Isolation and characterization of microsatellite loci in Phalaenopsis gigantea

This paper describes the characterization of the population structure of an ex situ population of Phalaenopsis gigantea, an endangered epiphytic orchid endemic to Sabah, Malaysia, using 30 polymorphic microsatellite loci. The objective of this investigation was the estimation of the genetic distance between accessions within an ex situ population on the basis of which a conservation strategy aimed at selective breeding and restoration was developed.     

Isolation and characterization of microsatellite loci in Shortia rotundifolia (Diapensiaceae), an endangered relict plant on the Ryukyu Islands and Taiwan

Ten microsatellite loci were isolated and characterized for Shortia rotundifolia, a relict temperate plant on the subtropical Ryukyu Islands and Taiwan. The number of alleles ranged from 2 to 21. The expected (H _E) and observed (H _O) heterozygosities were 0.027–0.877 and 0.027–0.622, respectively, from 74 individuals on Iriomotejima Island of Ryukyus. One locus exhibited significantly fewer heterozygotes than expected under Hardy–Weinberg equilibrium (P < 0.05). These primers amplifying microsatellites in this species may provide a useful tool for population genetics to establish conservation strategy.     

Isolation and characterisation of microsatellite loci in Calystegia soldanella (Convolvulaceae), an endangered coastal plant isolated in Lake Biwa,Japan

Eight microsatellite loci of Calystegia soldanella useful for comparisons of the genetic structure of isolated populations in the ancient Lake Biwa and coastal populations in Japan were isolated and characterised. The number of alleles ranged from 2 to 5. The expected (H _E) and observed (H _O) heterozygosities were 0.097–0.583 and 0.000–0.380, respectively, from 100 individuals from Lake Biwa and coastal populations. Seven of the eight loci exhibited significantly fewer heterozygotes than expected based on the Hardy–Weinberg equilibrium (P < 0.05). These primers amplifying microsatellites in C. soldanella may provide a population genetics tool useful in the establishment of a conservation strategy.     

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 15 microsatellite loci from Caridina gracilipes (Atyidae, Decapoda)

Caridina gracilipes, an atyid shrimp of Taiwan, inhabits the fresh and brackish waters. Its life history can be classified into amphidromous and landlocked types. Recent human’s transportation has caused concerns of genetic contamination between these long evolved lineages and ecological instabilities. Molecular markers with high sensitivities are required for detecting the possible introgression due to human disturbance. In the study, we described the development of 15 polymorphic microsatellite loci in C. gracilipes for genetic and ecological studies. These new markers were tested in 20 individuals of three populations. The number of alleles ranged from 6 to 17. Expected (H _E) and observed (H _O) heterozygosities ranged from 0.653 to 0.954, averaged at 0.865, and from 0.000 to 0.4, averaged at 0.146, respectively. All loci are significantly deviated from Hardy–Weinberg expectations due to the heterozygote deficiency. Significant genetic differentiation among populations was detected based on F _ST values, which were estimated from 0.82 to 0.88. Chiao-Chuan Han and Cheng-Sheng Chang are contributed equally to this work.     

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.     

Development and characterization of microsatellite loci in the endangered Tricyrtis ishiiana (Convallariaceae), a local endemic plant in Japan

Nine microsatellite loci were isolated and characterized for the endangered and tertiary relict perennial herb, Tricyrtis ishiiana. The number of alleles ranged from 2 to 33. The expected (H _E) and observed (H _O) heterozygosities were 0.207–0.944 and 0.215–0.813, respectively, from 96 individuals on one population. Five loci exhibited significantly fewer heterozygotes than expected under Hardy–Weinberg equilibrium (P < 0.05). These primers amplifying microsatellites in this species may provide a useful tool for population genetics to establish conservation strategy.     

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 <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 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 11 microsatellite loci for the Sichuan snub-nosed monkey,Rhinopithecus roxellana

We report on the isolation and characterization of 11 microsatellite loci in the Sichuan snub-nosed monkey (Rhinopithecus roxellana) from AC-enriched genomic libraries. 20 microsatellite loci were screened from the libraries, of which 11 were polymorphic. The number of observed alleles among 32 samples of snub-nosed monkey ranged from three to nine. The observed and expected heterozygosity were 0.071–0.815 and 0.201–0.819, respectively. The high variability revealed in this study should be useful tools for further study on social structure and population genetics of snub-nosed monkey in the future.     

