A Set of Novel Microsatellite Markers Developed for Luculia yunnanensis (Rubiaceae), an Endangered Plant Endemic to Yunnan, China

The genus Luculia Sweet contains about five species of small trees or shrubs and is a member of the family Rubiaceae (tribe Cinchoneae). Luculia yunnanensis is an endangered ornamental shrub endemic to southwest China. Only two natural populations of L. yunnanensis exist in the wild according to our field investigation. It can be inferred that L. yunnanensis is facing a very high risk of extinction in the wild and an urgent conservation strategy is required. By using a modified biotin-sterptavidin capture method, 24 primer sets were identified in two wild populations. Of these primers, 11 displayed polymorphisms and 13 were monomorphic. The number of alleles per locus ranged from two to four, values for observed and expected heterozygosities ranged from 0.000 to 0.833 and from 0.431 to 0.771, with averages of 0.389 and 0.614, respectively. These markers will be useful for further investigation of conservation of resources, selecting parental types in cross-breeding, evolution of this species at the molecular level and related research in Luculia species.     

Development of 16 Microsatellite Markers for Prince's Pine, Chimaphila japonica (Pyroleae, Monotropoideae, Ericaceae)

The perennial evergreen herb, Chimaphila japonica is found exclusively in East Asian temperate coniferous or sometimes in deciduous forests. By using the Fast Isolation by Amplified Fragment Length Polymorphism (AFLP) of Sequences Containing repeats (FIASCO) protocol, 20 microsatellite primer sets were identified in two wild populations. Of these primers, 16 displayed polymorphisms and 4 were monomorphic. The number of alleles per locus ranged from one to six among populations, values for expected and observed heterozygosities ranged from 0.000 to 0.848 and from 0.000 to 1.000, respectively. The new SSR markers will be useful in obtaining estimates of population-level genetic diversity and in phylogeographic studies of C. japonica.     

Development of Eighteen Microsatellite Markers in Anemone amurensis (Ranunculaceae) and Cross-Amplification in Congeneric Species

Polyploidy plays an important role in the evolution of plant genomes. To enable the investigation of the polyploidy events within the genus Anemone, we developed eighteen microsatellite markers from the hexaploid species A. amurensis (Ranunculaceae), and tested their transferability in five closely related species. The number of total alleles (N(A)) for each resulting locus varied from one to eight. The polymorphism information content (PIC) and Nei's genetic diversity (N(GD)) for these microsatellites ranged from 0.00 to 0.71 and 0.00 to 0.91, respectively. For each population, the N(A) was one to seven, and the values of PIC and N(GD) varied from 0.00 to 0.84 and 0.00 to 0.95, respectively. In addition, most of these microsatellites can be amplified successfully in the congeneric species. These microsatellite primers provide us an opportunity to study the polyploid evolution in the genus Anemone.     

Cross-Species,Amplifiable Microsatellite Markers for Neoverrucid Barnacles from Deep-Sea Hydrothermal Vents Developed Using Next-Generation Sequencing

Barnacles of the genus Neoverruca are abundant near deep-sea hydrothermal vents of the northwestern Pacific Ocean, and are useful for understanding processes of population formation and maintenance of deep-sea vent faunas. Using next-generation sequencing, we isolated 12 polymorphic microsatellite loci from Neoverruca sp., collected in the Okinawa Trough. These microsatellite loci revealed 2–19 alleles per locus. The expected and observed heterozygosities ranged from 0.286 to 1.000 and 0.349 to 0.935, respectively. Cross-species amplification showed that 9 of the 12 loci were successfully amplified for Neoverruca brachylepadoformis in the Mariana Trough. A pairwise F_ST value calculated using nine loci showed significant genetic differentiation between the two species. Consequently, the microsatellite markers we developed will be useful for further population genetic studies to elucidate genetic diversity, differentiation, classification, and evolutionary processes in the genus Neoverruca.     

