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.     

Isolation and characterization of microsatellite loci in <Emphasis Type="Italic">Taxus sumatrana</Emphasis> (Taxaceae) using PCR-based isolation of microsatellite arrays (PIMA)

Taxus sumatrana (Miq.) de Laub. (Taxaceae) is an endangered conifer with a scattered distribution in central Taiwan. In this study, we described the development of 12 microsatellite loci in T. sumatrana for genetic studies. These new markers were tested in nine individuals of the rare species. The number of alleles ranged from 3 to 13 and expected heterozygosity from 0.627 to 0.948. Eleven of twelve loci are significantly deviated from Hardy–Weinberg expectations due to the heterozygote deficiency.     

Isolation and characterization of 11 polymorphic microsatellite loci from <Emphasis Type="Italic">Fatsia polycarpa</Emphasis> (Araliaceae), an element of evergreen forests in Taiwan

Fatsia polycarpa Hayata (Araliaceae) is an element of evergreen forests at middle elevations in Taiwan, also representing the southern limitation in distribution of the genus. In this study, we described the development of 11 microsatellite loci from F. polycarpa in Taiwan for genetic studies. These new markers were tested in 20 individuals of 5 populations of the woody species. The number of alleles ranged from 9 to 18. The expected (H _E ) and observed (H _O ) heterozygosities were 0.80–0.95 and 0.10–0.75, respectively. Six of the 11 microsatellite loci are significantly deviated from Hardy–Weinberg expectations likely due to the population structure within samples. No linkage disequilibrium were observed from pairwise comparisons of loci. Chi-Chun Huang, Kuo-Hsiang Hung, and Tsai-Wen Hsu contributed equally to this article.     

Isolation and characterization of microsatellite loci in <Emphasis Type="Italic">Pedicularis verticillata</Emphasis> L. using PCR-based isolation of microsatellite arrays (PIMA)

Pedicularis verticillata L. is a highly valuable herb for traditional Chinese medical treatment. In this report, 11 microsatellite loci from P. verticillata were isolated. The simple sequence repeat (SSR) markers were screened in 23 samples of wild populations of P. verticillata, and eight samples from its sister P. ikomai. The number of alleles ranged from 4 to 13, and value of expected (H _E) and observed (H ₀) heterozygosity was 0.62609–0.89662 and 0–0.95652, respectively. All loci were significantly deviated from Hardy–Weinberg expectations due to the heterozygote deficiency, indicating a dramatic loss of genetic polymorphisms in the restrictedly distributed species. The markers amplified well in the two species are useful for examining genetic diversity and population genetic structure, which, in turn, can provide information for establishing conservation strategy for these endangered species.     

Isolation and characterization of polymorphic microsatellite loci in <Emphasis Type="Italic">Ardisia crenata</Emphasis> (Myrsinaceae)

Ardisia crenata, an evergreen shrub native to East Asia, has been a serious invasive plant to the southeastern USA. Here 13 polymorphic microsatellite loci were isolated and characterized from an enrichment genomic library of A. crenata. The average allele number of these microsatellites was four per locus, ranging from two to seven. The ranges of observed and expected heterozygosity were 0.000–1.000 and 0.239–0.789, respectively. These microsatellite markers will be useful for investigating population genetics and reproductive ecology of A. crenata.     

Isolation and characterization of 12 microsatellite loci from <Emphasis Type="Italic">Suzukia shikikunensis</Emphasis> (Lamiaceae), a genus endemic to Taiwan and Ryukyus

Suzukia shikikunensis Kudo is an endemic plant in Taiwan and suffers habitat destruction caused by human overexploitation. In this study, we developed 12 microsatellite primer pairs for genetic study. These markers were screened for 24 samples collected from wild populations distributed in Taiwan, and for a sister species S. luchuensis collected from Yonaguni and Lutao Islands. The number of alleles ranged from 5 to 14. The expected (H _E) and observed (H _O) heterozygosities were 0.65–0.922 and 0–0.625, respectively. All loci were significantly deviated from Hardy–Weinberg equilibrium due to the heterozygote deficiency. These primers amplifying microsatellites in the two species may provide a useful tool for population genetics to establish conservation strategy.     

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.     

Polymorphic microsatellite loci for the cardinal fish (<Emphasis Type="Italic">Apogon imberbis</Emphasis>)

Eight polymorphic microsatellite loci were isolated and characterized for the Cardinal fish (Apogon imberbis), a coastal-reef fish endemic to the Mediterranean Sea. Characterization of 30 Cardinal fish individuals form the western Mediterranean showed moderate to high allelic diversity ranging from 6 to 19 alleles per locus. Two loci showed significant departures from Hardy-Weinberg equilibrium presumably due to null alleles. No evidence of linkage disequilibrium was found for any locus pairwise comparasions. This microsatellite set could be useful for any basic population genetic studies of this species.     

