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.     

A set of microsatellite markers for use in the endangered sea urchin <Emphasis Type="Italic">Strongylocentrotus nudus</Emphasis> developed from <Emphasis Type="Italic">S. purpuratus</Emphasis> ESTs

The first set of nine microsatellite markers for the endangered sea urchin Strongylocentrotus nudus was developed from EST databases of S. purpuratus. Number of alleles per locus ranged from two to thirteen. The observed and expected heterozygosities ranged from 0.000 to 0.645 and from 0.063 to 0.912, respectively. These informative microsatellite markers will be useful in studies of population genetic structure for this species.     

Isolation and characterization of twelve novel microsatellite loci in the ark shell <Emphasis Type="Italic">Scapharca broughtonii</Emphasis>

Scapharca broughtonii is an over-exploited bivalve in China. In order to promote a conservation program for this species, it is necessary to evaluate its genetic diversity and population genetics. For this purpose, we isolated twelve novel polymorphic microsatellite markers and examined their allelic variation using 40 wild individuals collected from one locality along the Chinese coast. The number of alleles ranged from 2 to 22 per locus with the expected heterozygosities ranging from 0.444 to 0.944. These markers will be useful for the assessment of genetic variation for this species.     

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 microsatellite DNA loci for <Emphasis Type="Italic">Pugionium dolabratum</Emphasis> (Brassicaceae), an endangered psammophyte

Pugionium (Brassicaceae) is a small Central-Asian endemic genus with four tentative species described before. All of them grow in the desert habitat and have been considered as important psammophytes for evolutionary studies and ecological restorations. However, the wild resources of these species have been declined because of overexploitation. In this study, 12 polymorphic microsatellite loci have been developed for P. dolabratum Maxim, and the performance of primer pairs amplifying these loci in related three species has also been tested. The number of alleles in 18 individuals from 13 geographically distant populations ranged from three to seven per locus. The observed and expected heterozygosities varied from 0.17 to 0.30 and from 0.32 to 0.48, respectively. We further performed cross-priming tests in the remaining three species of this genus, P. cornutum, P. calcaratum and P. cristatum. These newly recovered microsatellite loci will be useful for studying the population genetics and adaptive evolution of these endangered psammophytes in the desert.     

Isolation and characterization of microsatellite loci in the endangered freshwater fish <Emphasis Type="Italic">Pararasbora moltrechti</Emphasis> (Cyprinidae) using PCR-based isolation of microsatellite arrays (PIMA)

Pararasbora moltrechti (Cyprinidae) is an endangered freshwater fish with a scattered distribution in the central Taiwan. In order to undertake a conservation program for this species in Taiwan, it is essential to evaluate its genetic diversity and population structure. For this purpose, we described the development of eight microsatellite loci in P. moltrechti for genetic studies. These new molecular markers were tested in 36 individuals of the rare species. The number of alleles per locus ranged from 8 to 14. As the expected heterozygosity varied from 0.770 to 0.932, all microsatellite loci, however, were significantly deviated from Hardy–Weinberg expectations due to the heterozygote deficiency. Hung-Du Lin and Teh-Wang Lee contributed equally.     

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.     

Development of 10 polymorphic microsatellite loci primers for Codonopsis pilosula Nannf. (Campanulaceae)

Codonopsis pilosula Nannf., as an important traditional Chinese medicinal plant species, has been used to treat fatigue, thirst and loss of appetite. In this study, we developed 10 new microsatellite loci primers from the genome of this species using the combined biotin capture method. The polymorphism of each locus was further assessed through 27 individuals from four geographically distant populations. The number of alleles per locus ranged from 6 to 11. The observed and expected heterozygosity ranged from 0.15 to 0.24 and 0.27 to 0.40, respectively. We further performed cross-priming tests of these loci in the other three congeneric species. These microsatellite markers will be useful for investigating genetic diversity within and between populations of these species.     

Microsatellite DNA loci from the drought desert plant Calligonum mongolicum Turcz. (Polygonaceae)

Calligonum mongolicum Turcz. (Polygonaceae) is a typical drought desert plant which can tolerate extreme drought, cold, and other stress in the northern extreme drought desert of China. They are playing an important role in maintaining the local ecosystems in these regions. In order to understand the adaptive evolution and develop effective conservation methods of this species in the arid habitats, it is important to know its genetic structure. In this study, 14 polymorphic microsatellite loci were isolated and characterized from an enrichment genomic library of this species. We further assessed the polymorphisms of each locus in 18 individuals from six geographically distant populations. The number of alleles per locus ranged from three to six. The observed and expected heterozygosity ranged from 0.24 to 0.35 and 0.38 to 0.57, respectively. These markers have been crossed checked in another congeneric species, C. mongolicum. These microsatellite markers will be useful for investigating the genetic structure and devising conservation strategies of this species, and morphological divergence between and it and the closely related species.     

