Development of 10 microsatellite loci for <Emphasis Type="Italic">Rheum tanguticum</Emphasis> (Polygonaceae)

Rhubarb is an important Traditional Chinese Medicine. However, the wild resource has been declining. In order to design appropriate conservation methods for the official species across their natural distributions, it is important to characterize their genetic diversity. Here, we describe the development of 10 new microsatellite loci for AC/TG/CCA in Rheum tanguticum. The microsatellites were enriched using the combined biotin capture method. The polymorphism of each locus was further assessed in 12 individuals from four geographically distinct populations of this species. The number of alleles ranged from three to seven and the expected heterozygosity ranged from 0.53 to 0.73. All markers have been checked in the other three species in the genus and two of them together comprise the official medicinal rhubarb resource with R. tanguticum. These microsatellite markers could provide a useful tool for genetic and conservation studies of the rhubarb species.     

Development of ten microsatellite loci for <Emphasis Type="Italic">Gentiana crassicaulis</Emphasis> (Gentianaceae)

Genetiana crassicaulis is one of famous Chinese medicinal plant. The over-collection for its root has caused its dramatic reduction. In order to devise adequate conservation and management strategies for this species, it is important to characterize its genetic diversity and understand its population structure. Here, 10 polymorphic microsatellite markers have been developed. AC/TG microsatellite was enriched by combining biotin capture method. Polymorphism of each locus was assessed in 30 individuals from six populations. The number of alleles ranged from 2 to 9 and the expected heterozygosity ranged from 0.32 to 0.78.     

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.     

Characterization of novel microsatellite loci in rare minnow (<Emphasis Type="Italic">Gobiocypris rarus</Emphasis>) and amplification in closely related species in Gobioninae

Rare minnow (Gobiocypris rarus) is an endangered small fish endemic to upper reach of the Yangtze River. From a (GT)n enriched genomic library, 32 microsatellites were isolated and characterized. Nineteen of these loci were polymorphic in a test population with alleles ranging from 2–7, and observed and expected heterozygosities from zero to 0.8438, and 0.2679 to 0.8264, respectively. In the cross-species amplifications, 13 out of 19 polymorphic loci were found to be also polymorphic in at least one of the 7 closely related species of the subfamily Gobioninae. These polymorphic microsatellite loci should provide sufficient level of genetic diversity to evaluate the fine-scale population structure in rare minnow and its closely related species for the conservation purpose.     

Polymorphic microsatellite loci in the Australian tree frog, <Emphasis Type="Italic">Litoria peronii</Emphasis>

Eleven novel polymorphic microsatellite loci developed from a microsatellite enriched genomic library, are presented for the Australian tree frog Litoria peronii. We screened 29 individuals from a single population and detected high levels of polymorphism for all 11 loci with the number of alleles/locus ranging from 9 to 24. Values of expected and observed heterozygosities ranged from 0.789–0.955 and 0.207–1.00, respectively. These microsatellite markers should prove useful in determining levels of genetic diversity, measuring gene flow and migration, assigning individuals to their most likely population of origin, and in the assignment of paternity.     

Development and characterization of new microsatellite markers for ginseng (<Emphasis Type="Italic">Panax ginseng</Emphasis> C. A. Meyer)

Panax ginseng C.A. Meyer, commonly known as Korean or Asian ginseng, is a perennial herb native to Korea and China. Its roots are highly prized for several medicinal properties. The present study describes development and characterization of twenty-two polymorphic microsatellite markers for this species. A total of 99 alleles were detected with an average of 4.5 alleles per locus across 20 accessions. Values for observed (H _O ) and expected (H _E ) heterozygosities ranged from 0.05 to 1.00 and from 0.18 to 0.73, respectively. Eleven loci deviated from Hardy–Weinberg equilibrium (P < 0.001). Significant (P < 0.05) heterozygote deficiency was observed at 13 loci. Exact test for linkage disequilibrium showed significant values (P < 0.05) between 12 pairs of loci. These microsatellite markers provide powerful tools for understanding population and conservation genetics of this species and also for genetic differentiation and authentication of different Panax species being used in commercial ginseng products.     

