses in A. opalus. Cross-species transferability was also assayed, supporting their potential use in other eight Acer species.     

Isolation via enrichment and characterization of 14 dinucleotide microsatellite loci in one catfish, Glyptosternum maculatum and cross-amplification in other related taxa

A total of 14 novel dinucleotide microsatellite loci were developed using genomic DNA enrichment protocol and characterized for one Glyptosternoid catfish, Glyptosternum maculatum. Polymorphism was explored using 36 individuals from one natural population. The observed number of alleles ranged from 2 to 9. The ranges of observed and expected heterozygosity were 0.0278 to 1 and 0.4319 to 0.8498, respectively and the average of PIC was 0.5754. In addition, successful cross-species amplification of these set of microsatellite markers in the related taxa, Pseudechenneis sulcatus (McClelland) suggested that they would provide a useful tool for the genetic and conservation studies of Sisoridae species.     

Nortriterpenoids and lignans from Schisandra sphenanthera

Nortriterpenoids, sphenadilactone C (1) and sphenasin A (2), together with four known lignans (3-6), were isolated from the leaves and stems of Schisandra sphenanthera. Their structures were elucidated by extensive analysis of 1D and 2D NMR spectroscopic data and compound 2 was further confirmed by single-crystal X-ray diffraction. Compound 1 features a partial enol moiety and an acetamide group in its structure. In addition, compounds 1, 3-6 showed weak anti-HIV-1 activity with EC₅₀ values in the range of 15.5-29.5 μg/mL.     

Isolation and characterization of eight polymorphic microsatellite loci for the critically endangered Arenaria nevadensis (Caryophyllaceae)

We report on the isolation and characterization of eight microsatellite markers from enriched libraries for Arenaria nevadensis, one of the most critically endangered plant species in the Iberian Peninsula. These are the first microsatellite loci reported for Arenaria species. The number of alleles ranged from two to eight, and the expected heterozygosity from 0.067 to 0.873. These markers will be useful for characterizing the genetic diversity in A. nevadensis and understanding its population structure, and will provide important genetic data for the conservation and recovery of this species.     

Isolation and characterization of microsatellite loci in Ostryopsis davidiana (Betulaceae)

Ostryopsis (Betulaceae) is a samll genus endemic to China with only two species. Both of them play an important role in restoring the local ecosystems. The distribution of genetic diversity between and within populations in each species are important to further utilize the wild genetic resources and explore the interspecific divergence. In this study, we developed 10 microsatellite loci from O. davidiana by the combining biotin capture method for the first time. A total of 27 microsatellite sequences were recovered through screening the library and 10 of them are polymorphic. The number of alleles per locus in 18 sampled individuals ranged from 3 to 6, expected heterozygosity and observed heterozygosity ranged from 0.2958 to 0.4767 and from 0.1591 to 0.2997, respectively. In addition, all markers have been crossly checked in the other congeneric species. These microsatellite markers would together provide a useful tool for investigating the genetic diversity and structure of both species and speciation mechanism between them.     

Isolation and characterization of polymorphic microsatellite loci from black sea bass (Centropristis striata) and cross-species amplification

Black sea bass (Centropristis striata) is an economically important serranid species. A number of 39 microsatellite loci were isolated from two enriched genomic library of C. striata. Eleven of these loci were polymorphic in a test population with alleles per locus ranging from 3 to 8, and observed and expected heterozygosities per locus from 0.26 to 1.00 and from 0.61 to 0.84, respectively. Four loci significantly deviated from Hardy–Weinberg equilibrium after Bonferroni correction and no significant linkage disequilibrium was found between pairs of loci. Cross-species amplification of these polymorphic microsatellite loci was performed in additional two related species. These polymorphic microsatellite loci would be useful for investigating genetic resource of C. striata and other related 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.     

Twelve polymorphic microsatellite loci for <Emphasis Type="Italic">Achnatherum inebrians</Emphasis> (Poaceae)

Microsatellites are often highly variable and abundant in most complex genomes, therefore are widely used in population genetic studies. In this study, twelve polymorphic microsatellite loci were isolated and characterized for the Achnatherum inebrians, a plant abundant in grasslands of Northwest China. Characterization of 24 A. inebrians individuals form four geographically distant populations (Gansu, Qinghai, Xinjiang and Inner Mongolia provinces) showed moderate to high allelic diversity ranging from 3 to 13 alleles per locus, and the expected heterozygosity ranging from 0.41 to 0.67. No evidence of linkage disequilibrium was found for any locus pairwise comparisons. The markers described here will be useful for investigating the genetic diversity, genetic structure and gene flow of this species. Na Chen and Yan-Zhuo Yang contributed equally to this work.     

