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 twelve novel microsatellites in yellow catfish, Pelteobagrus fulvidraco

The yellow catfish Pelteobagrus fulvidraco is a freshwater fish species. Due to overfishing and pollution of freshwater ecosystems, the wild stocks of this fish reduced substantially. We isolated and characterized 12 polymorphic microsatellites of this species. The number of alleles at the 12 microsatellite loci ranged from four to eight, with an average of 6.6/locus. The average observed heterozygosity was 0.72, whereas the expected heterozygosity ranged from 0.60 to 0.86 (average: 0.80). All 12 microsatellites conformed to Hardy–Weinberg Equilibrium and were in linkage equilibrium. These 12 novel microsatellites could facilitate studies of genetic diversity and population structure of the yellow catfish to supply necessary information of conservation of the yellow catfish.     

Isolation and characterization of polymorphic microsatellite markers of Anopheles sinensis,a malaria vector mosquito in the East Asia region

Eleven polymorphic dinucleotide (GA and CA) microsatellites were isolated and characterized in the mosquito Anopheles sinensis; this species is distributed over the East Asia region and is a primary vector of malaria, particularly in Korea. The number of alleles per locus ranged from 4 to 13. The observed heterozygosity (H_O) and expected heterozygosity (H_E) varied from 0.30 to 0.89 and from 0.59 to 0.90, respectively. These microsatellites could be useful in studying the evolution of the widely distributed A. sinensis in diverse environments.     

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.     

Characterization of 14 tetranucleotide microsatellite loci derived from Pacific herring

Spawning aggregations of Pacific herring (Clupea pallasi) often exhibit significant interannual variation in allele frequencies of neutral gene markers. We isolated 14 tetranucleotide microsatellites to examine hypothetical processes that may produce this unique genetic signal. We developed and tested primer pairs for each locus and then estimated locus variability in samples (n = 60) from two populations. The number of alleles per locus ranged from five to 49. The expected heterozygosity across loci and populations ranged from 0.20 to 0.96. These microsatellites will be useful for estimating genetic variation in herring on a fine geographical scale.     

Development and characterization of 13 microsatellite markers of Chanodichthys mongolicus (Cypriniformes: Cyprinidae) by RAD-seq

This study developed and characterized 13 novel polymorphic microsatellite markers from Mongolian redfin Chanodichthys mongolicus by restriction-site associated DNA sequencing (RAD-seq). Variability was tested on 36 individuals randomly collected from Qiantang River, Zhejiang Province, China. The number of alleles per locus ranged from 2 (MGB_1990, MGB_3456, MGB_6007, MGB_6539, MGB_7273, MGB_8632) to 9 (MGB_8632). Observed heterozygosity ranged from 0.000 (MGB_7273) to 0.972 (MGB_5057), whereas the expected heterozygosity ranged from 0.028 (MGB_6007) to 0.795 (MGB_4875). Polymorphism Information Content ranged from 0.027 (MGB_6007) to 0.755 (MGB_4875). Moreover, seven microsatellites were in agreement with Hardy-Weinberg equilibrium. Four pairwise tests in seven microsatellite loci (MGB_1498, MGB_1990, MGB_4875, MGB_5057, MGB_6539, MGB_7273, MGB_8632) indicated linkage disequilibrium among loci. These microsatellites can evaluate Mongolian redfin populations’ genetic parameters to provide precious information for a conservation management system.     

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.     

Characterization of Microsatellites in the IGF-2 and GH Genes of Asian Seabass (Lates calcarifer)

Four microsatellites were identified by screening the DNA sequences of Asian seabass (Lates calcarifer) deposited to GenBank. Two markers each are located in the growth hormone gene (GH) and in the insulin-like growth factor II gene (IGF-2), respectively. The markers were characterized by genotyping 34 Asian seabass individuals. All 4 microsatellites showed polymorphism: the number of alleles per locus ranged from 2 to 11 (average, 5.0), while the expected heterozygosity ranged from 0.51 to 0.85 (average, 0.63) at the 4 loci. Cross-priming with all 4 primer pairs was tested in species belonging to 5 different genera, but no bands were amplified. These microsatellites are the first genomic DNA markers characterized in L. calcarifer; thus they may be valuable for research and aquaculture production of this species. Received April 10, 2000; accepted July 13, 2000.     

