Twelve novel polymorphic microsatellites in a marine fish species,yellow croaker Larimichthys polyactis

We isolated 12 polymorphic microsatellites from an important marine food fish Larimichthys polyactis and characterized them in 32 unrelated individuals. Among the 12 microsatellites, four were tetranucleotide repeats and eight were dinucleotide repeats. The allele number ranged from five to 25 with an average of 15.4/locus; average expected heterozygosity was 0.81, ranging from 0.57 to 0.95, whereas the observed heterozygosity ranged from 0.34 to 1.00 (average: 0.78). Nine of the 12 markers conformed to Hardy–Weinberg equilibrium and showed no sign of linkage. These microsatellites will be useful for population genetic studies and selective breeding programs of this species.     

Isolation and characterization of dinucleotide microsatellites in greater amberjack, <Emphasis Type="Italic">Seriola dumerili</Emphasis>

Thirteen nuclear-encoded dinucleotide microsatellites were characterized from a genomic DNA library of greater amberjack, Seriola dumerili. The microsatellites include 12 perfect-repeat motifs and one imperfect-repeat motif. The number of alleles at the 13 microsatellites among a sample of 29 fish ranged from 3 to 25; gene diversity (expected heterozygosity) ranged from 0.296 to 0.948, while observed heterozygosity ranged from 0.276 to 0.897. Following Bonferroni correction, genotypes at all 13 microsatellites fit expectations of Hardy-Weinberg equilibrium. One pairwise comparison of microsatellites deviated significantly from expectations of genotypic equilibrium, suggesting that these two microsatellites may be linked. Greater amberjack support commercial and recreational fisheries along both the Atlantic and Gulf coasts of the U.S. and represent a species with potential for worldwide aquaculture. The microsatellites developed will be useful for conservation and population genetic studies of ‘wild’ and domesticated populations of greater amberjack.     

Description of 21 microsatellites for the Chinese bitterling,Rhodeus sinensis Günther, 1868

Chinese bitterling (Rhodeus sinensis) is a freshwater fish endemic to China. During the past several decades, natural populations of this species have become endangered due to habitat fragmentation and water pollution. To help conserve and restore R. sinensis, Illumina Miseq sequencing was used to identify 21 polymorphic microsatellites. The number of alleles varied from 3 to 13, and the observed heterozygosity and expected heterozygosity ranged from 0.3067 to 0.9145 and 0.3845 to 0.9521, respectively. The polymorphism information content ranged from 0.3173 to 0.9105. These novel microsatellites will be useful in both conservation and management of R. sinensis.     

Isolation and characterization of microsatellites in a marine food fish species,golden trevally Gnathanodon specious

The first set of six polymorphic microsatellites was isolated from a partial genomic DNA library of an important marine food fish Gnathanodon specious, and was characterized in 28 unrelated individuals. The allele number ranged from three to 12 with an average of seven/locus; average expected heterozygosity was 0.57 ranging from 0.10 to 0.87, whereas the observed heterozygosity ranged from 0.11 to 0.96 (average: 0.57). All six markers conform to Hardy–Weinberg and linkage equilibria. These markers will be useful for accessing genetic population structure of wild populations and for selective breeding program in fish farms.     

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.     

Characterization of microsatellite markers derived from sequence databases for the European flounder (Platichthys flesus)

The European flounder (Platichthys flesus; Pleuronectidae) is an important commercial fish species. Nevertheless, no microsatellites for this species have been published to date. The sequence of flounder genes present in the public database was searched for short tandem repeats. Out of 12 microsatellite pairs derived from Sequenced Databases, five amplified successfully with an expected heterozygosity ranging from 0.43 to 0.75. The average allele number of these microsatellites was 4.8 per locus with a range between two and nine.     

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.     

Isolation and characterization of microsatellite DNA primers for <Emphasis Type="Italic">Sinadoxa corydalifolia</Emphasis> (Adoxaceae)

Sinadoxa corydalifolia is the only species of Sinadoxa (Adoxaceae) with the aberrant morphology. This species has become extremely endangered in the Qinghai-Tibetan Plateau. To provide a population-level genetic profile for investigation and conservation of genetic diversity of this species, we developed 10 new microsatellite loci for this species by the combining biotin capture method. About 31 microsatellites were screened from the library, 10 of the screened microsatellites are polymorphic. The number of alleles per locus in 18 individuals ranged from 3 to 11, expected heterozygosity and observed heterozygosity ranged from 0.3071 to 0.6243 and from 0.1675 to 0.4357, respectively. We further performed cross-priming tests of these primers in another species of the Adoxaceae: Adoxa moschatellina and found 9 of 10 successfully amplified the targeted sequences. These newly developed loci provide a useful tool to investigate the genetic diversity and design the conversation measures of S. corydalifolia and study the genetic divergence and the initial speciation pattern between it and the related species in the Adoxaceae.     