Isolation and characterization of polymorphic microsatellite markers in <Emphasis Type="Italic">Cupressus </Emphasis><Emphasis Type="Italic">chenggiana</Emphasis> S. Y. Hu (Cupressaceae)

Cupressus chenggiana S. Y. Hu (Cupressaceae) is an endemic and endangered conifer species in southwest China. In order to study the population genetics and design the effective conservation methods, we aimed to develop microsatellite primers for this species in the present study. We developed eight new microsatellite loci for this species through biotin capture method. Polymorphism of each locus was further assessed in 18 individuals from three geographically distant populations. The number of alleles per locus ranged from 6 to 11 with an average of 8.13. The observed and expected heterozygosities ranged from 0.219 to 0.296 and from 0.374 to 0.470, with averages of 0.254 and 0.417, respectively. We further found that three of nine microsatellite loci developed previously for another congeneric species showed polymorphic banding patters. We performed primer-crossing tests of these loci in the other two congeneric species which are closely related to C. chenggiana (C. gigantea and C. duclouxiana). These microsatellite markers would be effective for analyzing genetic diversity and population genetic structure of this species and its morphological differentiation with the close relatives.     

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.     

Identification of microsatellite markers in <Emphasis Type="Italic">Hieracium pilosella</Emphasis> L.

In order to investigate the genetic diversity and structure of Hieracium pilosella L., we developed eleven pairs of primers for nuclear microsatellites through screening of three genomic DNA enriched libraries. About 127 individuals from six locations of the Trentino region (Italy) were analysed. These loci were all polymorphic and displayed 5–33 alleles per locus. These microsatellite markers constitute an efficient tool in investigating the genetic patterns of H. pilosella L. populations.     

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 microsatellite loci in <Emphasis Type="Italic">Cryptocarya chinensis</Emphasis> in lower subtropical China

We report on the isolation and characterization of eight microsatellite markers from repetitive DNA enriched libraries for Cryptocarya chinensis from lower subtropical China. These are the first microsatellite loci reported for Cryptocarya species. The number of alleles ranged from three to nine. Observed and expected heterozygosities ranged from 0.0518 to 0.9910 and 0.5241 to 0.7935 for polymorphic loci, respectively. These markers will allow analyses of the baseline genetic variability and population structure of C. chinensis to enrich our scientific understanding of forest fragmentation on genetic health of this species and provide strategies for effective conservation and management in this area.     

Development and characterization of microsatellite markers for an endangered shrub, <Emphasis Type="Italic">Ammopiptanthus mongolicus</Emphasis> (Leguminosae) and cross-species amplification in <Emphasis Type="Italic">Ammopiptanthus nanus</Emphasis>

We developed 11 polymorphic microsatellite markers for an endangered shrub Ammopiptanthus mongolicus using the Fast Isolation by AFLP of Sequences Containing repeats protocol. Polymorphism of each locus was assessed in 24 individuals from five natural populations of A. mongolicus. The average allele number of these markers was 4.2, ranging from 2 to 7. The observed (H _O) and expected (H _E) heterozygosities were 0.044–0.956 and 0.044–0.827, respectively. Further assessment in the congeneric species A. nanus revealed that nine of eleven loci were successfully amplified, but only two loci showed polymorphism. These markers provide powerful tools for the conservation genetics studies of A. mongolicus.     

Polymorphic microsatellite loci for population studies of the razor clam, <Emphasis Type="Italic">Sinonovacula constricta</Emphasis>

The razor clam (Sinonovacula constricta) is an important aquacultured bivalve in China. The natural populations of this species are decreasing quickly. To facilitate studies on genetic diversity and population structure of wild populations, microsatellites were isolated from a CA enriched genomic library. Eight microsatellite loci were polymorphic in 30 individuals from Chongming in Shanghai, China. The number of alleles per polymorphic locus varied from 6 to 13 and the values of observed heterozygosity and expected heterozygosity ranged from 0.350 to 1.000 and from 0.602 to 0.902, respectively. These microsatellites are being used in studying population differentiation and genetic diversity for effective conservation and management genetic resources of S. constricta.     

