Development of polymorphic microsatellite markers in barfin flounder (<Emphasis Type="Italic">Verasper moseri</Emphasis>) and spotted halibut (<Emphasis Type="Italic">Verasper variegatus</Emphasis>) by the cross-species amplification

Barfin flounder (Verasper moseri) and spotted halibut (Verasper variegatus) are two commercially important flatfish species in the Northeast Asia. In the present study, we reported polymorphic microsatellite markers in V. moseri and V. variegatus by the cross-species amplification of microsatellite primers developed previously in two other related marine fish species. A total of 244 polymorphic microsatellite markers were selected for cross-species amplification in V. moseri and V. variegatus, of which 182 markers deriving from Atlantic halibut (Hippoglossus hippoglossus) and 62 markers deriving from Japanese flounder (Paralichthys olivaceus). A sample of 10 individuals were detected. As a result, a total of 67 loci showed polymorphisms in V. moseri, and 62 loci showed polymorphisms in V. variegatus, with the observed number of alleles per locus ranging from two to five in V. moseri, and from two to seven in V. variegatus, respectively. This paper provided more candidate microsatellite markers which could be useful for construction of genetic linkage maps, evaluation of population genetic structure and stock management of V. moseri and V. variegatus.     

Genetic sex identification and the potential evolution of sex determination in Pacific halibut (Hippoglossus stenolepis)

The discovery of genetic markers linked to physiological traits in wild populations is increasingly desired for ecological and evolutionary studies, as well as to inform management decisions. However, identifying such markers often requires a large investment of both time and money. Serendipitously, in a recent microsatellite survey, we discovered three out of 16 microsatellite loci that were correlated to the female sex in Pacific halibut (Hippoglossus stenolepis). These three loci were screened in 550 Pacific halibut to determine their accuracy at identifying sex. Genetic assignment successfully identified sex in 92% of individuals from sample collections spanning 3,000 km and 9 years. All but two of 287 females had one copy of a characteristic allele for at least one of the three microsatellite loci, resulting in consistent heterozygote excess in females. This pattern is consistent with the hypothesis that females are the heterogametic sex in Pacific halibut, which thus may have a different sex-determination pattern than the closely related Atlantic halibut (Hippoglossus hippoglossus). A rapid divergence of sex-determining mechanisms could be either a cause or consequence of speciation between Pacific and Atlantic halibut. In either case, the ability to genetically identify sex in individual Pacific halibut provides a new tool for ecological studies, fisheries management, and insight into the evolution of sex determination in flatfish.     

Development and characterization of microsatellite loci for lotus (Nelumbo nucifera)

This paper reports the development of microsatellite primers for Nelumbo nucifera Gaerten. By screening genomic libraries enriched with 10 kinds of probes, Seventeen polymorphic loci were isolated and primers were designed. Polymorphism of these 17 loci was assessed in 24 individuals. All the 17 loci are polymorphic and the number of alleles ranged from two to seven. Observed heterozygosity and expected heterozygosity ranged from 0.0000 to 0.9176 and from 0.2837 to 0.7917 respectively. These microsatellite loci should be useful for studying the genetic diversity of N. nucifera.     

Exploitation of a turbot (Scophthalmus maximus L.) immune-related expressed sequence tag (EST) database for microsatellite screening and validation

In this study, we identified and characterized 160 microsatellite loci from an expressed sequence tag (EST) database generated from immune-related organs of turbot (Scophthalmus maximus). A final set of 83 new polymorphic microsatellites were validated after the analysis of 40 individuals of Atlantic origin including both wild and farmed individuals. The allele number and the expected heterozygosity ranged from 2 to 18 and from 0.021 to 0.951, respectively. Evidences of null alleles at moderate-high frequencies were detected at six loci using population data. None of the analysed loci showed deviations from Mendelian segregation after the analysis of five full-sib families including approximately 92 individuals/family. The markers are used to consolidate the turbot genetic map, and because they are mostly EST-derived, they will be very useful for comparative genomic studies within flatfishes and with model fish species. Using an in silico approach, we detected significant homologies of microsatellite sequences with the EST databases of the flatfish species with highest genomic resources (Senegalese sole, Atlantic halibut, bastard halibut) in 31% of these turbot markers. The conservation of these microsatellites within Pleuronectiformes will pave the way for anchoring genetic maps of different species and identifying genomic regions related to productive traits.     

Characterization of six microsatellite loci in the Baltic blue mussel <Emphasis Type="Italic">Mytilus trossulus</Emphasis> and cross-species amplification in North Sea <Emphasis Type="Italic">Mytilus edulis</Emphasis>

The blue mussel Mytilus trossulus occurs in the Pacific and in the North Atlantic. We developed and characterized six microsatellite loci for Baltic M. trossulus. Seventeen microsatellite loci were screened, of which six were polymorphic. The number of alleles among 50 individuals ranged from 3 to 13 and the observed and expected heterozygosity were 0.09–0.46 and 0.34–0.86, respectively. The loci were also tested for cross amplification in M. edulis, in which four of the six microsatellite loci were successfully amplified.     