Isolation and characterization of microsatellites from a marine foodfish species ribbonfish Trichiurus haumela

The ribbonfish, Trichiurus haumela, is an important marine foodfish species. Supply of this fish relies on capture from the wild. No information about genetic diversity and population structure of wild stocks is available. Therefore, we developed the first set of 10 polymorphic microsatellites. The average allele number was 13.2 per locus ranging from six to 20. The expected heterozygosity was 0.84, whereas the observed heterozygosity was 0.71. This set of microsatellites provides a tool for assessing genetic diversity and population structure of this species.     

Polymorphic microsatellite loci for population studies of the razor clam, <Emphasis Type="Italic">Sinonovacula constricta</Emphasis>

The razor clam (Sinonovacula constricta) is an important aquacultured bivalve in China. The natural populations of this species are decreasing quickly. To facilitate studies on genetic diversity and population structure of wild populations, microsatellites were isolated from a CA enriched genomic library. Eight microsatellite loci were polymorphic in 30 individuals from Chongming in Shanghai, China. The number of alleles per polymorphic locus varied from 6 to 13 and the values of observed heterozygosity and expected heterozygosity ranged from 0.350 to 1.000 and from 0.602 to 0.902, respectively. These microsatellites are being used in studying population differentiation and genetic diversity for effective conservation and management genetic resources of S. constricta.     

PCR primers for trinucleotide and tetranucleotide microsatellites in greater amberjack,Seriola dumerili

Eighteen nuclear‐encoded microsatellites from a genomic DNA library of greater amberjack, Seriola dumerili, were isolated and characterized. The microsatellites include 13 perfect (five tetranucleotide and eight trinucleotide) and five imperfect (three tetranucleotide, one trinucleotide and one combination dinucleotide/trinucleotide) repeat motifs. The number of alleles at the 18 microsatellites among a sample of 29 fish ranged from two to 20; gene diversity (expected heterozygosity) ranged from 0.068 to 0.950, whereas observed heterozygosity ranged from 0.069 to 0.966. Following Bonferroni correction, genotypes at all 18 microsatellites fit expectations of Hardy–Weinberg equilibrium, and all pairwise comparisons of microsatellites did not deviate significantly from genotypic equilibrium. Greater amberjack support commercial and recreational fisheries along both the Atlantic and the Gulf coasts of the USA and represent a species with potential for worldwide aquaculture. The microsatellites developed will be useful for population genetic studies of ‘wild’ populations and breeding studies of domesticated populations.     

Isolation and characterization of polymorphic microsatellite loci in a traditional Chinese medicinal plant, Schisandra sphenanthera

Schisandra sphenanthera (Schisandraceae) is a traditional Chinese medicinal plant. In this study, nine polymorphic microsatellite loci were developed and characterized for the species. A genomic DNA enrichment protocol was used to isolate microsatellite loci and polymorphism was explored using 36 individuals sampled from a natural population. The observed number of alleles ranged from 2 to 10 with an average of 5.2. Expected and observed heterozygosity ranged from 0.30 to 0.82 and 0.33 to 0.97, respectively. These microsatellites have been directly applied to both population and conservation genetic studies of S. sphenanthera.     

Development and Characterization of RAPD and Microsatellite Markers for Genetic Variation Analysis in the Critically Endangered Yellow Catfish <Emphasis Type="Italic">Horabagrus nigricollaris</Emphasis> (Teleostei: Horabagridae)

Random-amplified polymorphic DNA (RAPD) and microsatellite markers were developed and used for the analysis of genetic variability in the critically endangered yellow catfish Horabagrus nigricollaris, sampled from the Chalakkudy River, Kerala, India. Eight RAPD and five microsatellite markers were detected to genotype the species. In RAPD, the 73 fragments were 20.55% polymorphic, whereas 4 polymorphic loci (80%) were obtained in microsatellites. In microsatellites, the number of alleles across the 5 loci was 1-5, and the range of heterozygosity was 0.25-0.5. The mean observed number of alleles was 2.4, and the effective number was 1.775 per locus. The average heterozygosity across all investigated samples was 0.29, indicating a significant deficiency of heterozygotes in this species. RAPD and microsatellite methods report a low degree of gene diversity and lack of genetic heterogeneity in the population of H. nigricollaris, emphasizing the need for fishery management, conservation, and rehabilitation of this species.     

Isolation and characterization of novel microsatellite markers in commercial selected golden Malaysian arowana fish, Scleropages formosus (Osteoglossidae)

Malaysian arowana (dragonfish; Scleropages formosus) is an ancient osteoglossid fish from southeast Asia. Due to the high demand of the ornamental fish trade and because of habitat loss, the species is close to extinction. We isolated and characterized 10 polymorphic microsatellites of this species, using 5'-anchored PCR. The number of alleles at the 10 microsatellite loci ranged from 2 to 28, with a mean of 7.8/locus. The observed heterozygosity ranged from 0.03 to 0.93 (mean: 0.39), whereas the expected heterozygosity ranged from 0.03 to 0.94 (mean: 0.46). Seven microsatellites deviated from Hardy-Weinberg equilibrium, and three conformed to Hardy-Weinberg equilibrium and were in linkage equilibrium. These 10 novel microsatellites should facilitate studies of genetic diversity and population structure of arowana to help plan actions for the conservation of the indigenous Malaysian arowana.     