Isolation and Characterization of New 24 Microsatellite DNA Markers for Golden Cuttlefish (Sepia esculenta)

Twenty-four microsatellite DNA markers were isolated and characterized for golden cuttlefish (Sepia esculenta) from a (GT)(13)-enriched genomic library. Loci were tested in 48 individuals from Jiaozhou bay of China. The numbers of alleles per locus ranged from two to 25 with an average of 10.3. The observed and expected heterozygosities ranged from 0.063 to 0.896 and from 0.137 to 0.953, with averages of 0.519 and 0.633, respectively. Six loci significantly deviated from Hardy-Weinberg equilibrium after Bonferroni's correction and no significant linkage disequilibrium between loci pairs was detected. These microsatellite markers would be useful for analyzing the population genetic structure to make conservation and management decisions for S. esculenta.     

Comparison between Wild and Hatchery Populations of Korean Pen Shell (Atrina pectinata) Using Microsatellite DNA Markers

Pen shell (Atrina pectinata) is a popular food source with a high commercial value in a number of Asian Pacific areas. The natural A. pectinata population has been declining continuously over the past several decades. Microsatellite DNA markers are a useful DNA-based tool for monitoring the genetic variation of pen shell populations. In this study, 20 polymorphic microsatellite (MS) DNA markers were identified from a partial genomic pen shell DNA library enriched in CA repeats, and used to compare allelic variation between wild and hatchery pen shell populations in Korea. A total of 438 alleles were detected at the 20 MS loci in the two populations. All loci were easily amplified and demonstrated allelic variability, with the number of alleles ranging from 5 to 35 in the wild population and from 5 to 22 in the farmed population. The average observed and expected heterozygosities were 0.69 and 0.82, respectively, in the hatchery samples and 0.69 and 0.83, respectively, in the wild samples. Statistical analysis of fixation index (F(ST)) and analysis of molecular variance (AMOVA) showed minor, but significant, genetic differences between the wild and hatchery populations (F(ST) = 0.0106, CI(95%) = 0.003-0.017). These microsatellite loci may be valuable for future aquaculture and population genetic studies for developing conservation and management plans. Further studies with additional pen shell samples are needed to conclusively determine the genetic diversity between the wild and hatchery populations.     

Isolation and Characterization of 11 New Microsatellite Loci in Erigeron breviscapus (Asteraceae), an Important Chinese Traditional Herb

Erigeron breviscapus (Vant.) Hand.-Mazz. (Asteraceae) is a species endemic to southwestern China and an important traditional Chinese herb for cardiovascular and cerebral vessel diseases. Applying a modified biotin-streptavidin capture method, 11 microsatellite loci were discovered. Polymorphism of each locus was assessed in 24 individuals collected from five wild populations. The number of alleles per locus ranged from 2 to 7, with an average of 4.273. The observed (HO) and expected (HE) heterozygosities varied from 0.250 to 0.958 and from 0.337 to 0.786, respectively. Over half of these loci were successfully amplified in two congeneric species. The developed microsatellite markers will be useful for future population genetics and conservation studies, as well as accurate identification of different varieties.     

Development of Microsatellite Markers Using Pyrosequencing in Galium trifidum (Rubiaceae), a Rare Species in Central Europe

We identify a large number of microsatellites from Galium trfidum, a plant species considered rare and endangered in Central and Western Europe. Using a combination of a total enriched genomic library and small-scale 454 pyrosequencing, we determined 9755 contigs with a length of 100 to 6192 bp. Within this dataset, we identified 153 SSR motifs in 144 contigs. Here, we tested 14 microsatellite loci in 2 populations of G. trifidum. The number of alleles and expected heterozygosity were 1-8 (mean 3.2) and 0.00-0.876 (0.549 on average), respectively. The markers described in this study will be useful for evaluating genetic diversity within and between populations, and gene flow between G. trifidum populations. These markers could also be applied to investigate the biological aspects of G. trifidum, such as the population dynamics and clonal structure, and to develop effective conservation programs for the Central European populations of this species.     