Microsatellites from genes show polymorphism in two related Oreochromis species

Six microsatellites were detected in the IGF‐II, GH2, prolactin I and insulin genes of Mozambique tilapia (Oreochromis mossambicus) and Nile tilapia (Oreochromis niloticus) by screening DNA sequences deposited to GenBank. Genotyping of 24 individuals from each species revealed that all six microsatellites were polymorphic in both species. In Mozambique tilapia the number of alleles ranged from 3 to 17 (average 9.8), whereas in Nile tilapia the range was 4–21 (average 10.5). The range of expected heterozygosity values were 0.44–0.95 (average 0.79) in Mozambique tilapia, and 0.52–0.96 (average 0.73) in Nile tilapia.     

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 polymorphic microsatellite loci in Bornean treeshrews (Tupaia spp.)

In this study, we developed five microsatellite loci from an enriched genomic library constructed for the pygmy treeshrew (Tupaia minor), and adapted another two from a previous study on the common treeshrew (Tupaia glis), for use in studying mating and dispersal patterns in Bornean treeshrews. We screened 32 plain treeshrew (Tupaia longipes) and 54 large treeshrew (Tupaia tana) individuals at these loci. Polymorphism ranged from two to 13 alleles, and heterozygosity ranged from 0.29 to 0.88. These results indicate the general utility of these microsatellites for genetic analyses in other Tupaia spp.     

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.     

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 nurse shark (Ginglymostoma cirratum)

We report on the development of nine polymorphic microsatellite loci in nurse shark (Ginglymostoma cirratum) using a combination of enriched and unenriched subgenomic libraries. Based on the small percentage of positive clones in the unenriched library (0.4%) it appears that microsatellites are very scarce in nurse shark genomes. Numbers of alleles at polymorphic loci ranged from two to 15; observed heterozygosity ranged from 0.17 to 0.90. We expect these loci to be useful for studies of breeding structure and paternity.     

Isolation and characterization of polymorphic microsatellites from Chinese mitten crab,Eriocheir sinensis

Chinese mitten crab (Eriocheir sinensis) has great commercial significance in Asia, but is an invasive organism in Europe and America. We isolated five novel microsatellites for E. sinensis. All five loci were polymorphic. The average allele number was 16.8 per locus with a range of 13 to 21, while the expected heterozygosity ranged from 0.82 to 0.91 with an average of 0.88. These markers could be applicable to studies of the genetic diversity and population structure of E. sinensis.     

Development and characterization of microsatellites in Vanda varieties

The orchid genus Vanda includes more than 70 monopodial species and numerous hybrids. The aim of this study was to develop microsatellite markers for this horticultural valuable genus. Thirteen polymorphic microsatellite markers were isolated from the variety Vanda Miss Joaquim and were characterized in four Vanda species, 11 Vanda hybrids, and one Aranda intergenus hybrid. Populations from three species were also analysed. Number of alleles ranged from two to 19. For the V. sumatrana population, the expected heterozygosity ranged from 0 to 0.76 (mean 0.31) and the observed heterozygosity ranged from 0 to 1 (mean 0.36). All the varieties tested were distinct from one another (similarity index < 0.8). These microsatellites could be used for studying genetic diversity and population structure of wild populations within the orchid genus Vanda, as well as for distinguishing cultured Vanda varieties.     

Development of a set of highly polymorphic genomic microsatellites (gSSRs) in Sitka spruce (Picea sitchensis (Bong.) Carr.)

