Development of eighteen polymorphic microsatellite markers in <Emphasis Type="Italic">Scylla paramamosain</Emphasis> by 5′ anchored PCR technique

The mud crab Scylla paramamosain plays a significant role in fishery resources in China. In this study, we developed 18 polymorphic microsatellite markers in this important crab by 5′ anchored PCR technique. A total of 125 alleles were detected in a single population of 32 individuals of S. paramamosain. The number of alleles per locus ranged from five to nine, with the allele size ranging from 166 to 316 bp. The polymorphism information content (PIC), observed and expected heterozygosity ranged from 0.39 to 0.88, from 0.33 to 0.92 and from 0.42 to 0.86, respectively. Three loci (Scypa13, Scypa14 and Scypa15) deviated significantly from Hardy–Weinberg equilibrium (HWE) after Bonferroni correction (P < 0.0028), and no linkage disequilibrium was found between loci pairs. These polymorphic microsatellite markers will be useful for the study of population genetic structure, construction of genetic linkage maps and mapping of economically quantitative trait loci (QTL) in S. paramamosain.     

Characterization of 40 single nucleotide polymorphism (SNP) via T m-shift assay in the mud crab (Scylla paramamosain)

In this study, single nucleotide polymorphism (SNP) were identified, confirmed and genotyped in the mud crab (Scylla paramamosain) using T _m-shift assay. High quality sequences (13, 311 bp long) were obtained by re-sequencing that contained 91 SNPs, with a density of one SNP every 146 bp. Of all 91 SNPs, 40 were successfully genotyped and characterized using 30 wild specimens by T _m-shift assay. The minor allele frequency per locus ranged from 0.017 to 0.500. The observed and expected heterozygosity, and polymorphism information content (PIC) ranged from 0.000 to 0.600, from 0.033 to 0.509, and from 0.033 to 0.375, respectively, with an average of 0.142, 0.239 and 0.198 per locus. Seventeen SNPs were significantly deviated from Hardy–Weinberg equilibrium. No significant linkage disequilibrium between pairs of loci was detected after sequential Bonferroni correction (P > 0.00125). Seventeen SNPs were related with known function genes. This study provided new molecular markers for investigation of population genetic diversity, construction of genetic linkage maps and molecular marker-assisted selection in this important crustacean species.     

Isolation and characterization of gene-derived single nucleotide polymorphism (SNP) markers in Scylla paramamosain

Single nucleotide polymorphisms (SNPs) are thought to be well suitable for genetic and evolutionary studies. In this study, we reported the first set of SNP markers in a commercially important crab species, Scylla paramamosain. A total of 12,500 base pairs high quality DNA sequences were obtained from 15 genes, and thirty-seven SNPs were identified, representing one SNP every 338 base pairs. Twenty-four SNPs were successfully genotyped in a single population. All loci had two alleles and the minor allele frequency ranged from 0.02 to 0.44. The observed and expected heterozygosity ranged from 0.04 to 0.59 and from 0.04 to 0.50, respectively. No significant departures from Hardy–Weinberg equilibrium at each locus was found. The linkage disequilibrium was detected in six loci pairs, but absent after sequential Bonferroni correction. These SNP markers will provide a useful addition to the genetic tools for genetic and evolutionary studies for S. paramamosain.     

Polymorphic microsatellites in largemouth bronze gudgeon (Coreius guichenoti) developed from repeat‐enriched libraries and cross‐species amplifications

Largemouth bronze gudgeon (Coreius guichenoti) is a medium‐sized fish endemic from the upper Yangtze River of China and its survival is threatened by the construction of the Three Gorges Dam. This study reports 20 new polymorphic microsatellites from a repeat‐enriched genomic library with a mean number allele of 5.2, and observed and expected heterozygosities ranging from 0.035 to 1, and from 0.13 to 0.917, respectively. In a cross‐species amplification test, nine of the 37 tested loci were found to be also polymorphic in a congeneric species, brass gudgeon (C. heterodon). In addition, other four loci from common carp (Cyprinus carpio) were also polymorphic in C. guichenoti. Out of these 24 polymorphic microsatellites, only three loci significantly deviated from Hardy–Weinberg equilibrium in the sampled population (P < 0.0025), and all pairwise tests for linkage disequilibrium among loci were nonsignificant after applying sequential Bonferroni correction (P > 0.0026). These novel microsatellites provide sufficient levels of polymorphism for studies on population genetics and conservation in C. guichenoti and its related species.     