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.     

Isolation and characterization of microsatellite markers for the endangered <Emphasis Type="Italic">Taxus yunnanensis</Emphasis>

Eleven polymorphic microsatellite loci were characterized for the endangered conifer Taxus yunnanensis. Eight loci were isolated through SSR-anchored PCR, one locus was developed by cross-species amplification tests, while the last two loci were obtained from cross-species microsatellite sequences available in GenBank. Variability of these markers was tested in 48 individuals collected from its main distribution range in Southwest China. The number of alleles per locus ranged from 2 to 9, and the observed and expected heterozygosity varied from 0.000 to 0.625 and from 0.062 to 0.853, respectively. Ten of these eleven loci were significantly deviated from Hardy–Weinberg expectation, except TS07 which showed a distinct heterozygote excess. The availability of these new polymorphic microsatellite markers will provide an ideal marker system for detailed population genetics studies in T. yunnanensis and potentially also for closely related species.     

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.     

Isolation and characterization of polymorphic microsatellite markers for the endangered tree <Emphasis Type="Italic">Handeliodendron bodinieri</Emphasis> (Sapindaceae)

Nine polymorphic microsatellite loci were developed and characterized for Handeliodendron bodinieri, an endangered tree endemic to karst forest in southwest China. Each locus displayed between 4 and 11 alleles. The observed heterozygosity ranged from 0.345 to 0.931 and expected heterozygosity varied from 0.414 to 0.824. The combined probabilities of paternity exclusion over all loci were 0.931 for P _r(Ex₁) and 0.993 for P _r(Ex₂), respectively. These loci represent a powerful tool in investigating gene flow and population structure in natural populations of H. bodinieri.     

<Emphasis Type="Italic">Tiliqua rugosa</Emphasis> microsatellites: isolation via enrichment and characterisation of loci for multiplex PCR in <Emphasis Type="Italic">T. rugosa</Emphasis> and the endangered <Emphasis Type="Italic">T. adelaidensis</Emphasis>

We used an enrichment technique to isolate 18 novel di and tri microsatellites for the socially monogamous lizard Tiliqua rugosa. These loci were amplified in conjunction with previously described loci in two and three PCR multiplexes for T. rugosa and the endangered T. adelaidensis, respectively. The loci were highly polymorphic in both species, exhibiting between 2 and 32 alleles with observed heterozygosity ranging from 0.43 to 0.96. These markers will be useful for population-level analyses and can contribute to a genetic foundation for conservation strategies for the endangered T. adelaidensis.     

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 of microsatellite loci from the endemic and endangered Adriatic sturgeon (<Emphasis Type="Italic">Acipenser naccarii</Emphasis>)

With the aim of elaborating a breeding plan on a captive stock of the highly endangered Adriatic sturgeon (Acipenser naccarii), a total of 10 polymorphic microsatellite loci were isolated from an enriched library. The results of cross amplification of additional 8 loci previously isolated from A. oxyrinchus, A. fulvescens and Scaphyrinchus platorhynchus are also reported. Given the tetraploid condition of the species the genetic variability was estimated basing on the number of alleles per individuals and the average band sharing.     

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.     

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.     

Development and characterization of microsatellite loci in <Emphasis Type="Italic">Ixeridium dentatum</Emphasis> (Asteraceae,Lactuceae)

We isolated and characterized seven microsatellite loci for the perennial herb Ixeridium dentatum ssp. dentatum, an apomictic triploid distributed throughout the lowland areas of East Asia. The number of alleles ranged from two to seven in 32 screened individuals of I. dentatum ssp. dentatum from Japan. The observed and expected heterozygosities were 0.000–0.950 and 0.000–0.891, respectively, calculated using genotypes of 20 individuals of I. dentatum ssp. nipponicum. One locus (msid4) deviated significantly from Hardy–Weinberg equilibrium (P = 0.0001). These microsatellites were also tested for cross-amplification in 11 other taxa of Lactuceae, including five endangered taxa. These primers should be useful genetic tools not only for Ixeridium but also for other Lactuceae taxa.     

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 species of Australian freshwater cod, <Emphasis Type="Italic">Maccullochella</Emphasis>

The Australian freshwater cod genus, Maccullochella is represented by three species: Murray cod, M. peelii peelii, eastern freshwater cod, M. ikei, and trout cod, M.macquariensis. Seven novel microsatellite loci from M. ikei and six previously published loci from M. peelii peelii were tested on wild populations of Murray, eastern and trout cod. Levels of polymorphism varied between species with 13 loci polymorphic in Murray cod, 9 in trout cod and 7 in eastern cod. Observed heterozygosities ranged from 0.053 to 0.842. This suite of microsatellite loci will facilitate future studies of the genetic status of wild and hatchery bred populations of Maccullochella.