Isolation and characterization of cross-amplification microsatellite panels for species of procapra (bovidae; antilopinae)

The three Procapra species, Tibetan gazelle (P. picticaudata), Mongolian gazelle (P. gutturosa) and Przewalski's gazelle (P. przewalskii) are endemic to Asia. Several intraspecific genetic issues have been studied with species-specific microsatellite loci in these Asian gazelles. However, cross-species microsatellite panels are absent, which inhibits comparative conservation and evolutionary studies of the Procapra. In this study, we isolated 20 cross-species microsatellite loci for Procapra from both related species and the genomic library of P. przewalskii. Fifty-three samples of the three gazelles were used to characterize the markers. Allele numbers ranged from three to 20, with a mean of 7.93 per locus. Observed heterozygosity (H(O)) averaged 0.680 and expected heterozygosity (H(E)) 0.767. The mean polymorphic information content (PIC) was 0.757 for P. picticaudata, 0.803 for P. gutturosa and 0.590 for P. przewalskii. Nine loci were significantly deviated from Hardy-Weinberg (H-W) equilibrium in the three species. Significant linkage disequilibrium was detected in four pairs of loci in P. przewalskii, five pairs in P. gutturosa and 51 pairs in P. picticaudata. Considering the abundance of published loci and their high success rates of cross-amplification, testing and utilization of loci from related species is efficient for wild species of Bovidae. The cross-species microsatellite loci we developed will facilitate further interspecies genetic studies in Procapra.     

Isolation and Characterization of Microsatellite Loci in Pistacia weinmannifolia (Anacardiaceae)

Fourteen polymorphic microsatellite loci were isolated from the genomic DNA of Pistacia weinmannifolia, using the Fast Isolation by AFLP of Sequences Containing repeats (FIASCO) method, and screened on 12 individuals from each of two wild populations. The 14 polymorphic loci had an average of 4.1 alleles per locus varying from 1 to 9. The observed (H(o)) and expected (H(e)) heterozygosities across the two populations ranged from 0.000 to 0.933 and from 0.000 to 0.906, respectively. Tests for departure from Hardy-Weinberg equilibrium (HWE) and genotypic linkage disequilibrium (LD) were conducted for each of the two populations separately. It was found that no locus significantly deviated from HWE proportions and no significant LD was detected between loci (p < 0.001). In the test of cross-species utility, we successfully amplified nine (64.2%) of 14 loci in P. chinensis and four (28.6%) in P. mexicana. The relatively high level of polymorphism for these markers will facilitate further studies of gene flow, population structure and evolutionary history of P. weinmannifolia and its congeners.     

Development and characterization of 20 microsatellite mrkers for Chinese back sleeper, Bostrychus sinensis

Twenty microsatellite markers were isolated and characterized from the Chinese black sleeper, Bostrychus sinensis. Loci were screened in 30 individuals from Taiwan. For each locus, the number of alleles varied from 4 to 22 with mean expected and observed heterozygosity of 0.79 and 0.66, respectively. One locus significantly deviated from Hardy-Weinberg equilibrium after Bonferroni correction and no significant linkage disequilibrium was detected. This set of microsatellites will provide a suitable tool for population genetic studies of Chinese black sleeper.     

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.     

A Molecular Genetic Linkage Map of Eucommia ulmoides and Quantitative Trait Loci (QTL) Analysis for Growth Traits

Eucommia ulmoides is an economically important tree species for both herbal medicine and organic chemical industry. Effort to breed varieties with improved yield and quality is limited by the lack of knowledge on the genetic basis of the traits. A genetic linkage map of E. ulmoides was constructed from a full-sib family using sequence-related amplified polymorphism, amplified fragment length polymorphism, inter-simple sequence repeat and simple sequence repeat markers. In total, 706 markers were mapped in 25 linkage groups covering 2133 cM. The genetic linkage map covered approximately 89% of the estimated E. ulmoides genome with an average of 3.1 cM between adjacent markers. The present genetic linkage map was used to identify quantitative trait loci (QTL) affecting growth-related traits. Eighteen QTLs were found to explain 12.4%–33.3% of the phenotypic variance. This genetic linkage map provides a tool for marker-assisted selection and for studies of genome in E. ulmoides.     

Isolation and Characterization of 13 Microsatellite Loci from a Korean Endemic Species, Sophora koreensis (Fabaceae)

To evaluate the population genetics structure as a means of devising conservation strategies, we developed microsatellite primers for Sophora koreensis, a narrowly endemic and endangered species in Korea. Thirteen polymorphic microsatellite markers were developed in Korean populations of S. koreensis. Genetic diversity was analyzed in 40 individuals from two populations. The number of alleles per locus ranged from 4 to 14, with observed and expected heterozygosities ranging from 0.200 to 1.000 and from 0.189 to 0.864, respectively. The microsatellite markers described here are valuable tools for the population genetics research of S. koreensis. They can be used to obtain information for creating suitable management strategies to conserve this endemic and endangered species.     