Twelve polymorphic microsatellite loci from a dinucleotide-enriched genomic library of Japanese Spanish mackerel (Scomberomorus niphonius)

In the study, 34 microsatellite loci were isolated from a dinucleotide-enriched genomic library of Japanese Spanish mackerel (Scomberomorus niphonius). And 12 microsatellite loci were found to be polymorphic between 3 and 8 alleles. The number of observed and expected heterozygosity per locus in 23 individuals ranged from 0.6087 to 1.0000 and 0.8908 to 0.9773, respectively. Two loci significantly deviated from Hardy–Weinberg equilibrium after Bonferroni correction analysis and there was no significant linkage disequilibrium found between pairs of loci. As a result, 12 microsatellite loci probably located on different chromosome pairs and these polymorphic microsatellite loci should provide sufficient level of genetic diversity to investigate the fine-scale population structure and evaluate the breeding strategy in S. niphonius. The authors Shi-Chao Xing and Gen-Bo Xu contributed equally.     

Isolation and characterization of microsatellites in Chinese white olive (Canarium album) and cross-species amplification in Canarium pimela

We developed 11 polymorphic microsatellite primers from Canarium album, which is a famous Chinese fruit tree with diversified economy values. The observed and expected heterozygosities (H _o and H _e) ranged from 0.133 to 0.833 and 0.128 to 0.810, with averages of 0.607 and 0.629, respectively. Four loci were deviated from Hardy–Weinberg equilibrium after Bonferroni correction (P < 0.01), but no linkage disequilibrium was detected among any pair of loci after Bonferroni correction (P < 0.05). Cross-species amplification in another important cultured species Canarium pimela showed that 10 of 11 primers were polymorphic, with number of alleles per locus ranging from 2 to 5. The primers will be useful to explore further studies about introgressive hybridization and conversation of wild genetic resources of these two species.     

Isolation and characterization of fourteen novel microsatellite loci in the Hong Kong oyster, Crassostrea hongkongensis

Fourteen polymorphic microsatellite markers were isolated from the Hong Kong oyster, Crassostrea hongkongensis, with a partial genomic library enriched for tandem repeat sequences of (CA)₁₂, (GA)₁₂, (ATG)₆ and (TAGA)₄. Polymorphism of these loci was assessed in a sample of 48 wild unrelated individuals. The average allelic number of these polymorphic loci was 6.36 per locus, with a range of 4–16. The observed and expected heterozygosity varied from 0.208 to 0.729 (averaging 0.502) and from 0.193 to 0.789 (averaging 0.615), respectively. After Bonferroni correction (P > 0.0036), 11 of the 14 loci accorded with Hardy–Weinberg equilibrium, and the rest three were detected significant departure from HWE. Additionally, two loci (Ch103 and Ch104) showed significant linkage disequilibrium (P < 0.01). This is the first set of microsatellite loci developed in this species and would be useful for studies of population genetics, stock management and other relevant research in C. hongkongensis.     

Isolation and characterization of 12 microsatellite loci from cutlassfish (Trichiurus haumela)

The cutlassfish (Trichiurus haumela) is an important commercial fish species. In the present study, we report 12 polymorphic microsatellite DNA markers for cutlassfish. The number of alleles per locus ranged from 2 to 9 in a sample of 26 individuals. Observed and expected heterozygosities per locus varied from 0.2727 to 0.9583 and from 0.4059 to 0.7926, respectively. Two loci (Trha25 and Trha27) showed significant departure from Hardy–Weinberg equilibrium after sequential Bonferroni correction (P < 0.0042). No significant linkage disequilibrium between pairs of loci was found. These microsatellite markers provide powerful tools for investigating genetic population structure, population history and conservation management of cutlassfish. Jin-Zhen Bi and Chang-Wei Shao contributed equally to this work.     

Development of twenty-two polymorphic microsatellite loci in the noble scallop, Chlamys nobilis

Twenty-two polymorphic microsatellites were isolated from the noble scallop, Chlamys nobilis with a partial genomic library enriched for tandem repeat sequences of (CA)₁₂ and (GA)₁₂. Polymorphism of these loci was assessed in a sample of 32 unrelated individuals. The average allele number was 7.23 per locus with a range of 3–19, and the observed and expected heterozygosity varied from 0.032–0.875 (averaging 0.452) and 0.315–0.939 (averaging 0.646), respectively. After Bonferroni correction (P > 0.0023), 11 of the 22 markers accorded with Hardy–Weinberg equilibrium, and the rest showed significant departure from HWE. Three pairs of loci showed significant linkage disequilibrium (P < 0.01). A poor transferability of these loci to other two cultured species, C. farreri and Patinopecten yessoensis was detected. These markers would be useful for studies of population genetics, linkage mapping and other relevant research in C. nobilis.     