Robust, polymorphic microsatellite DNA markers (simple sequence repeats—SSRs) are valuable tools for a range of tree conservation and breeding applications. SSRs are routinely used in the study of population genetic structure and diversity, pedigree reconstruction and genetic linkage mapping. Their abundance in the genome, co-dominant inheritance and potential for cross-species amplification make microsatellites highly prized markers. This paper characterises 22 novel genomic polymorphic microsatellite loci for Sitka spruce (Picea sitchensis (Bong.) Carr.). Amplification of DNA from Sitka spruce material was carried out both with a set of unrelated trees to obtain diversity statistics for each locus, and with the progeny of a full-sib family to test simple Mendelian inheritance. Observed heterozygosity ranged from 0.38 to 0.91 and allele number per locus ranged from 6 to 21, with a mean of 12.2. In addition, the primer pairs were tested with DNA from Norway spruce (P. abies) and white spruce (P. glauca) to investigate their potential for cross-species amplification and ten loci amplified in all three species. The results from these genomic microsatellites are compared to data generated from microsatellites derived from Picea EST libraries. In summary, this novel, highly polymorphic markers represent a significant addition to the rapidly expanding Picea genomics tool-box. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

A multiplex set of microsatellite markers for the scarlet rosefinch (Carpodacus erythrinus)

Cardueline finches have become important models in studies of sexual selection and evolution of carotenoid‐based ornamentation. Here, we describe eight new polymorphic microsatellites isolated from the Scarlet rosefinch (Carpodacus erythrinus) and four from the House finch (Carpodacus mexicanus). Together with the cross‐species amplification of additional loci, originally published for two species of songbirds, we optimized a multiplex panel for C. erythrinus allowing genotyping of 22 polymorphic loci. Number of alleles and heterozygosity per locus in a sample of 34 individuals ranged from three to 38 and from 0.27 to 0.94, respectively.     

Microsatellite markers of genetic diversity and population structure of Carica papaya

We assessed the genetic diversity of 96 papaya accessions by molecular characterisation using microsatellite markers. Fifteen polymorphic primers were selected. Accessions, which were classified as Common, Formosa and Solo according to fruit types, were evaluated for allele frequency, heterozygosity, polymorphism information content (PIC), inbreeding coefficient (f) and the genetic diversity structure. Fifteen primers amplified 68 alleles with an average of 4.53 per locus. PIC values ranged from 0.19 to 0.69. The observed heterozygosity (H_O) was low for all selected microsatellites. High f estimates (0.58) and excess of homozygotes indicated inbreeding, mainly caused by the tendency to select hermaphrodite plants for succeeding generations. Analysis of molecular variance showed that most of the variation (98%) resides within subpopulation. The genetic analysis based on Bayesian statistics proved to be sensitive enough to detect relationships among the papaya accessions, grouping them into six clusters, irrespective of their classification types.     

Microsatellite Variation Among Red Snapper (Lutjanus campechanus) from the Gulf of Mexico

Allelic variation at a total of 20 nuclear-encoded microsatellites was examined among adult red snapper (Lutjanus campechanus) sampled from 4 offshore localities in the Gulf of Mexico. The number of alleles at the 20 microsatellites ranged from 5 to 20; average (± SE) direct count heterozygosity values ranged from 0.148 ± 0.025 to 0.902 ± 0.008. No significant departures from expectations of Hardy-Weinberg equilibrium were found for any locus within samples, and genotypes at pairs of microsatellites appeared to be randomly associated, i.e., in genotypic equilibrium. Tests of homogeneity in allele distributions among the 4 localities were nonsignificant for 19 of the microsatellites. Allele distribution at microsatellite Lca 43 was heterogeneous among localities before (but not after) Bonferroni corrections for multiple tests executed simultaneously. Tests of homogeneity in the distribution of individual alleles at Lca 43 gave similar results: one low frequency allele was distributed heterogeneously among samples before, but not after, Bonferroni correction. Molecular analysis of variance indicated that more than 99% of variation at each microsatellite was distributed within sample localities. These results generally are consistent with the hypothesis of a single population (stock) of red snapper in the northern Gulf of Mexico. Received September 25, 2000; accepted January 16, 2001