Isolation and characterization of polymorphic microsatellite loci from a dinucleotide-enriched genomic library of obscure puffer (Takifugu obscurus) and cross-species amplification

Obscure puffer (Takifugu obscurus) is an anadromous fish species in China. Here, we reported 10 polymorphic microsatellite loci isolated from a dinucleotide-enriched genomic library of T. obscurus. The number of alleles, observed and expected heterozygosity per locus in 30 individuals ranged from four to 10, from 0.57 to 0.86 and from 0.68 to 0.90, respectively. Three 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 T. obscurus.     

Isolation and cross‐species amplification of microsatellite loci from the fucoid seaweeds Fucus vesiculosus,F. serratus and Ascophyllum nodosum (Heterokontophyta,Fucaceae)

The Fucaceae is a family of brown seaweeds that dominate and frequently co‐occur on North Atlantic rocky shores. We developed nine polymorphic microsatellite markers for the fucoid seaweeds Fucus vesiculosus, F. serratus and Ascophyllum nodosum using a combined, enriched library. Six of these loci were polymorphic in at least two species, showing from two to eight alleles with heterozygosities ranging from 0.41 to 0.85. Loci were also tested on F. spiralis, revealing five polymorphic microsatellite loci in this species.     

Isolation and characterization of polymorphic microsatellite loci from a dinucleotide-enriched genomic library of seven-band grouper (Epinephelus septemfasciatus) and cross-species amplification

Seven-band grouper (Epinephelus septemfasciatus) is a commercially important fishery species. Sixty-six microsatellite loci were isolated from a dinucleotide-enriched genomic library of E. septemfasciatus. Twelve of these loci were polymorphic in a test population with alleles per locus ranging from two to five, and observed and expected heterozygosities per locus from 0.04 to 1.00 and from 0.28 to 0.76, respectively. Three loci significantly deviated from Hardy–Weinberg equilibrium and no significant linkage disequilibrium was found between pairs of loci after Bonferroni correction. Cross-species amplification of these polymorphic microsatellite loci was performed in additional two related species. These polymorphic microsatellite loci would be useful for investigating genetic resource of E. septemfasciatus and other related species. Lili Zhao and Changwei Shao have contributed equally.     

Characterization of tetranucleotide microsatellite loci and development and validation of multiplex reactions for the study of right whale species (genus Eubalaena)

A North Atlantic right whale (Eubalaena glacialis) genomic library was developed and screened with a (GATA)₈ probe to identify tetranucleotide microsatellite loci. Sixteen characterized loci were polymorphic in North Atlantic and/or South Atlantic (Eubalaena australis) right whales, 12 being polymorphic in E. glacialis, and 15 in E. australis. Fourteen of these were combined with 21 other previously identified loci for a suite of 35 loci which can be used to increase resolution of genetic analyses of these species. Multiplex reactions were developed for genotyping samples at these loci, providing a method that is rapid, reliable and cost‐effective.     

Isolation and characterization of polymorphic microsatellite loci from black sea bass (Centropristis striata) and cross-species amplification

Black sea bass (Centropristis striata) is an economically important serranid species. A number of 39 microsatellite loci were isolated from two enriched genomic library of C. striata. Eleven of these loci were polymorphic in a test population with alleles per locus ranging from 3 to 8, and observed and expected heterozygosities per locus from 0.26 to 1.00 and from 0.61 to 0.84, respectively. Four loci significantly deviated from Hardy–Weinberg equilibrium after Bonferroni correction and no significant linkage disequilibrium was found between pairs of loci. Cross-species amplification of these polymorphic microsatellite loci was performed in additional two related species. These polymorphic microsatellite loci would be useful for investigating genetic resource of C. striata and other related species.     

Isolation and Characterization of Twenty Microsatellite Loci in Japanese Sea Cucumber (Stichopus japonicus)

Twenty microsatellite markers were first developed from the Japanese sea cucumber Stichopus japonicus using an enrichment protocol. Of the 20 microsatellite loci, 19 loci were polymorphic in the population examined. At these polymorphic loci, the number of alleles per locus varied from 2 to 15, and the observed heterozygosities ranged from 0.03 to 0.97, which is considerably higher than those previously found for allozymes. The high variability of the microsatellite markers identified in this study will make them excellent tools for genetic analyses of S. japonicus.     

Identification and characterisation of nine new gene-associated microsatellite markers for Atlantic cod (<Emphasis Type="Italic">Gadus morhua</Emphasis> L.)

Nine polymorphic microsatellite markers were identified by screening of 2464 ESTs derived from a cDNA library of Atlantic cod (Gadus morhua L.). About 35 novel microsatellite loci were selected and characterised in 96 individual cod. Nine markers were successfully amplified with number of alleles from 3 to 18 per locus and the average heterozygosity was 0.57 in the panel examined (range 0.29–0.86). All loci followed the Hardy–Weinberg expectation and no significant linkage disequilibrium was found in a test including all pairwise combinations. The gene identity was determined at four of the loci, confirming the associated microsatellites as Type I markers.     