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.     

Isolation and Characterization of Microsatellite Loci for Hibiscus aridicola (Malvaceae), an Endangered Plant Endemic to the Dry-Hot Valleys of Jinsha River in Southwest China

Hibiscus aridicola (Malvaceae) is an endangered ornamental shrub endemic to the dry-hot valleys of Jinsha River in southwest China. Only four natural populations of H. aridicola exist in the wild according to our field investigation. It can be inferred that H. aridicola is facing a very high risk of extinction in the wild and an urgent conservation strategy is required. By using a modified biotin-streptavidin capture method, a total of 40 microsatellite markers were developed and characterized in H. aridicola for the first time. Polymorphisms were evaluated in 39 individuals from four natural populations. Fifteen of the markers showed polymorphisms with two to six alleles per locus; the observed heterozygosity ranged from 0.19 to 0.72. These microsatellite loci would be useful tools for population genetics studies on H. aridicola and other con-generic species which are important to the conservation and development of endangered species.     

Isolation and Characterization of Microsatellite Loci in the Chinese Cobra Naja atra (Elapidae)

We characterize thirteen polymorphic microsatellite loci isolated from Naja atra genomic libraries, which were enriched for AC-motif microsatellites. The thirteen loci were screened on a group of 48 individuals from two populations, one in Yong'an and the other in Ganzhou. These markers revealed a relatively high degree of genetic diversity (4-12 alleles per locus) and heterozygosity (Ho ranged from 0.213-0.854 and He ranged from 0.301-0.838). Tests for departure from Hardy-Weinberg equilibrium and for linkage disequilibrium were conducted for each of the two populations separately. After sequential Bonferroni correction, none of the 13 loci showed significant departures from Hardy-Weinberg equilibrium. Hierarchical analysis of molecular variance indicated that a small but significant (P < 0.001) proportion (16.0%) of the total variation in the microsatellite DNA data were attributable to differences among populations, indicating geographical structuring and restricted gene flow. It could be attributable to the Wuyi mountains in the area having a sufficiently isolating effect to significantly reduce gene flow. Our microsatellite data also showed a low N(m) (1.31) value in the two populations from mainland China. Thus, the Yong'an and Ganzhou populations could be treated as distinct evolutionarily significant units (ESUs). The high level of polymorphism revealed by these microsatellite markers will be useful for the study of gene flow, population structure and evolutionary history of N. atra.     

Isolation of Bacteria with Antifungal Activity against the Phytopathogenic Fungi Stenocarpella maydis and Stenocarpella macrospora

Stenocarpella maydis and Stenocarpella macrospora are the causal agents of ear rot in corn, which is one of the most destructive diseases in this crop worldwide. These fungi are important mycotoxin producers that cause different pathologies in farmed animals and represent an important risk for humans. In this work, 160 strains were isolated from soil of corn crops of which 10 showed antifungal activity against these phytopathogens, which, were identified as: Bacillus subtilis, Pseudomonas spp., Pseudomonas fluorescens, and Pantoea agglomerans by sequencing of 16S rRNA gene and the phylogenetic analysis. From cultures of each strain, extracellular filtrates were obtained and assayed to determine antifungal activity. The best filtrates were obtained in the stationary phase of B. subtilis cultures that were stable to the temperature and extreme pH values; in addition they did not show a cytotoxicity effect against brine shrimp and inhibited germination of conidia. The bacteria described in this work have the potential to be used in the control of white ear rot disease.     

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 Polymorphic Microsatellite Loci from Metapenaeopsis barbata Using PCR-Based Isolation of Microsatellite Arrays (PIMA)

The red-spot prawn, Metapenaeopsis barbata, is a commercially important, widely distributed demersal species in the Indo-West Pacific Ocean. Overfishing has made its populations decline in the past decade. To study conservation genetics, eight polymorphic microsatellite loci were isolated. Genetic characteristics of the SSR (simple sequence repeat) fingerprints were estimated in 61 individuals from adjacent seas of Taiwan and China. The number of alleles, ranging from 2 to 4, as well as observed and expected heterozygosities in populations, ranging from 0.048 to 0.538, and 0.048 and 0.654, respectively, were detected. No deviation from Hardy-Weinberg expectations was detected at either locus. No significant linkage disequilibrium was detected in locus pairs. The polymorphic microsatellite loci will be useful for investigations of the genetic variation, population structure, and conservation genetics of this species.     