Isolation and characterization of polymorphic microsatellite loci from a dinucleotide-enriched genomic library of starry flounder (Platichthys stellatus) and cross-species amplification

Starry flounder (Platichthys stellatus) is a rare fish species in China. Here, we reported 12 polymorphic microsatellite loci isolated from a dinucleotide-enriched genomic library of starry flounder (P. stellatus). The number of alleles, observed and expected heterozygosity per locus in 30 individuals ranged from two to six, from 0.2500 to 1.0000 and from 0.4512 to 0.7667, respectively. One locus significantly deviated from Hardy–Weinberg equilibrium after Bonferroni correction and no significant linkage disequilibrium between pairs of loci was found. Cross-species amplification of these microsatellite loci in additional three fish species was performed. These polymorphic microsatellite loci would be useful for investigating genetic population structure and construction of genetic linkage map in P. stellatus. Guidong Miao and Changwei Shao have contributed equally.     

Identification of polymorphic microsatellite loci in the mud crab Scylla serrata (Brachyura: Portunidae)

Five microsatellite loci are identified and characterized from the genome of Scylla serrata, a widespread and commercially important species of coastal marine crab. The loci were detected by randomly screening for di‐ and tri‐nucleotide repeat units within a partial genomic library developed for the species. The five loci consist of dinucleotide repeats and are both co‐dominant and polymorphic within the species. A sample (N = 36) of S. serrata from one Australian population has an average observed heterozygosity of 0.875 and provides no evidence of either linkage among loci or significant deviation from random mating expectations across loci. PCR products for each of the five loci were also observed from a small sample of three other species within the Scylla genus. These markers may provide genetic information that will be useful for both aquaculture and studies of natural populations of the genus.     

Isolation and characterization of polymorphic microsatellite loci from a dinucleotide-enriched genomic library of spotted maigre (Nibea albiflora)

In the present study, we reported 13 polymorphic microsatellite loci isolated from a dinucleotide-enriched genomic library of Nibea albiflora. The number of alleles, observed and expected heterozygosity per locus in 44 individuals ranged from 2 to 13, from 0.0909 to 0.9773 and from 0.0886 to 0.9073, respectively. Three loci significantly deviated from Hardy–Weinberg equilibrium after Bonferroni correction. Analysis of linkage disequilibrium showed non-significant among the two pairs of loci. As a result, 13 microsatellite loci probably located on different chromosome pairs and these polymorphic microsatellite loci should provide sufficient level of genetic diversity to investigate the fine-scale population structure and evaluate the breeding strategy in Nibea albiflora. Shichao Xing, Changwei Shao contributed equally to this work.     

Isolation and characterization of polymorphic microsatellite loci from a repeat-enriched genomic library of stone flounder (Kareius bicoloratus) and cross-species amplification

Stone flounder (Kareius bicoloratus) is a rare fish species in China. Here, we reported 11 polymorphic microsatellite loci isolated from a repeat-enriched genomic library of stone flounder. The number of alleles, observed and expected heterozygosity per locus in 32 individuals ranged from 3 to 6, from 0.1613 to 0.8667 and from 0.1549 to 0.7932, respectively. Two loci significantly deviated from Hardy–Weinberg equilibrium after Bonferroni correction and no significant linkage disequilibrium between pairs of loci was found. Cross-species amplification of these microsatellite loci in additional three fish species was performed. These polymorphic microsatellite loci would be useful for investigating genetic population structure and construction of genetic linkage map in Kareius bicoloratus. Yong-Sheng Tian and Gui-Dong Miao are contributed equally.     

A first set of gene-derived polymorphic microsatellite markers in half-smooth tongue sole (<Emphasis Type="Italic">Cynoglossus semilaevis</Emphasis>)

Half-smooth tongue sole (Cynoglossus semilaevis) is a commercially important marine fish species in China. A set of type I microsatellite markers were identified through bioinformatic mining of the GenBank database. Thirteen of these markers showed polymorphisms through genotyping a sample of 30 individuals. A total of 47 alleles were detected, with an average of 3.6 alleles per locus. The number of alleles, observed and expected heterozygosity per locus ranged from two to five, from 0.14 to 0.93 and from 0.27 to 0.77, respectively. Three loci significantly deviated from Hardy-Weinberg equilibrium after Bonferroni correction (P < 0.0038) and no significant linkage disequilibrum between pairs of loci was found. The markers identified in this study will contribute to construction of genetic linkage maps and mapping of quantitative trait loci (QTL) of C. semilaevis.     

