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.     

Development of New Microsatellite DNA Markers from Apostichopus japonicus and Their Cross-Species Application in Parastichopus parvimensis and Pathallus mollis

Twenty microsatellite DNA markers were developed for sea cucumber and used to investigate polymorphisms of 60 wild Apostichopus japonicus individuals collected from China. It revealed that all the markers were polymorphic. A total of 164 alleles were detected at 20 loci. The number of alleles per locus varied from 3 to 17 with an average of 8.2, and the expected heterozygosities of each locus ranged from 0.03 to 0.89 with an average of 0.64. Cross-species amplification was also conducted in Parastichopus parvimensis collected from the United States and Pathallus mollis collected from Peru. The result showed that 17 loci amplified Parastichopus parvimensis DNAs while only 4 loci could amplify Pathallus mollis DNAs. All of the polymorphic markers would be useful for future genetic breeding and the assessment of genetic variation within sea cucumbers.     

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 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.     

Rapid Development of Microsatellite Markers with 454 Pyrosequencing in a Vulnerable Fish, the Mottled Skate, Raja pulchra

The mottled skate, Raja pulchra, is an economically valuable fish. However, due to a severe population decline, it is listed as a vulnerable species by the International Union for Conservation of Nature. To analyze its genetic structure and diversity, microsatellite markers were developed using 454 pyrosequencing. A total of 17,033 reads containing dinucleotide microsatellite repeat units (mean, 487 base pairs) were identified from 453,549 reads. Among 32 loci containing more than nine repeat units, 20 primer sets (62%) produced strong PCR products, of which 14 were polymorphic. In an analysis of 60 individuals from two R. pulchra populations, the number of alleles per locus ranged from 1-10, and the mean allelic richness was 4.7. No linkage disequilibrium was found between any pair of loci, indicating that the markers were independent. The Hardy-Weinberg equilibrium test showed significant deviation in two of the 28 single-loci after sequential Bonferroni's correction. Using 11 primer sets, cross-species amplification was demonstrated in nine related species from four families within two classes. Among the 11 loci amplified from three other Rajidae family species; three loci were polymorphic. A monomorphic locus was amplified in all three Rajidae family species and the Dasyatidae family. Two Rajidae polymorphic loci amplified monomorphic target DNAs in four species belonging to the Carcharhiniformes class, and another was polymorphic in two Carcharhiniformes species.     

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 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.     

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.     

Rapid Development of Microsatellite Markers for the Endangered Fish Schizothorax biddulphi (Günther) Using Next Generation Sequencing and Cross-Species Amplification

Tarim schizothoracin (Schizothorax biddulphi) is an endemic fish species native to the Tarim River system of Xinjiang and has been classified as an extremely endangered freshwater fish species in China. Here, we used a next generation sequencing platform (ion torrent PGM™) to obtain a large number of microsatellites for S. biddulphi, for the first time. A total of 40577 contigs were assembled, which contained 1379 SSRs. In these SSRs, the number of dinucleotide repeats were the most frequent (77.08%) and AC repeats were the most frequently occurring microsatellite, followed by AG, AAT and AT. Fifty loci were randomly selected for primer development; of these, 38 loci were successfully amplified and 29 loci were polymorphic across panels of 30 individuals. The H_o ranged from 0.15 to 0.83, and H_e ranged from 0.15 to 0.85, with 3.5 alleles per locus on average. Cross-species utility indicated that 20 of these markers were successfully amplified in a related, also an endangered fish species, S. irregularis. This study suggests that PGM™ sequencing is a rapid and cost-effective tool for developing microsatellite markers for non-model species and the developed microsatellite markers in this study would be useful in Schizothorax genetic analysis.     

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.     

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.     

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 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.     

Isolation and Characterization of Novel Genomic and EST-SSR Markers in Coreoperca whiteheadi Boulenger and Cross-Species Amplification

We described and characterized 11 expressed sequence tag (EST)-derived simple sequence repeats (SSR) and seven genomic (G)-derived SSRs in Coreoperca whiteheadi Boulenger. The EST-SSRs comprised 62.2% di-nucleotide repeats, 32.2% tri-nucleotide repeats and 5.5% tetra-nucleotide repeats, whereas the majority of the G-SSRs were tri-nuleotide repeats (81.4%). The number of alleles for the 18 loci ranged from 3 to 6, with a mean of 3.8 alleles per locus. The observed (Ho) and expected heterozygosities (He) values ranged from 0.375 to 1.000, and 0.477 to 0.757, respectively. The polymorphic information content (PIC) values ranged from 0.466 to 0.706. The mean values number of alleles, Ho, He, and PIC of EST-SSRs were higher than those of the G-SSRs. Four microsatellite loci deviated significantly from Hardy-Weinberg equilibrium (HWE) after Bonferroni correction and no significant deviations in linkage disequilibrium (LD) were observed. These loci are the first to be characterized in C. whiteheadi and should be useful in the investigation of a genetic evaluation for conservation. Compared with 11 loci in C. whiteheadi, 37 potential polymorphic EST-SSRs were found in Siniperca chuatsi (Basilewsky), which will provide a valuable tool for mapping studies and molecular breeding programs in S. chuatsi.     

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.     

The Possible Physical Barrier and Coastal Dispersal Strategy for Japanese Grenadier Anchovy,Coilia nasus in the East China Sea and Yellow Sea: Evidence from AFLP Markers

In order to ascertain the taxonomic status of the Ariake Sea population of Japanese grenadier anchovy, Coilia nasus, and assess the contemporary possible genetic barrier between the west and east coastal waters of the East China Sea, we used amplified fragment length polymorphism (AFLP) markers to detect the genetic structure of C. nasus, in the East China Sea and Yellow Sea. Eighty-one individuals of C. nasus were collected from five locations and 12 individuals of Coilia mystus were sampled from the Yangtze River Estuary. A total of 371 loci were detected by five primer combinations, 310 of which were polymorphic (83.56%). Analysis of molecular variation (AMOVA) and pairwise fixation index (F_ST) revealed significant genetic differentiation among five samples, indicating limited gene flow among populations. The dendrogram for populations by neighbor-joining (NJ) cluster analysis provided evidence of a clear relationship between genetic and geographic patterns, supporting significant genetic differentiation between China coastal populations and Ariake Sea populations. Compared to the genetic divergence between C. nasus and C. mystus, the level of genetic differentiation between China and the Ariake Sea populations of C. nasus is obvious below the species level, indicating isolated populations of C. nasus in the Ariake Sea. Isolation by distance analysis revealed that direct ocean distance with deep-water at the continental slope and high salinity between west and east coastal waters of the East China Sea served as major physical barrier to C. nasus, supporting the coastal dispersal pattern in this estuarine species, and rejecting offshore dispersal strategy.     

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 twelve polymorphic microsatellite Loci for the cocoa mirid bug sahlbergella singularis

Mirids are the primary pests affecting cocoa production in Africa, but no genetic studies have been conducted on these insects. Here we report the isolation and characterization of 12 polymorphic microsatellite loci for Sahlbergella singularis. A microsatellite-enriched genomic DNA library was developed and screened to identify marker loci. Twelve polymorphic loci were identified by screening 28 individuals collected from one presumed population in cocoa plantations in Southern Cameroon. The number of alleles ranged from 5 to 25, whereas the observed and the expected heterozygosities ranged from 0.179 to 0.786 and from 0.671 to 0.946, respectively. Tests showed significant deviations from HW equilibrium for four loci, but no linkage disequilibrium was detected at any of the loci. No cross-species amplification was observed in two other mirid pests in Africa.     

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.