Application of Novel Polymorphic Microsatellite Loci Identified in the Korean Pacific Abalone (Haliotis diversicolor supertexta (Haliotidae)) in the Genetic Characterization of Wild and Released Populations

The small abalone, Haliotis diversicolor supertexta, of the family Haliotidae, is one of the most important species of marine shellfish in eastern Asia. Over the past few decades, this species has drastically declined in Korea. Thus, hatchery-bred seeds have been released into natural coastal areas to compensate for the reduced fishery resources. However, information on the genetic background of the small abalone is scarce. In this study, 20 polymorphic microsatellite DNA markers were identified using next-generation sequencing techniques and used to compare allelic variation between wild and released abalone populations in Korea. Using high-throughput genomic sequencing, a total of 1516 (2.26%; average length of 385 bp) reads containing simple sequence repeats were obtained from 86,011 raw reads. Among the 99 loci screened, 28 amplified successfully, and 20 were polymorphic. When comparing allelic variation between wild and released abalone populations, a total of 243 different alleles were observed, with 18.7 alleles per locus. High genetic diversity (mean heterozygosity = 0.81; mean allelic number = 15.5) was observed in both populations. A statistical analysis of the fixation index (F_ST) and analysis of molecular variance (AMOVA) indicated limited genetic differences between the two populations (F_ST = 0.002, p > 0.05). Although no significant reductions in the genetic diversity were found in the released population compared with the wild population (p > 0.05), the genetic diversity parameters revealed that the seeds released for stock abundance had a different genetic composition. These differences are likely a result of hatchery selection and inbreeding. Additionally, all the primer pair sets were effectively amplified in another congeneric species, H. diversicolor diversicolor, indicating that these primers are useful for both abalone species. These microsatellite loci may be valuable for future aquaculture and population genetic studies aimed at developing conservation and management plans for these two abalone species.     

Isolation and Characterization of Sixteen Polymorphic Microsatellite Loci in the Golden Apple Snail Pomacea canaliculata

We report the characterization of 16 polymorphic microsatellite markers in the golden apple snail, Pomacea canaliculata, a pest registered in the list of "100 of the world's worst invasive alien species". The fast isolation by AFLP (Amplified Fragment Length Polymorphism) of sequences containing repeats (FIASCO) method was used to isolate microsatellite loci, and polymorphism was explored with 29 individuals collected in an invasive region from China. These primers showed a number of alleles per locus ranging from three to 13. The ranges of observed and expected heterozygosity were 0.310-0.966 and 0.523-0.898, respectively. These microsatellite markers described here will be useful for population genetic studies of P. canaliculata.     

Isolation and Characterization of 13 New Polymorphic Microsatellite Markers in the Phaseolus vulgaris L. (Common Bean) Genome

In this study, 13 polymorphic microsatellite markers were isolated from the Phaseolus vulgaris L. (common bean) by using the Fast Isolation by AFLP of Sequence COntaining Repeats (FIASCO) protocol. These markers revealed two to seven alleles, with an average of 3.64 alleles per locus. The polymorphic information content (PIC) values ranged from 0.055 to 0.721 over 13 loci, with a mean value of 0.492, and 7 loci having PIC greater than 0.5. The expected heterozygosity (H_E) and observed heterozygosity (H_O) levels ranged from 0.057 to 0.814 and from 0.026 to 0.531, respectively. Cross-species amplification of the 13 prime pairs was performed in its related specie of Vigna unguiculata L. Seven out of all these markers showed cross-species transferability. These markers will be useful for future genetic diversity and population genetics studies for this agricultural specie and its related species.     

Novel Polymorphic Microsatellite Loci for the Korean Black Scraper (Thamnaconus modestus), and Their Application to the Genetic Characterization of Wild and Farmed Populations

In this study, we developed 20 polymorphic microsatellite markers for the Korean black scraper, Thamnaconus modestus (Günther, 1877), Monacanthidae, and used them to compare allelic variation between wild and hatchery populations in Korea. All loci were readily amplified and demonstrated allelic variability, with the number of alleles ranging from 5-35 in the wild population and 5-22 in the farmed population. The average observed and expected heterozygosities were estimated, respectively, as 0.74 and 0.80 in the hatchery samples and 0.78 and 0.81 in the wild ones. These results indicate lower genetic variability in the hatchery population than in the wild population and minor, but significant, genetic differentiation between the two populations (F(ST) = 0.005, P < 0.01). Additionally, cross-amplification was tested in another monacanthid species, Stephanolepis cirrhifer; many loci were found that yielded useful information. The high degree of polymorphism exhibited by the 20 microsatellites will be useful in future aquaculture and population genetic studies for developing conservation and management plans.     