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 polymorphic microsatellite loci from bluefin leatherjacket (Navodon septentrionalis Gunther, 1877)

The first set of 18 polymorphic microsatellite markers were developed from bluefin leatherjacket (Navodon septentrionalis Gunther, 1877). From a (GT)_n-enriched genomic library, we got 121 microsatellites, of which 60 were randomly selected for designing microsatellite primers. Eighteen of these loci were polymorphic in a test population of 32 individuals with alleles ranging from 2 to 9, and expected and observed heterozygosities from 0.1463 to 0.8517 and from 0.1562 to 1.0000, respectively. No significant linkage disequilibrium between pairs of loci was found, but three loci significantly deviated from Hardy–Weinberg equilibrium after Bonferroni correction. These polymorphic microsatellite loci should provide sufficient level of genetic diversity to investigate population structure in bluefin leatherjacket.     

Isolation and charaterization of polymorphic microsatellite loci from so-iuy mullet (Mugil soiuy Basilewsky 1855)

The first set of polymorphic microsatellite markers were developed from so-iuy mullet (Mugil soiuy Basilewsky 1855). From a (GT)n-enriched genomic library, 53 microsatellites were selected for designing microsatellite primers, of which 36 gave working primer pairs. Ten of these loci were polymorphic in a test population of 24 individuals with alleles ranging from 3 to 9, and observed and expected heterozygosities from 0.2083 to 0.9167 and from 0.2651 to 0.8812, respectively. No significant linkage disequilibrium between pairs of loci was found, but two loci significantly deviated from Hardy–Weinberg equilibrium after Bonferroni correction. These polymorphic microsatellite loci should provide sufficient level of genetic diversity to investigate the fine-scale population structure and evaluate the breeding strategy in Mullet. Genbo Xu and Changwei Shao Contributed equally.     

Isolation and characterization of polymorphic microsatellite loci in <Emphasis Type="Italic">Achnatherum sibiricum</Emphasis> (Poaceae)

Achnatherum sibiricum is a threaten and toxic perennial bunchgrass mainly in the north of China. We developed 11 polymorphic microsatellite loci from A. sibiricum by combining biotin capture method. After validating and scoring, these loci were polymorphic in a test population with the range of alleles from four to 13 per locus. The observed and expected heterozygosities ranged from 0.1649 to 0.5624 and from 0.3071 to 0.8826, respectively. All 11 microsatellite markers were in Hardy–Weinberg equilibrium. These polymorphic microsatellites will be useful for genetic diversity analysis and population structure construction for A. sibiricum.     

Isolation and characterization of microsatellite markers for the endangered <Emphasis Type="Italic">Taxus yunnanensis</Emphasis>

Eleven polymorphic microsatellite loci were characterized for the endangered conifer Taxus yunnanensis. Eight loci were isolated through SSR-anchored PCR, one locus was developed by cross-species amplification tests, while the last two loci were obtained from cross-species microsatellite sequences available in GenBank. Variability of these markers was tested in 48 individuals collected from its main distribution range in Southwest China. The number of alleles per locus ranged from 2 to 9, and the observed and expected heterozygosity varied from 0.000 to 0.625 and from 0.062 to 0.853, respectively. Ten of these eleven loci were significantly deviated from Hardy–Weinberg expectation, except TS07 which showed a distinct heterozygote excess. The availability of these new polymorphic microsatellite markers will provide an ideal marker system for detailed population genetics studies in T. yunnanensis and potentially also for closely related species.     

Twelve novel polymorphic microsatellite loci for the Yellow grouper (Epinephelus awoara) and cross-species amplifications

Yellow grouper (Epinephelus awoara) is a commercially important marine fish species. A dinucleotide-enriched genomic library of E. awoara was constructed using the method of FIASCO. Twelve loci were polymorphic in a test population with alleles per locus ranging from two to eight, and observed and expected heterozygosities per locus from 0.13 to 1.00 and from 0.20 to 0.86, respectively. Five loci significantly deviated from Hardy–Weinberg equilibrium and no significant linkage disequilibrium was found between pairs of loci. Cross-species amplification of these polymorphic microsatellite loci was performed in additional two related species. These polymorphic microsatellite loci would be useful for investigating genetic diversity of E. awoara and related species. L. Zhao and C. Shao have contributed equally to this work.     

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 charaterization of 12 dinucleotide microsatellite loci from Belenger’s jewfish (Johnius belengerii Cuvier 1830)

We describe the first isolation of 12 polymorphic microsatellite markers from Belenger’s jewfish (Johnius belengnerii Cuvier 1830). From a (GT)n-enriched genomic library, 54 microsatellites were selected for designing microsatellite primers, of which 36 gave working primer pairs. 12 of these loci were polymorphic in a test population of 21 individuals with alleles ranging from 3 to 18, and expected and observed heterozygosities from 0.5772 to 0.9449 and from 0.4286 to 0.9231, respectively. No significant linkage disequilibrium between pairs of loci was found, however, loci Jobe24 significantly deviated from Hardy–Weinberg equilibrium after Bonferroni correction. These polymorphic microsatellite loci should provide sufficient level of genetic diversity to investigate population structure in Belenger’s jewfish.