Isolation and Identification of Fourteen Microsatellite Markers in Clivia miniata and Clivia nobilis (Amaryllidaceae)

Clivia is a genus of great horticultural importance and has been widely cultivated as ornamental plants in all over the world. In order to assess the phylogenetic relationships and genetic diversity of the wild Clivia species and cultivars, we isolated AC-enriched repeats using FIASCO from a single clone each of C. miniata Regel. and Clivia nobilis Lindl. Of the fourteen repeats, 10 were polymorphic and 4 were monomorphic. The polymorphic marker loci were characterized using 61 Clivia accessions. The number of alleles ranged from two to six, observed heterozygosity ranged from 0.04 to 1.00 and expected heterozygosity ranged from 0.04 to 0.83. These microsatellite marker loci provide tools for future studies of Clivia species and cultivars.     

Development of Novel Microsatellite Markers in the Omei Treefrog (Rhacophorus omeimontis)

Eleven novel microsatellite markers were developed and characterized for the Omei treefrog (Rhacophorus omeimontis) using the fast isolation by AFLP of sequences containing repeats method. Polymorphism of each locus was tested in 24 individuals from two wild populations. The number of alleles per locus ranged from 4 to 15, the average observed and expected heterozygosity per locus ranged from 0.250 to 0.839 and from 0.562 to 0.914, respectively. Two of the 11 microsatellite loci showed significant deviations from Hardy-Weinberg equilibrium. Two locus pairs showed significant linkage disequilibrium. Neither evidence of scoring error due to stuttering nor evidence of large allele dropout was found at all of the 11 loci, but evidence of null alleles was indicated at two loci because of general excess of homozygotes for most allele size classes. These polymorphic loci will be useful markers in studying mate choice of the Omei treefrog.     

Isolation and Characterization of Novel Microsatellite Markers in Pomegranate (Punica granatum L.)

Pomegranate (Punica granatum L.) has been cultivated from ancient times for its economic, ornamental and medicinal properties globally. Here, we report the isolation and characterization of 12 polymorphic microsatellite markers from a repeat-enriched genomic library of Punica granatum L. The genetic diversity of these loci was assessed in 60 genotypes of Punica granatum L. All loci were variable: the number of polymorphic alleles per locus ranged from two to five (average 2.9). The observed and expected heterozygosities ranged from 0.15 to 0.87 and 0.29 to 0.65, respectively. The polymorphic information content ranged from 0.26 to 0.61 (average: 0.43). To the best of our knowledge, this is the first time that polymorphic microsatellite markers have been reported for P. granatum L. These new markers should allow studies of the population structure and genetic diversity of pomegranate to be performed in the future.     

Characterization of 42 Microsatellite Markers from Poison Ivy,Toxicodendron radicans (Anacardiaceae)

Poison ivy, Toxicodendron radicans, and poison oaks, T. diversilobum and T. pubescens, are perennial woody species of the Anacardiaceae and are poisonous, containing strong allergens named urushiols that cause allergic contact dermatitis. Poison ivy is a species distributed from North America to East Asia, while T. diversilobum and T. pubescens are distributed in western and eastern North America, respectively. Phylogreography and population structure of these species remain unclear. Here, we developed microsatellite markers, via constructing a magnetic enriched microsatellite library, from poison ivy. We designed 51 primer pairs, 42 of which successfully yielded products that were subsequently tested for polymorphism in poison oak, and three subspecies of poison ivy. Among the 42 loci, 38 are polymorphic, while 4 are monomorphic. The number of alleles and the expected heterozygosity ranged from 1 to 12 and from 0.10 to 0.87, respectively, in poison ivy, while varied from 2 to 8 and, from 0.26 to 0.83, respectively in poison oak. Genetic analysis revealed distinct differentiation between poison ivy and poison oak, whereas slight genetic differentiation was detected among three subspecies of poison ivy. These highly polymorphic microsatellite fingerprints enable biologists to explore the population genetics, phylogeography, and speciation in Toxicodendron.     