Polymorphic Microsatellite Loci Isolated from the Squalidus argentatus Using PCR-Based Isolation of Microsatellite Arrays (PIMA)

Squalidus argentatus (Sauvage and Dabry de Thiersant 1874) is a small-sized freshwater fish which is distributed in Mainland China, Hainan Island and Taiwan. The populations of S. argentatus have dropped sharply probably due to overharvesting and water pollution recently. Eleven polymorphic microsatellite markers were developed for the cyprinid fish S. argentatus. These new markers were tested on 43 individuals collected from Yangtze River and Qiantang River. The number of alleles, observed and expected heterozygosity per locus, in two populations ranged from 3 to 14, from 0.333 to 0.954 and from 0.480 to 0.928, respectively. Only two loci are significantly deviated from Hardy-Weinberg expectations due to the heterozygote deficiency. No significant linkage disequilibrium was detected between the pairwise comparisons of these loci. These polymorphic microsatellite loci will enable us to study the genetic variation, population structure, and conservation genetics of this species in the future.     

Eleven Novel Polymorphic Microsatellite Loci for Oval Squid Sepioteuthis Lessoniana (Shiro-Ika Type)

The oval squid Sepioteuthis lessoniana is one of the most economically important squid species in Japan; however, its population structure is poorly understood due to the lack of hypervariable markers. Such information is critical for managing sustainable fisheries, as well as for ensuring the existence of wild S. lessoniana stocks. Eleven candidate microsatellite loci were isolated from a small insert genomic DNA library. Polymorphisms in these 11 loci were screened in 24 wild individuals. The number of alleles per locus was found to range from 5 to 19 alleles, and the observed heterozygosity ranged from 0.292 to 0.958. No evidence for linkage disequilibrium was detected among all the loci. The genotypic proportions conformed to Hardy-Weinberg equilibrium, except at one locus. In conclusion, these polymorphic microsatellite loci may be used to develop a genetic framework to manage S. lessoniana in the future.     

Isolation and Characterisation of 11 Polymorphic Microsatellite Markers in Papaver rhoeas L. (Corn Poppy), a Major Annual Plant Species from Cultivated Areas

Papaver rhoeas, an annual plant species in the Papaveraceae family, is part of the biodiversity of agricultural ecosystems and also a noxious agronomic weed. We developed microsatellite markers to study the genetic diversity of P. rhoeas, using an enriched microsatellite library coupled with 454 next-generation sequencing. A total of 13,825 sequences were obtained that yielded 1795 microsatellite loci. After discarding loci with less than six repeats of the microsatellite motif, automated primer design was successful for 598 loci. We tested 74 of these loci for amplification with a total of 97 primer pairs. Thirty loci passed our tests and were subsequently tested for polymorphism using 384 P. rhoeas plants originating from 12 populations from France. Of the 30 loci, 11 showed reliable polymorphism not affected by the presence of null alleles. The number of alleles and the expected heterozygosity ranged from 3 to 7.4 and from 0.27 to 0.73, respectively. A low but significant genetic differentiation among populations was observed (F_ST = 0.04; p < 0.001). The 11 validated polymorphic microsatellite markers developed in this work will be useful in studies of genetic diversity and population structure of P. rhoeas, assisting in designing management strategies for the control or the conservation of this species.     

Genetic Diversity of the Critically Endangered Thuja sutchuenensis Revealed by ISSR Markers and the Implications for Conservation

Thuja sutchuenensis Franch. is a critically endangered plant endemic to the North-East Chongqing, China. Genetic variation was studied to assess the distribution of genetic diversity within and among seven populations from the single remnant locations, using inter-simple sequence repeat (ISSR) markers. A total of 15 primers generated 310 well defined bands, with an average of 20.7 bands per primer. The seven populations revealed a relatively high level of genetic diversity in the species. The percentage of polymorphic bands, Nei’s gene diversity and Shannon’s information index at the population and species level were 76.1%, 0.155, 0.252 and 100%, 0.165, 0.295, respectively. A low level of genetic differentiation among populations (G_ST = 0.102), in line with the results of Analyses of Molecular Variance (AMOVA), and a high level of gene flow (N_m = 4.407) were observed. Both the Unweighted Pair Group Method with Arithmatic Mean (UPGMA) cluster analysis and Principal Coordinates Analysis (PCoA) supported the grouping of all seven populations into two groups. In addition, Mantel test revealed no significant correlation between genetic and geographical distances (r = 0.329, p = 0.100). The low genetic differentiation among populations implies that the conservation efforts should aim to preserve all the extant populations of this endangered species.