Isolation and characterization of 31 polymorphic microsatellite markers in barfin flounder (Verasper moseri) and the cross-species amplification in spotted halibut (Verasper variegatus)

The construction of a genetic linkage map and evaluation of population genetic diversity both require large numbers of polymorphic molecular markers. In the present study, we report 31 polymorphic microsatellite markers, of which 28 were isolated from two repeat-enriched libraries constructed from genomic DNA, and three were detected in two genes (MCH-R1 and MCH-R2) of barfin flounder (Verasper moseri). A total of 94 alleles were detected with an average of 3.0 alleles per locus. The number of alleles, observed and expected heterozygosity per locus ranged from two to six, from 0.30 to 1.00 and from 0.33 to 0.78, respectively. Three loci significantly deviated from Hardy-Weinberg equilibrium after Bonferroni correction (P < 0.0016) and no significant linkage disequilibrum between pairs of loci was found. Cross-species amplification of these markers was evaluated using the closely related species spotted halibut (Verasper variegatus). This study will potentially be useful for stock management, constructing of a genetic linkage map, mapping economically important quantitative trait loci (QTL), and studying the population genetic diversity of barfin flounder (Verasper moseri).     

Development of 12 polymorphic microsatellite markers in <Emphasis Type="Italic">Coilia ectenes</Emphasis> Jordan and Seale, 1905 (Clupeiformes: Engraulidae) and cross-species amplification in <Emphasis Type="Italic">Coilia mystus</Emphasis> Linnaeus, 1758

In this study, twelve polymorphic microsatellite markers were developed for Coilia ectenes (synonym C. nasus). In a sample of 30 C. ectenes individuals in a Wuhan population from Yangtze River, a total of 78 alleles were detected, and the number of alleles per locus ranged from 3 to 12. The observed and expected heterozygosities ranged from 0.100 to 1.000 and from 0.098 to 0.899, respectively. Six loci significantly deviated from the Hardy-Weinberg equilibrium after Bonferroni correction (P < 0.004), and no significant linkage disequilibrium between pairs of loci was found. The applicability of these markers in a closely related species, C. mystus, was evaluated by cross-species amplification. These microsatellite markers will be useful in studies on population genetics, conservation genetics, and fishery management and in the construction of genetic linkage maps of C. ectenes and C. mystus.     

Isolation and characterization of ten polymorphic microsatellite markers for the blue mussel (Mytilus edulis)

In this study, we isolated 10 novel polymorphic microsatellite markers in Mytilus edulis by using the magnetic beads enrichment procedure. The characteristics of these loci were estimated by using a sample of 32 individuals of M. edulis. The number of alleles at 10 polymorphic microsatellite loci ranged from 2 to 15 with an average of 5.667. HO and HE ranged from 0.2667 to 1.0000 (0.6800 in average) and from 0.4723 to 0.9226 (0.6190 in average), The PIC value of 6 loci was more than 0.5, and that of the other 4 was between 0.25 and 0.50, Significant deviation from Hardy–Weinberg Equilibrium was observed at ME8, ME115 and ME153 after Bonferroni correction (P < 0.004, adjusted value), which possibly was due to the presence of null alleles. This study will be useful for the analysis of population genetic diversity, and the management of this important M. edulis resource.     

Isolation and characterization of microsatellite markers for the heavily exploited rockfish Sebastes schlegeli, and cross-species amplification in four related Sebastes spp.

Here we report development and characterization of 14 polymorphic microsatellite loci from Sebastes schlegel. Polymorphism at these loci revealed from 3 to 23 alleles. The observed heterozygosity ranged from 0.34 to 1.00, and the expected heterozygosity ranged from 0.31 to 0.95. No linkage disequibrium was found. Two loci were significantly deviated from HWE (P < 0.01). The 14 loci were also surveyed in four other Sebastes species and 12 loci successfully amplified, where allelic diversity ranged from highly polymorphic to monomorphic. These results demonstrate these microsatellite markers can be used for the study of intra- and inter-specific genetic diversity.     

Isolation and characterization of ten new polymorphic microsatellite loci in the mud crab, Scylla paramamosain

We developed polymerase chain primers for ten microsatellite loci in the mud crab, Scylla paramamosain. All markers were obtained from a (CA)₁₅ and (CT)₁₅-enrichment DNA library, and characterized in 30 individuals from one wild population. The number of alleles per locus varies between 8 and 18, and the observed and expected heterozygosities ranged from 0.6207 to 0.9333 and from 0.5886 to 0.9243, respectively. These polymorphic loci provide a valuable tool for population genetic analysis and parentage determination in this species.     

Ten species specific microsatellite loci for Lahontan cutthroat trout,Oncorhynchus clarki henshawi

Ten polymorphic microsatellite loci (containing tri and tetra‐nucleotide repeats) were developed for the Lahontan cutthroat trout (Oncorhynchus clarki henshawi), a subspecies of cutthroat trout listed as threatened under the United States Endangered Species Act. Polymorphism was assessed for 445 individuals from 12 populations representing eight watersheds spread throughout the three Distinct Population Segments defined for this subspecies. All loci were polymorphic (X? = 19, range 7–26 alleles). All loci were in Hardy–Weinberg equilibrium (HWE) except for one locus (OCH 9) in a single population (P < 0.00014 after Bonferroni correction for multiple tests).