Isolation and Characterization of Microsatellite Markers in the Domestic Ferret (Mustela putorius furo)

The domestic ferret (Mustela putorius furo) is an important model organism for the study of avian influenza and other diseases of humans and animals, as well as a popular pet animal. In order to evaluate genetic diversity and study disease relationships in ferrets, 22 nuclear microsatellite loci (17 dinucleotide and 5 tetranucleotide) were developed from ferret genomic libraries and organized into seven multiplex sets. Polymorphism was preliminarily assessed in one population in Australia and one in the USA, sampled with 25 individuals each. The loci displayed allelic diversity ranging from 1 to 5 alleles, and expected and observed heterozygosities ranging from 0.04 to 0.65 and 0.04 to 0.76, respectively. Additionally, the loci amplified products in 15 samples from the wild ancestor, European polecat (Mustela putorius) and domestic ferret-polecat hybrids. In polecat/hybrid samples, allelic diversity ranged from 3 to 8 alleles, and expected and observed heterozygosities ranged from 0.13 to 0.81 and 0.13 to 0.80 respectively. These markers will be useful for molecular assessments of genetic diversity and applications to evolution, ecology, and health in domestic ferrets and wild polecats.     

Isolation and Characterization of Eleven Polymorphic Microsatellite Loci for the Valuable Medicinal Plant Dendrobium huoshanense and Cross-Species Amplification

Dendrobium huoshanense (Orchidaceae) is a perennial herb and a widely used medicinal plant in Traditional Chinese medicine (TCM) endemic to Huoshan County town in Anhui province in Southeast China. A microsatellite-enriched genomic DNA library of D. huoshanense was developed and screened to identify marker loci. Eleven polymorphic loci were isolated and analyzed by screening 25 individuals collected from a natural population. The number of alleles per locus ranged from 2 to 5. The observed and expected heterozygosities ranged from 0.227 to 0.818 and from 0.317 to 0.757, respectively. Two loci showed significant deviations from Hardy-Weinberg equilibrium and four of the pairwise comparisons of loci revealed linkage disequilibrium (p < 0.05). These microsatellite loci were cross-amplified for five congeneric species and seven loci can be amplified in all species. These simple sequence repeats (SSR) markers are useful in genetic studies of D. huoshanense and other related species and in conservation decision-making.     

Isolation and Characterization of 15 New Microsatellite Markers in Oncomelania hupensis, the Snail Intermediate Host of Schistosoma japonicum in Mainland China

Oncomelania hupensis is the unique intermediate host of Schistosoma japonicum, which plays a key role during the transmission of schistosomiasis. It is mainly found in the Yangtze River valley and mountains or hills in southwest China. In this paper, we described 15 new microsatellite makers in O. hupensis. Polymorphism of each locus was assessed in 80 individuals from four wild populations (n = 20 per population). The number of alleles per locus ranged from 6 to 29, with an average of 15.8. The observed (H(O)) and expected (H(E)) heterozygosities varied from 0.397 to 0.851 and from 0.696 to 0.948, respectively. These microsatellite markers will be useful for population genetic studies and genome mapping in O. hupensis.     

Development of 20 Microsatellite Markers for Solenocera crassicornis and Their Cross-Species Application in Solenocera melantho

Twenty microsatellite markers were isolated and characterized for Solenocera crassicornis from a (GT)13-enriched genomic library. Their polymorphisms were investigated using 44 wild individuals from the South Yellow Sea. Our investigation revealed that all the markers were polymorphic. The number of alleles per locus varied from 6 to 19 with an average of 12.35. The observed and expected heterozygosities ranged from 0.400 to 0.977 and from 0.609 to 0.940, with averages of 0.788 and 0.859, respectively. Four loci significantly deviated from Hardy-Weinberg equilibrium after Bonferroni's correction. Cross-species amplification was also conducted in Solenocera melantho collected from the East China Sea. The result showed that 14 loci could be amplified from Solenocera melantho DNAs. These polymorphic markers would be useful for assessment of genetic variation and population structure of S. crassicornis and S. melantho.     

