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 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.     

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.     

Characterization of 12 polymorphic microsatellite loci in the Japanese bush warbler Cettia diphone

Japanese bush warblers, Cettia diphone, are a common species in Japan and have a polygynous breeding system. In the breeding ground some males have their own territories with more than one female. Male floaters are also not uncommon in the breeding ground. To understand breeding strategy in this species, exact parentage should be elucidated. In order to obtain a tool for this purpose, we isolated 34 microsatellite loci from a genomic library in this species and developed primers for 12 loci. These primers were tested in the Japanese bush warbler and successfully amplified. In analyses of 49 unrelated individuals, allelic numbers ranged from two to 22, and observed heterozygosity (H_O) ranged from 0.27 to 0.854 except for two loci (Cdi29 and Cdi35a) with H_O < 0.1.     

Genetic variation and population structure of endemic yellow catfish, <Emphasis Type="Italic">Horabagrus</Emphasis><Emphasis Type="Italic">brachysoma</Emphasis> (Bagridae) among three populations of Western Ghat region using RAPD and microsatellite markers

Random amplified polymorphic DNA (RAPD) and microsatellite markers were applied to evaluate the genetic variation in endemic and endangered yellow catfish, Horabagrus brachysoma sampled from three geographic locations of Western Ghat, South India river systems. In RAPD, of 32 10-mer RAPD primers screened initially, 10 were chosen and used in a comparative analysis of H. brachysoma collected from Meenachil, Chalakkudy and Nethravathi River systems. Of the 124 total RAPD fragments amplified, 49 (39.51%) were found to be shared by individuals of all 3 populations. The remaining 75 fragments were found to be polymorphic (60.48%). In microsatellites, six polymorphic microsatellite loci were identified by using primers developed for Pangasius hypophthalmus, Clarias macrocephalus and Clarias gariepinus. The identified loci were confirmed as microsatellite by sequencing after making a clone. The nucleotide sequences of 6 loci were published in NCBI genbank. The number of alleles across the six loci ranged from 4 to 7 and heterozygosities ranged from 0.07 to 0.93. The mean number of alleles and effective number of alleles per locus were 5.00 and 3.314, respectively. The average heterozygosity across all investigated samples was 0.72, indicating a significant deficiency of heterozygotes in this species. RAPD and microsatellite methods reported a high degree of gene diversity and genetic distances depicted by UPGMA dendrograms among the populations of H. brachysoma.     

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 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.     

Microsatellite loci for studies on population differentiation and connectivity of the red‐bellied yellow tail fusilier,Caesio cuning (Caesionidae)

Ten polymorphic microsatellites were isolated from the red‐bellied yellow tail fusilier Caesio cuning, a reef‐associated fish which occurs widely in the Indo‐Pacific region. The species is exploited by both small‐scale and commercial fisheries. Fifty individuals from six populations were genotyped using primers that reliably amplified 10 polymorphic loci. The number of alleles per locus ranged from three to 11. Observed heterozygosity (H_O) and expected heterozygosity (H_E) ranged between 0.400 to 0.880 and 0.337 to 0.843, respectively. These microsatellite loci may be used to study population structure, genetic diversity and connectivity of C. cuning in the range of its distribution.     

Development and characterization of 13 novel microsatellite loci from the flat-headed bat (Tylonycteris pachypus) with cross-species amplification in closely related taxa

13 novel microsatellite loci was isolated using the enriched library method from genomic DNA of the flat-headed bat (Tylonycteris pachypus). These loci showed high levels of genetic polymorphism testing on 54 individuals sampled from Guangxi province, China. The number of observed alleles per locus ranged from 2 to 15. The expected and observed heterozygosity values ranged from 0.170 to 0.900 and from 0.185 to 0.944, respectively. One loci departed significantly from Hardy-Weinberg equilibrium (HWE) after Bonferroni correction and no Linkage disequilibrium was found between any pairs of loci. In addition, these loci were tested in the sister species, Tylonycteris robustula, seven loci amplified successfully and were also polymorphic.     

High variability and disomic segregation of microsatellites in the octoploid Fragaria virginiana Mill. (Rosaceae)

The objectives of the present study were to develop microsatellite markers for the wild strawberry, Fragaria virginiana, to evaluate segregation patterns of microsatellite alleles in this octoploid species, and assess genetic variability at microsatellite loci in a wild population. A genomic library was screened for microsatellite repeats and several PCR primers were designed and tested. We also tested the use of heterologous primers and found that F. virginiana primers amplified products in cultivated strawberry, Fragaria × ananassa Duch. and Fragaria chiloensis. Similarly, microsatellite loci developed from cultivated strawberry also successfully amplified F. virginiana loci. We investigated four microsatellite loci in detail, three developed from F. virginiana and one from cultivated strawberry. A survey of 100 individuals from a population of F. virginiana in Pennsylvania demonstrated high heterozygosities (H_e or gene diversity ranged from 0.80 to 0.88 per locus) and allelic diversity (12–17 alleles per locus), but individual plants had no more than two alleles per locus. Segregation patterns in parents and progeny of two controlled crosses at these four loci were consistent with disomic Mendelian inheritance. Together these findings suggest that the genome of F. virginiana is "highly diploidized" and at least a subset of microsatellite loci can be treated as codominant, diploid markers. Significant heterozygote deficiencies were found at three of the four loci for hermaphroditic individuals but for only one locus among females in this gynodioecious species.Communicated by J. Dvorak     

Characterization of microsatellite loci developed for <Emphasis Type="Italic">Amaranthus hypochondriacus</Emphasis> and their cross-amplifications in wild species

The present study reports isolation and characterization of 12 polymorphic microsatellite markers for Amaranthus hypochondriacus. A total of 92 alleles were detected across the 20 accessions, with an average of 7.7 alleles per locus. The observed (H _O ) and expected (H _E ) heterozygosity values ranged from 0 to 0.95 and from 0.49 to 0.92, respectively. At significance threshold (P < 0.05), nine loci deviated from Hardy-Weinberg equilibrium (HWE), whereas significant linkage disequilibria (LD) were observed between five pairs of loci. The 12 loci were successfully amplified in 18 other amaranth species representing cultivated grain and vegetable species, their putative progenitors and wild species. These results demonstrate wide potential applicability of these markers for the study of intra- and inter-specific genetic diversity as well as evolutionary relationships among cultivated and wild amaranths.     