Isolation and characterization of microsatellite loci in the Japanese pipistrelle (Pipistrellus abramus)

We describe the first set of ten microsatellite markers isolated in Pipistrellus abramus. The number of alleles per locus ranged from 7 to 13. The observed and expected heterozygosities values ranged from 0.486 to 0.971 and from 0.752 to 0.876, respectively. Three loci revealed significant departure from Hardy–Weinberg equilibrium and no linkage disequilibrium was found between loci pairs. These informative microsatellite markers will be a powerful molecular tool for studying the population genetic structure of P. abramus, as well as other species of this genus.     

Isolation and characterization of microsatellite loci in the Brazilian orchid <Emphasis Type="Italic">Epidendrum fulgens</Emphasis>

Epidendrum fulgens has a patchy distribution along the Atlantic Rainforest in the Brazilian coast, due to the destruction of its native habitat. Here, we report on both the development of nine new microsatellite markers isolated from this species and the characterization of their allele variability in two distant and unrelated populations. The number of alleles observed for each locus ranged from 2 to 17 with an average of 6.4 alleles per locus. These microsatellites should be valuable tools for studying the effect of habitat fragmentation on the genetic structure of E. fulgens populations.     

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 microsatellite DNA primers in Juniperus przewalskii Kom (Cupressaceae)

Juniperus przewalskii (Cupressaceae) is a dominant tree species endemic to the northeast Qinghai-Tibetan Plateau. This species plays an important role in maintaining the arid ecosystem in this region. However, natural distributions of this species have been declined. In order to develop effective conservation methods, it is important to know the distribution of the genetic diversity within and among populations. In this study, we developed nine new microsatellite loci for this species. We used the combining biotin capture method to enrich AG/CT/AC/GGT microsatellites. The polymorphisms of each locus were further assessed in 12 individuals from four geographically distant populations. The number of alleles per locus varied from three to six and expected heterozygosity ranged from 0.58 to 0.70. These loci together provide a useful tool to investigate the genetic diversity of this species. In addition, all markers have been crossly checked in the other four congeneric species.     

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.     

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.     

Microsatellite analysis of genetic diversity in the Chinese alligator (<Emphasis Type="Italic">Alligator sinensis</Emphasis>) Changxing captive population

Chinese alligator (Alligator sinensis) is a critically endangered species endemic to China. In this study, the extent of genetic variation in the captive alligators of the Changxing Reserve Center was investigated using microsatellite markers derived from American alligators. Out of 22 loci employed, 21 were successfully amplified in the Chinese alligator. Sequence analysis showed loci in American alligators had a bigger average size than that of the Chinese alligators and the longest allele of an individual locus almost always existed in the species with longer stretch of repeat units. Eight of the 22 loci were found to be polymorphic with a total of 26 alleles present among 32 animals scored, yielding an average of 3.25 alleles per polymorphic locus. The expected heterozygosity (H _E) ranged at a moderate level from 0.4385 to 0.7163 in this population. Compared to that in the American alligators, a lower level of microsatellite diversity existed in the Changxing population as revealed by about 46% fewer alleles per locus and smaller H _E at the homologous loci. The average exclusion power and the ability to detect shared genotypes and multiple paternity were evaluated for those markers. Results suggested that when the polymorphic loci were combined, they could be sensitive markers in genetic diversity study and relatedness inference within the Chinese alligator populations. The level of genetic diversity present in the current Changxing population indicated an important resource to complement reintroductions based on the individuals from the other population. In addition, the microsatellite markers and their associated diversity characterized in this population could be utilized to further investigate the genetic status of this species.     

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.     

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 polymorphic microsatellite DNA markers for Sasa senanensis (Poaceae: Bambuseae)

Sasa senanensis is a dwarf bamboo species distributed on the floors of cool temperate forests in Japan and adjacent regions. We isolated eight polymorphic microsatellite loci from this species. The number of alleles ranged from two to eight and the observed heterozygosity per locus from 0.13 to 0.74. Seven of the eight loci were also polymorphic in Sasa nipponica. Most of these markers were successfully amplified in other dwarf bamboo species. These markers will be useful for investigating clonal structure and population genetics in some dwarf bamboo species.     

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.