Isolation and characterization of polymorphic nuclear microsatellite loci in <Emphasis Type="Italic">Taxus baccata</Emphasis> L.

Seven polymorphic nuclear microsatellite markers for Taxus baccata L. (English yew) were developed using an enriched-library method. An additional polymorphic SSR was obtained by testing eight primer pairs from the congeneric species Taxus sumatrana. Mendelian inheritance for the seven Taxus baccata SSRs was proved by genotyping 17 individuals and 124 megagametophytes (conifer seed haploid tissue). A total of 96 individuals from 5 different populations (10–26 samples per population) were used to estimate genetic diversity parameters. High levels of genetic diversity, with values ranging from 0.533 to 0.929 (6–28 alleles per SSR) were found. No linkage disequilibrium between pairs of loci was detected. All loci but one showed significant departures from Hardy–Weinberg equilibrium. Excess of homozygosity was probably due to high inbreeding in English yew populations, an outcome of low effective population size and/or fragmented distribution. Highly polymorphic SSRs will be used to conduct population genetic studies at different geographical scales and to monitor gene flow.     

Isolation and characterization of 18 microsatellite loci in the Egyptian vulture (Neophron percnopterus)

We developed 18 new microsatellite loci for the endangered Egyptian vulture (Neophron percnopterus). Microsatellite loci were screened for variation in two different populations belonging to separate subspecies: the nominal N. p. percnopterus and the Canarian N. p. majorensis. Mean expected heterosygosities were respectively 0.51 and 0.46, while the mean number of alleles per locus was 4.7 and 3.9. These new markers allow further genetic studies for the endangered Canarian Egyptian Vulture.     

Isolation and characterization of thirteen polymorphic microsatellite loci for Lithocarpus glaber

We isolated and characterized polymorphic microsatellite loci in Lithocarpus glaber (Fagaceae), an evergreen broadleaved monoecious tree, to provide tool for analyzing genetic structure and diversity. Thirteen polymorphic microsatellite loci were developed and tested in two L. glaber populations. The number of alleles per locus varied from 2 to 19. The observed and expected heterozygosities within populations were 0.037–0.833 and 0.316–0.931, respectively. Four loci significantly deviated from Hardy–Weinberg equilibrium after Bonferroni correction in each population and no significant linkage disequilibrium between pairs of loci was found. These polymorphic loci showed high levels of polymorphism within tested populations and will be useful in further population genetic studies.     

Isolation and characterization of polymorphic microsatellite DNA markers in the rock bream (Oplegnathus fasciatus)

From a (GT)₁₃-enriched genomic library of Oplegnathus fasciatus, 14 polymorphic microsatellite were isolated and characterized in a test population with alleles ranging from two to nine, the observed and expected heterozygosities from 0.0000 to 1.0000, and from 0.1726 to 0.8507, respectively. Five loci deviated from the Hardy–Weinberg equilibrium, and linkage disequilibrium between two loci was significant. Two loci were also polymorphic in Pagrosomus major assessed for cross-species amplification. These polymorphic microsatellites will be useful for genetic diversity analysis and molecule-assisted breeding for O. fasciatus. Changwei Shao contributed equally.     

Development and characterization of highly polymorphic microsatellite loci for the Western Spadefoot toad, Pelobates cultripes

Nine highly polymorphic microsatellite markers were isolated and characterized for the Western Spadefoot, Pelobates cultripes. Remarkably, for this amphibian species high numbers of microsatellites were found as part of larger repeat containing regions, making primer design difficult. For nine loci, primers were designed successfully and genotyping of individuals was reliable and consistent. Number of alleles and heterozygosity for these loci ranged from 9 to 34 and from 0.72 to 0.94, respectively. The high levels of polymorphism revealed by our developed loci should provide insight into population genetic structure and levels of dispersal for this typical Mediterranean temporary pond-breeding amphibian.     

Development and characterization of microsatellite loci in Chinese medicinal plant Akebia trifoliate ssp. australis and cross-species amplification in closely related taxa

Eleven polymorphic microsatellite loci were isolated and characterized from an AC-enriched genomic library of Akebia trifoliate ssp. australis. The number of allele per locus ranged from 3 to 14. The observed heterozygosity and expected heterozygosity at population level were 0.196–1.000 and 0.522–0.902, respectively. In addition, this set of microsatellites produced robust cross-species amplification in other two related taxa, suggesting these microsatellite markers should provide a useful tool for genetic and conservation studies of the Akebia species.