Genetic analysis of forest species <Emphasis Type="Italic">Eugenia uniflora</Emphasis> L. through of newly developed SSR markers

Nine microsatellite loci for genetic analysis of three populations of the tropical tree Eugenia uniflora L. (pitanga or Brazilian cherry) from fragments of semideciduous forest were developed. We used the technique of building a (GA) _n and (CA) _n microsatellite-enriched library by capture with streptavidin-coated magnetic beads. We assessed the polymorphism of seven microsatellites in 84 mature trees found in three areas (Ribeirão Preto, Tambaú and São José do Rio Pardo), highly impacted by the agricultural practices, in a large region among Pardo river and Mogi-Guaçu river basins, in state of São Paulo, Brazil. All loci were polymorphic, and the number of alleles was high, ranging from 6 to 24, with a mean of 14.4. All stands showed the same high level of genetic diversity (mean H _E  = 0.83) and a low genetic differentiation (mean F _ST = 0.031), indicating that genetic diversity was higher within rather than among populations. Seven of the nine loci were highly variable, and sufficiently informative for E. uniflora. It was concluded that these new SSR markers can be efficiently used for gene flow 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.     

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.     

Characterization of 21 microsatellite marker loci in the ring-tailed lemur (<Emphasis Type="Italic">Lemur catta</Emphasis>)

Ring-tailed lemur (Lemur catta) is the only species in the Genus Lemur, distributed in the deciduous and spiny forests of southwestern Madagascar. This species is listed as endangered due to the loss and fragmentation of its natural habitat, a consequence of deforestation. Twenty-one nuclear microsatellite loci were isolated from a genomic DNA derived from a free-ranging ring-tailed lemur population from the Tsimanampetsotsa National Park, Madagascar. We report various parameter estimates and measures to establish the utility of this marker suite as screened among individuals this single forest fragment.     

Isolation and characterization of microsatellite DNA markers for the flagfish <Emphasis Type="Italic">Jordanella </Emphasis><Emphasis Type="Italic">floridae</Emphasis>

The flagfish (Jordanella floridae) is commonly used in studies of wetlands ecology. Here we describe the isolation of ten microsatellite loci, six of which were polymorphic. The observed number of alleles ranged from 4 to 24 and observed heterozygosities ranged from 0.59 to 0.81 for the polymorphic loci. The isolation of these markers will enable estimations of genetic diversity in natural populations.     

Isolation and characterization of nine microsatellite loci for the takin (<Emphasis Type="Italic">Budorcas taxicolor</Emphasis>)

We report on the isolation and characterization of nine microsatellite markers in the takin (Budorcas taxicolor) from genomic DNA-enriched libraries. Twenty-eight microsatellites were screened from the libraries, and nine of the screened microsatellites were polymorphic. The number of observed alleles for each locus in 28 individuals ranged from two to seven, and the expected and observed heterozygosity was 0.105–0.758 and 0.071–0.821, respectively. Four loci (TK01, TK02, TK04 and TK08) of nine deviated from Hardy-Weinberg expectation and no significant linkage association was found among all these loci. These microsatellite markers provide useful tool for population genetic studies of the takin.     

Assessing Genetic Diversity in <Emphasis Type="Italic">Olea europaea</Emphasis> L. Using ISSR and SSR Markers

Olea europaea L. is one of the most economically important crops in the Mediterranean area, and known for having large genetic variability. In order to assess the genetic diversity, DNA from 41 olive cultivars, present in the protected denomination of origin (PDO) region of Trás-os-Montes, was screened using inter simple sequence repeat (ISSR) and microsatellite (SSR) markers. Eleven ISSR primers amplified 135 reproducible bands of which 108 were polymorphic. The percentage of polymorphic bands detected by ISSR was 79%. The highest number of polymorphic bands was obtained by the use of primers UBC807 (15) and UBC809 (16). A total of 67 alleles were detected by six SSR primers, with an average of 11 alleles per primer. The number of alleles per locus ranged from five (ssrOeUaDCA05) to 15 (ssrOeUaDCA03). The observed heterozygosity ranged from 0.219 (ssrOeUaDCA05) to 0.900 (ssrOeUaDCA04), while the expected heterozygosity varied between 0.426 (ssrOeUaDCA05) and 0.887 (ssrOeUaDCA03). The polymorphism information content (PIC) values ranged from 0.392 (ssrOeUaDCA05) to 0.863 (ssrOeUaDCA03). The collection of primers selected gave a reasonable number of amplification products for the genetic diversity analysis. Based on the results, the genetic diversity among 41 olive cultivars is discussed. This study reveals the great importance of guaranteeing the differentiation of olive cultivars and their application for certification purposes.