Isolation and characterization of microsatellite loci in <Emphasis Type="Italic">Melicope quadrilocularis</Emphasis> (Rutaceae), an endemic plant species of the Bonin Islands,Japan, and cross-species amplification in closely related taxa

Twelve microsatellite loci were isolated and characterized for Melicope quadrilocularis, an insular endemic tree species of the Bonin Islands. The observed number of alleles at each locus ranged from 1 to 18. The range of expected heterozygosity was 0.0000–0.9445. The inter-specific applicability of these loci was evaluated by analyzing two other endemic species and one endemic variety of Melicope that are also distributed on the Bonin Islands. All primer pairs for the 12 loci tested successfully amplified the loci in all taxa, except for primers for four loci in M. nishimurae.     

Taxonomic composition and ploidy level among European water frogs (Anura: Ranidae: Pelophylax) in eastern Hungary

The western Palaearctic water frogs in the genus Pelophylax comprise several distinct species and three hybridogenetic hybrid forms. In this study, we focus on the Pelophylax esculentus complex, which consists of two sexual species, Pelophylax ridibundus and Pelophylax lessonae, and their hybridogenetic hybrid, Pelophylax esculentus. Specifically, we investigated taxonomic composition and ploidy level of water frogs sampled in three different types of wetland habitats in the Hortobágy National Park (HNP), eastern Hungary. Using variation in serum albumin intron 1 (SAI‐1) and 15 microsatellite loci, we detected the presence of all members of the P. esculentus complex in the studied localities. In one locality, all three taxa occurred syntopically, while in others water frog populations consisted of P. ridibundus and P. esculentus exclusively. The genomic composition of the 63 examined hybrid specimens analysed with microsatellites showed the occurrence of diploid genotypes only. We used a population genetic approach (allelic richness, gene diversity, multilocus genotypes and multilocus disequilibrium) to infer the breeding system of water frogs at HNP. Our data indicate that at least in two populations, hybrids form gametes with clonally transmitted P. ridibundus genome and produce a new hybrid generation by mating with P. lessonae.     

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 microsatellite loci from the orchid Serapias vomeracea (Orchidaceae) and cross‐priming to other Serapias species

In this paper we describe the isolation and characterization of six polymorphic microsatellite loci from the orchid Serapias vomeracea. This species is widely distributed in the Mediterranean region. Microsatellite loci were isolated from an enriched library and primer pairs were designed for 18 loci. Primer pairs for six loci amplified well and were tested on samples from southern Italy. Levels of genetic variability detected at these six loci are high, with numbers of alleles per locus ranging from 3 to 6, and observed heterozygosity (H_O) ranging from 0.35 to 0.86. All primer pairs tested amplified DNA from four other Serapias species, indicating that the primers are useful for population genetic studies throughout the genus.     

Isolation of novel microsatellite loci in the black goby Gobius niger and cross‐amplification in other gobiid species (Perciformes,Gobiidae)

Twelve microsatellite loci were isolated from and characterized for the black goby Gobius niger. These loci were tested on a total of 48 individuals from two geographically distant locations (Orbetello and Cattolica) and the number of alleles ranged from two to 18, with expected (H_e) and observed (H_o) heterozygosities ranging from 0·042 to 0·941 and from 0·042 to 0·917, respectively. The loci described were used to cross‐amplify other gobiid species belonging to Gobius, Zosterisessor, Lesueurigobius and Aphia.     

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.     

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.     

Ten polymorphic microsatellite loci for the Atlantic halibut (Hippoglossus hippoglossus) and cross-species application in related species

In the present study, 10 polymorphic microsatellite DNA loci from Atlantic halibut (Hippoglossus hippoglossus) were isolated and characterized. The number of alleles for these loci ranged from 2 to 4 in tested 24 individuals. Observed and expected heterozygosities per locus varied from 0.21 to 0.70 and from 0.31 to 0.65, respectively. Most of these 10 microsatellite loci were successfully amplified and showed polymorphic in five related species. These loci will be useful for the assessment of genetic diversity and population structure of Atlantic halibut. H. Ding and C. Shao contributed equally to this work.     

Development of 15 polymorphic microsatellite loci from <Emphasis Type="Italic">Garrulax morrisonianus</Emphasis> (Timaliidae), an endemic avian species of Taiwan

Garrulax morrisonianus, an endemic avian species of Taiwan, inhabits evergreen forests at high elevations of 2,000–3,952 m. In this study, we developed 15 microsatellite primer pairs for genetic study. These markers were screened for 52 samples collected from wild populations of different geographical regions. The number of alleles ranged from 4 to 13. The expected (H _E) and observed (H _O) heterozygosities were 0.430–0.725 and 0.000–0.500, respectively. All loci were significantly deviated from Hardy-Weinberg equilibrium due to the heterozygote deficiency. The authors Tzen-Yuh Chiang, Sao-Cheng Lao, Ya-Fu Lee contributed to the study equally.