Isolation and characterization of microsatellite loci in the xishishe clam <Emphasis Type="Italic">Coelomactra antiquata</Emphasis> (Bivalvia: Veneroida)

The xishishe clam Coelomactra antiquata is a commercially important bivalve species, but has been suffering from severe population decline due to over-exploitation and the deterioration of environmental conditions. To investigate the genetic variation and structures of this clam, nine polymorphic microsatellites were developed. The number of alleles ranged from two to 28 per locus with the observed heterozygosity ranging from 0.083 to 0.921. This set of microsatellites will be useful for the study of the genetic structures of C. antiquata.     

Genetic variation and phylogenetic relationship between two species of yellow catfish, <Emphasis Type="Italic">Horabagrus brachysoma</Emphasis> and <Emphasis Type="Italic">H. nigricollaris</Emphasis> (Teleostei: Horabagridae) based on RAPD and microsatellite markers

The two species of yellow catfish, Horabagrus brachysoma and H. nigricollaris are categorized as ‘endangered’ and ‘critically endangered’ respectively in their wild habitat. Proper knowledge of genetic structure and variability of these endangered species are highly essential for the management, conservation and improvement of fish stocks. Therefore, genetic variation and phylogenetic relationships between these species of yellow catfish sampled from Chalakkudy River in the hot spot of biodiversity-Western Ghats region, Kerala, India were analyzed by using Random amplified polymorphic DNA (RAPD) and microsatellite markers. 85 RAPD and five microsatellites loci were detected to analyze the genetic variation and phylogenetic relationships among these species. Out of 85 RAPD loci produced only 52.94% were polymorphic whereas in microsatellite, all 5 loci were polymorphic (100%). Species-specific RAPD bands were found in both species studied. In microsatellite, the number of alleles across the five loci ranged from 1 to 8. The observed heterozygosities in H. brachysoma and H. nigricollaris were 0.463 and 0.443, respectively. Here, both RAPD and microsatellite methods reported a low degree of gene diversity and lack of genetic heterogeneity in both species of Horabagrus which strongly emphasize the need of fishery management, conservation and rehabilitation of these species.     

Ten polymorphic microsatellite markers for Scaphoideus titanus,the vector of flavescence dorée phytoplasma

The leafhopper Scaphoideus titanus is a vector of flavescence dorée phytoplasma, the causal agent of the most important grapevine yellow disease in European vineyards. Ten polymorphic microsatellite loci were developed from a genomic library enriched for AC and AG repeats. Levels of polymorphism were evaluated in 106 individuals from S. titanus European and American populations. An average of 16 alleles per locus was detected and the observed heterozygosity ranged from 0.141 to 0.813. Cross‐species amplification was successful in four other Cicadellidae species. These 10 microsatellites are valuable markers for population genetic and phylogeographical studies of S. titanus.     

Microsatellites from Clarias batrachus and their polymorphism in seven additional catfish species

We isolated 18 novel microsatellite loci from the walking catfish (Clarias batrachus), and examined their cross‐amplification in seven additional catfish species from three families. Sixteen of the 18 microsatellites were polymorphic in the source species (allele number: 2–10/locus and expected heterozygosity: 0.30–0.87). Moreover, nine of these 18 primer pairs cross‐amplified specific and polymorphic products from the genome of at least six of the seven other catfish species tested. However, the success rate of cross‐species amplification varied from locus to locus, indicating that cross‐species amplification of microsatellites is locus‐dependent.     

Development and characterization of microsatellite loci in Chinese medicinal plant Akebia trifoliate ssp. australis and cross-species amplification in closely related taxa

Eleven polymorphic microsatellite loci were isolated and characterized from an AC-enriched genomic library of Akebia trifoliate ssp. australis. The number of allele per locus ranged from 3 to 14. The observed heterozygosity and expected heterozygosity at population level were 0.196–1.000 and 0.522–0.902, respectively. In addition, this set of microsatellites produced robust cross-species amplification in other two related taxa, suggesting these microsatellite markers should provide a useful tool for genetic and conservation studies of the Akebia species.     

Nine new microsatellite loci for the orange roughy (Hoplostethus atlanticus)

We isolated nine new dinucleotide microsatellites for the deep-sea fish Hoplostethus atlanticus. These loci are generally highly polymorphic, with allele number ranging from 3 to 31 and observed heterozygosity ranging from 0.067 to 0.933. In conjuction with previously published loci, these markers will be useful for fine-scale analysis of population structure in this commercially important and threatened species.