Development of Seven Microsatellite Markers Using Next Generation Sequencing for the Conservation on the Korean Population of Dorcus hopei (E. Saunders, 1854) (Coleoptera,Lucanidae)

We developed microsatellite markers for genetic structural analyses of Dorcus hopei, a stag beetle species, using next generation sequencing and polymerase chain reaction (PCR)-based genotyping for regional populations. A total of 407,070,351 base pairs of genomic DNA containing >4000 microsatellite loci except AT repeats were sequenced. From 76 loci selected for primer design, 27 were polymorphic. Of these 27 markers, 10 were tested on three regional populations: two Chinese (Shichuan and Guangxi) and one Korean (Wanju). Three markers were excluded due to inconsistent amplification, genotyping errors, and Hardy-Weinberg equilibrium (HWE). By multi-locus genotyping, the allele number, observed heterozygosity and polymorphism information content of seven microsatellite loci were ranged 2‒10, 0.1333‒1.0000, and 0.1228‒0.8509, respectively. In an analysis on the genetic differentiation among regional populations including one Japanese population and one cross-breeding population, the individual colored bar-plots showed that both Chinese populations were closer to each other than to the Far East Asian populations. In Far East Asian populations, Wanju and Nirasaki populations could not be distinguished from each other because the frequency of genetic contents was very similar in some individuals of two populations. Moreover, the cross-breeding population contained all patterns of genetic contents shown in Chinese, Korean, and Japanese populations, compared with the genetic content frequency of each regional population. As a result, we examined whether the cross-breeding population might be a hybrid population, and might contain a possibility of interbreeding with Chinese populations in parental generations. Therefore, these markers will be useful for analyses of genetic diversity in populations, genetic relationships between regional populations, genetic structure analyses, and origin tests.     

Development of 30 Novel Polymorphic Expressed Sequence Tags (EST)-Derived Microsatellite Markers for the Miiuy Croaker, Miichthys miiuy

Expressed sequence tags (ESTs) can be used to identify microsatellite markers. We developed 30 polymorphic microsatellite markers from 5053 ESTs of the Miichthys miiuy. Out of 123 EST derived microsatellites for which PCR primers were designed, 30 loci were polymorphic in 30 individuals from a single natural population with 2-13 alleles per locus. The observed and expected heterozygosities were from 0.1024 to 0.7917 and from 0.2732 to 0.8845, respectively. Nine loci deviated from the Hardy-Weinberg equilibrium, and linkage disequilibrium was significant between 22 pairs of loci. These polymorphic microsatellite loci will be useful for genetic diversity analysis and molecule-assisted breeding for M. miiuy.     

Isolation and Characterization of 21 Microsatellite Loci in Cardiocrinum giganteum var. yunnanense (Liliaceae), an Important Economic Plant in China

Twenty-one microsatellite markers from the genome of Cardiocrinum giganteum var. yunnanense, an important economic plant in China, were developed with a fast isolation protocol by amplified fragment length polymorphism of sequences containing repeats (FIASCO). Polymorphism within each locus was assessed in 24 wild individuals from Gaoligong Mountains in western Yunnan Province, China. The number of alleles per locus ranged from 2 to 4 with a mean of 2.9. The expected and observed levels of heterozygosity ranged from 0.042 to 0.726 and from 0.000 to 1.000, with averages of 0.44 and 0.31, respectively. These polymorphic microsatellite markers should prove useful in population genetics studies and assessments of genetic variation to develop conservation and management strategies for this species.     

Nuclear Microsatellite Primers for the Endangered Relict Fir,Abies pinsapo (Pinaceae) and Cross-Amplification in Related Mediterranean Species

Twelve nuclear microsatellite primers (nSSR) were developed for the endangered species Abies pinsapo Boiss. to enable the study of gene flow and genetic structure in the remaining distribution areas. Microsatellite primers were developed using next-generation sequencing (454) data from a single Abies pinsapo individual. Primers were applied to thirty individuals from the three extant localities. The number of alleles per locus ranged from one to four. Cross-amplification was tested for other Abies species from the Mediterranean Basin, and most of the loci showed higher polymorphisms in the Mediterranean species than in A. pinsapo. These microsatellite markers provide tools for conservation genetic studies in Abies pinsapo as well other Abies species from the Mediterranean Basin.