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.     

Establishing the foundation for an applied molecular taxonomy of otters in Southeast Asia

Four species of otters (Mustelidae, Lutrinae) occur in Southeast Asia and are considered to be of conservation concern: Aonyx cinerea (Asian small-clawed otter), Lutra lutra (Eurasian otter), Lutra sumatrana (Hairy-nosed otter), and Lutrogale perspicillata (Smooth-coated otter). Among these, L. sumatrana is endemic to the region, yet little is known about its biology, and the precise distribution of all four species in Southeast Asia is not well known. Furthermore, the taxonomy and systematics of L. sumatrana and L. perspicillata have been the subject of controversy, which has implications for the legal protection and for conservation programs of these taxa. To resolve these controversies, we used a multigene data set comprised of segments from 13 nuclear and 5 mitochondrial loci (11,180 nucleotides) to evaluate the phylogenetic relationships of Asian Old World otters. Phylogenies were also estimated using two mitochondrial loci (1,832 nucleotides) obtained from two or more individuals of the four Southeast Asian species. The results from maximum parsimony, maximum likelihood and Bayesian inference showed that L. sumatrana and L. lutra are sister taxa, whereas L. perspicillata is sister to A. cinerea. Furthermore, the results from the two-mitochondrial gene analyses indicate that L. sumatrana is reciprocally monophyletic with respect to L. lutra, supporting the specific validity of the former taxon. Signs such as tracks and feces are often used in field surveys to provide information on the distribution and abundance of otters, but the accuracy of these methods may be compromised when several closely related species occur sympatrically. Therefore, the two-gene data set was used to develop a provisional set of diagnostic nucleotides that can be potentially used to identify the four species of Southeast Asian otters from noninvasively collected biological samples, such as feces. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Eight new polymorphic microsatellite loci from the eastern phoebe (<Emphasis Type="Italic">Sayornis phoebe</Emphasis>)

We describe eight new polymorphic microsatellite loci (5 dinucleotide and three trinucleotide) for the eastern phoebe (Sayornis phoebe) to complement five previously published loci. The number of alleles per locus ranged from 3 to 15 and observed heterozygosities ranged from 0.33 to 0.91. Preliminary screening revealed that loci were polymorphic in other Tyrannidae: Empidonax virescens (n = 10), Tyrannus tyrannus (n = 10), Tyrannus vociferans (n = 5), and Tyrannus melancholicus (n = 28).     

Isolation and characterization of polymorphic microsatellite loci in Candidia barbata (Cyprinidae) using PCR-based isolation of microsatellite arrays (PIMA)

Candidia barbata is an endemic cyprinid fish in Taiwan and suffers habitat destruction caused by human overexploitation. In this study, a total of 14 polymorphic microsatellite loci from C. barbata were isolated and characterized using an optimized protocol to construct a microsatellite-enriched genomic library. The analysis of variability was performed in 30 specimens of Taiwan. The mean number of alleles across loci was 4.92 ± 1.44. The levels of expected and observed heterozygosities varied from 0.1266 to 0.5079, and from 0.0667 to 0.9667, respectively. Frequencies of null alleles of the 14 loci are not significantly greater than zero. No linkage disequilibrium was detected between loci in either population.     

Development and characterization of microsatellite loci for lotus (Nelumbo nucifera)

This paper reports the development of microsatellite primers for Nelumbo nucifera Gaerten. By screening genomic libraries enriched with 10 kinds of probes, Seventeen polymorphic loci were isolated and primers were designed. Polymorphism of these 17 loci was assessed in 24 individuals. All the 17 loci are polymorphic and the number of alleles ranged from two to seven. Observed heterozygosity and expected heterozygosity ranged from 0.0000 to 0.9176 and from 0.2837 to 0.7917 respectively. These microsatellite loci should be useful for studying the genetic diversity of N. nucifera.     

Isolation and characterization of microsatellite loci in endangered Cycas changjiangensis (Cycadaceae)

Eight microsatellite loci were isolated from repetitive DNA enriched libraries for Cycas changjiangensis, an endangered endemic species in Hainan Island, China. The number of allele ranged from three to seven. The observed (H _O) and expected (H _E) heterozygosities ranged from 0.0000 to 0.8750 and from 0.2359 to 0.7582, respectively. These microsatellite loci will enrich our scientific understanding for C. changjiangensis conservation.     

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.     

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 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.     