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 of Lycopodium fordii Bak. (Lycopodiaceae, Pteridophyta)

Lycopodium fordii Bak. (Lycopodiaceae, Pteridophyta) is a gardening plant with a native, fragmentary distribution in Taiwan. In this study, we described the development of eleven microsatellite loci in L. fordii for genetic studies. These new markers were tested in 16 individuals of three populations. The number of alleles ranged from 1 to 5 and the expected heterozygosity from 0.41746 to 0.72222. Four of the nine polymorphic loci were significantly deviated from Hardy-Weinberg expectations due to the heterozygote deficiency. The microsatellite markers have also been proved as informative genetic markers for other 15 Lycopodium species.     

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 eleven microsatellite loci in small abalone, Haliotis diversicolor Reeve

Eleven novel microsatellite markers were isolated from small abalone, Haliotis diversicolor Reeve. These loci were tested on 22 individuals from two different geographic populations. We identified a total of 162 alleles from the 11 microsatellite loci. All of the loci were highly polymorphic. Polymorphism information content (PIC) is ranging from 0.7276 to 0.9163. Observed and expected heterozygosities ranged from 0.2727 to 1.0000 and from 0.7738 to 0.9429, respectively. Three loci deviated significantly from Hardy–Weinberg equilibrium. No pairs of loci displayed linkage disequilibrium. These polymorphic markers will be used to analyze population structure, genetic diversity and construct a genetic linkage map. Xin Zhan and Hai-Yan Hu contribute equally to this study.     

Isolation and characterization of microsatellite loci in the Amami rabbit (Pentalagus furnessi)

We report on the isolation and characterization of eight polymorphic and five monomorphic microsatellites in the Amami rabbit (Pentalagus furnessi). Microsatellite polymorphism was determined using 25 individuals. There were 2–11 alleles for each polymorphic locus with heterozygosity ranging between 0.08 and 0.76. Linkage disequilibrium was not suggested between any pairs among the eight polymorphic loci. We suggest that these primers be used in future studies to monitor population size, determine dispersal patterns, and genetic diversity within and between populations of this and related species.     

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 polymorphic microsatellite loci from bluefin leatherjacket (Navodon septentrionalis Gunther, 1877)

The first set of 18 polymorphic microsatellite markers were developed from bluefin leatherjacket (Navodon septentrionalis Gunther, 1877). From a (GT)_n-enriched genomic library, we got 121 microsatellites, of which 60 were randomly selected for designing microsatellite primers. Eighteen of these loci were polymorphic in a test population of 32 individuals with alleles ranging from 2 to 9, and expected and observed heterozygosities from 0.1463 to 0.8517 and from 0.1562 to 1.0000, respectively. No significant linkage disequilibrium between pairs of loci was found, but three loci significantly deviated from Hardy–Weinberg equilibrium after Bonferroni correction. These polymorphic microsatellite loci should provide sufficient level of genetic diversity to investigate population structure in bluefin leatherjacket.     

Development and characterization of polymorphic microsatellite loci in the endangered Yangtze finless porpoise (Neophocaena phocaenoides asiaeorientalis)

The finless porpoise (Neophocaena phocaenoides) is one of the smallest cetacean species widely distributed in the shallow coastal waters of the Indo-Pacific Oceans. The population size of the Yangtze subspecies (N. p. asiaeorientalis) has sharply decreased in the last two decades and access to objective data on its population structure and genetic diversity would be of great assistance for their proper management. Here we report on the isolation of nine polymorphic microsatellite using the “Fast Isolation by AFLP of Sequences Containing repeats” (FIASCO) protocol. Polymorphism was assessed using 30 porpoise individuals randomly sampled in the Yangtze River. The number of alleles per locus varies from 2 to 9, with an average value of 5.56, whereas the ranges of observed and expected heterozygosities were 0.300–0.633 (mean 0.496) and 0.473–0.804 (mean 0.659), respectively.     

Isolation and characterization of microsatellite loci in the endangered freshwater fish <Emphasis Type="Italic">Varicorhinus alticorpus</Emphasis> (Cyprinidae)

Varicorhinus alticorpus (Cyprinidae) is an endangered freshwater fish with a scattered distribution in the southern and eastern regions of Taiwan. We described the development of nine polymorphic microsatellite loci in V. alticorpus for genetic studies. These new markers were tested in 20 individuals of the rare species. The number of alleles ranged from 4 to 13. Expected (H _E) and observed (H _O) heterozygosities ranged from 0.692 to 0.892 (averaged at 0.821) and from 0.000 to 0.350 (averaged at 0.088), respectively. All loci are significantly deviated from Hardy–Weinberg expectations due to the heterozygote deficiency. This suite of highly polymorphic microsatellites provides the first chance to undertake a conservation program for this species in Taiwan. It is essential to evaluate its genetic diversity and the fine-scale population structure. Tzen-Yuh Chiang, Teh-Wang Lee, and Feng-Jiau Lin contributed equally to this work.