Development and characterisation of microsatellite loci in Farfugium japonicum (Asteraceae)

Eight microsatellite loci for the perennial herb Farfugium japonicum, including the rheophytic variety luchuense endemic to riparian areas of the Ryukyu Islands, Japan, were isolated and characterised. The number of alleles ranged from 5 to 14. The expected (H _E) and observed (H _O) heterozygosities were 0.344–0.885 and 0.121–0.754, respectively, from 69 individuals in one population. Six loci exhibited significantly fewer heterozygotes than expected under Hardy–Weinberg equilibrium (P < 0.05). The primers amplifying microsatellite sequences in F. japonicum may provide a population genetics tool useful in the establishment of a conservation strategy.     

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 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 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.     

Fifteen polymorphic microsatellite loci from Jamaican streamertail hummingbirds (Trochilus)

We isolated and characterized 15 microsatellite loci from the endemic Jamaican streamertail hummingbird Trochilus polytmus. Loci were screened in 12 individuals of both T. polytmus and its sister species T. scitulus, also a Jamaican endemic. The number of alleles per locus ranged from 2 to 10, observed heterozygosity ranged from 0 to 1, and the probability of identity values ranged from 0.038 to 0.663. These new loci provide tools for characterizing the narrow hybrid zone between the two species.     

Isolation and characterization of microsatellite markers from reed parrotbill, Paradoxornis heudei (Aves: Timaliidae)

Eleven microsatellite markers were obtained from reed parrotbill, Paradoxornis heudei, using the fast isolation by AFLP of sequences containing repeats (FIASCO) method as part of an effort to compare levels of genetic diversity in different populations of China. Polymorphism levels ranged form 5 to 11 alleles (mean = 9.27) using 32 reed parrotbills, with the observed heterozygosity ranging from 0.037 to 0.80. Six loci were significant deviated from HWE and nine loci showed linkage equilibrium. The utility of these loci on two other Passeriformes species, vinous-throated parrotbill (P. webbianus) and oriental great reed warbler (Acrocephalus orientalis), were also tested.     

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.     

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.     

Twelve polymorphic microsatellite loci from a dinucleotide-enriched genomic library of Japanese Spanish mackerel (Scomberomorus niphonius)

In the study, 34 microsatellite loci were isolated from a dinucleotide-enriched genomic library of Japanese Spanish mackerel (Scomberomorus niphonius). And 12 microsatellite loci were found to be polymorphic between 3 and 8 alleles. The number of observed and expected heterozygosity per locus in 23 individuals ranged from 0.6087 to 1.0000 and 0.8908 to 0.9773, respectively. Two loci significantly deviated from Hardy–Weinberg equilibrium after Bonferroni correction analysis and there was no significant linkage disequilibrium found between pairs of loci. As a result, 12 microsatellite loci probably located on different chromosome pairs and these polymorphic microsatellite loci should provide sufficient level of genetic diversity to investigate the fine-scale population structure and evaluate the breeding strategy in S. niphonius. The authors Shi-Chao Xing and Gen-Bo Xu contributed equally.     

Novel microsatellite loci in the threatened European long-snouted seahorse (<Emphasis Type="Italic">Hippocampus guttulatus</Emphasis>) for genetic diversity and parentage analysis

The long-snouted seahorse Hippocampus guttulatus is one of the two European seahorse species. We describe the isolation of the first 12 microsatellite loci in this threatened species. These new markers were tested in non-invasive samples of 32 seahorses from NW Spain. The number of alleles ranged from 2 to 15 (mean: 6.3) and expected heterozygosity from 0.031 to 0.912 (mean: 0.500). All loci conformed to Hardy–Weinberg expectations and no genotypic disequilibrium was observed between any pair of loci. The theoretical exclusion probabilities for this set of loci, when no parental information exists or when one parent is known, were 0.973 and 0.998, respectively. This study indicates the usefulness of these novel loci for population analysis and kinship studies in Hippocampus guttulatus. Their potential application is extended to the other European seahorse species, since all loci were successfully cross-amplified in H. hippocampus.     

Isolation and characterization of microsatellite loci in the commercially important mudshrimp Austinogebia edulis (Upogebiidae) using PCR-based isolation of microsatellite arrays (PIMA)

Populations of mudshrimp, Austinogebia edulis, in the intertidal mud flat of western Taiwan have severely declined due to habitat destruction and overfishing in the past decades. Nine polymorphic microsatellite loci were isolated for this ecologically threatened species by using a polymerase chain reaction-based procedure. The number of alleles per locus ranged from five to 14 in 20 specimens from Central West Taiwan. Observed and expected heterozygosities ranged from 0 to 0.2500 (averaged at 0.0944) and 0.7333 to 0.9385 (averaged at 0.8524), respectively. There were significant departures from Hardy–Weinberg expectations. Significant LD was discovered in most loci. These primers may provide a tool for understanding population structure in A. edulis. Tzen-Yuh Chiang and Hung-Du Lin contributed to the study equally.