New polymorphic microsatellite markers in Pacific abalone Haliotis discus hannai and their application to genetic characterization of wild and aquaculture populations

Seven new microsatellite markers were developed for the Pacific abalone (Haliotis discus hannai, Haliotidae), and allelic variability was compared between a wild population and a hatchery population in Yeosu, Korea. All loci amplified readily and demonstrated allelic variability, with the number of alleles ranging from 6 to 15 in the wild population and from 3 to 12 in farmed populations. Average observed and expected heterozygosities were estimated at 0.65 and 0.77 in the hatchery samples, and 0.79 and 0.87 in the wild samples. These results indicated lower genetic variability in the hatchery population, as compared with the wild population and significant genetic differentiation between the wild population and the hatchery samples (F _ST=0.055, p<0.001). These microsatellite loci may be valuable for future population genetic studies and for tracking hatchery samples used in stock enhancement programs.     

Genetic differences between wild and hatchery populations of red sea cucumber (Stichopus japonicus) inferred from microsatellite markers: implications for production and stocking programs design

Red sea cucumber Stichopus japonicus is the most important and valuable commercial sea cucumber species in Korea. Its farming and stock enhancement started in the early 2000s and is still rapid expansion in Korea. Therefore, the analyses of genetic status of wild and hatchery populations are necessary to maintain the genetic diversity of this valuable marine resource. In this study, possible genetic similarity and differences between the wild population and hatchery population in Jeju, Korea were accessed using multiplex assays with eight highly polymorphic microsatellite loci. High levels of polymorphism were observed between the two populations. A total of 93 different alleles were found. Although a considerable loss of unique alleles and relatively high inbreeding coefficient value were observed in the hatchery samples, no statistically significant reductions were found in heterozygosity or allelic diversity in the hatchery population, compared with the wild population. However, significant genetic heterogeneity was found between two populations. These results suggest that genetic drift has probably promoted differentiation between populations, and stocking intensity in wild populations may correlate with loss of genetic integrity. Therefore, the sustainable exploitation plans of the fishery resource should be developed by applying basic genetic principles combined with molecular monitoring. This genetic baseline information of Korean red sea cucumber has important implications for designing of genetically sustainable restocking programs and more efficient conservation management guidelines for these valuable genetic materials.     

Comparative genetic diversity of wild and hatchery-produced Pacific oyster (Crassostrea gigas) populations in Korea using multiplex PCR assays with nine polymorphic microsatellite markers

The Pacific oyster, Crassostrea gigas, is the most important and valuable commercial fishery species in Korea. Its farming started 20 years ago and is still rapid expansion in Korea. In this study, to maintain the genetic diversity of this valuable marine resource, possible genetic similarity and differences between the wild population and hatchery population in Tongyeong, Korea were accessed using multiplex assays with nine highly polymorphic microsatellite loci. A total of 250 different alleles were found over all loci. Despite a long history of hatchery practices, very high levels of polymorphism (mean alleles = 22.89 and mean heterozygosity = 0.92) were detected between the two populations. No statistically significant reductions were found in heterozygosity or allelic diversity in the hatchery population compared with the wild population. However, significant genetic heterogeneity was found between two populations. These results provide no evidence to show that hatchery practice of Pacific oyster in Korea has significantly affected the genetic variability of the hatchery stock. Although further studies are needed for comprehensive determinations of the hatchery and wild populations with increased number of Pacific oyster sample collections, information on the genetic variation and differentiation obtained in this study can be applied for genetic monitoring of aquaculture stocks, genetic improvement by selective breeding and designing of more efficient conservation management guidelines for these valuable genetic materials.     

Wild and aquaculture populations of the eastern oyster compared using microsatellites

Five new microsatellite markers were developed for the eastern oyster (Crassostrea virginica), and allelic variability was compared between a wild Chesapeake Bay population (James River) and a hatchery strain (DEBY). All loci amplified readily and demonstrated allelic variability with the number of alleles ranging from 16 to 36 in the wild population and from 11 to 19 in the DEBY strain. Average observed and expected heterozygosities were estimated at 0.66 and 0.80 in the hatchery sample. The corresponding estimates were 0.91 and 0.75 in the wild sample. Results indicated lower genetic variability in the DEBY strain and significant genetic differentiation between the wild population and hatchery strain. These microsatellite loci will prove valuable for future population genetic studies and in tracking of hatchery strains used in restoration.     

Genetic differences between wild and hatchery populations of Korean spotted sea bass (Lateolabrax maculatus) inferred from microsatellite markers

The spotted sea bass, Lateolabrax maculatus, is popular in recreational fishing and aquaculture in Korea. Its natural population has declined during the past two decades; thus, beginning in the early 2000s stock-enhancement programs were introduced throughout western and southern coastal areas. In this study, genetic similarities and differences between wild and hatchery populations were assessed using multiplex assays with 12 highly polymorphic microsatellite loci; 96 alleles were identified. Although many unique alleles were lost in the hatchery samples, no significant reductions were found in heterozygosity or allelic diversity in the hatchery compared to the wild population. High genetic diversity (He = 0.724–0.761 and Ho = 0.723–0.743), low inbreeding coefficient (F _IS = 0.003–0.024) and Hardy–Weinberg equilibrium were observed in both wild and hatchery populations. However, the genetic heterogeneity between the populations was significant. Therefore, genetic drift likely promoted inter-population differentiation, and rapid loss of genetic diversity remains possible. Regarding conservation, genetic variation should be monitored and inbreeding controlled in a commercial breeding program.     

Comparative Genetic Diversity of Wild and Captive Populations of the Bare-Faced Curassow (Crax fasciolata) Based on Cross-Species Microsatellite Markers: Implications for Conservation and Management

The bare-faced curassow (Crax fasciolata) is a large Neotropical bird that suffers anthropogenic pressure across much of its range. A captive population is maintained for conservation management, although there has been no genetic screening of stocks. Based on the six microsatellite markers developed for Crax globulosa, the genetic variability of C. fasciolata and possible differences between a wild and a captive population were investigated. Only three loci were polymorphic, with a total of 27 alleles. More than half of these alleles were private to the wild (n = 8) or captive (n = 7) populations. Significant deviations from Hardy–Weinberg equilibrium were restricted to the captive population. Despite the number of private alleles, genetic drift has probably promoted differentiation between populations. Our results indicate that wild C. fasciolata populations are genetically impoverished and structured, but species-specific microsatellite markers will be necessary for a more reliable assessment of the species’ genetic diversity.     

Isolation and characterization of polymorphic microsatellite markers for the black rockfishSebastes inermis

Black rockfish (Sebastes inermis, Scorpaenidae) is a commercially important fisheries resource in Korea. However, no genetic diversity information is available for wild or released hatchery populations. To study the effects of stock enhancement programs and conservation biology, we developed microsatellite DNA markers fromS. inermis. We report the isolation and characterization of seven microsatellite loci, designated KSi221B, KSi239A, KSi25A, KSi252B, KSi193A, KSi259B, and KSi234, isolated using an enrichment method based on magnetic/biotin capture of microsatellite sequences from a size-selected genomic library. To characterize each locus, 30 individuals from a naturalS. inermis population in Yeosu, Korea, were genotyped. All loci except one, KSi193A, were polymorphic with an average of 9.5 alleles per locus (range 2–20). The mean observed and expected heterozygosities were 0.71 (range 0.43–1.00) and 0.76 (range 0.41–0.90), respectively. Significant deviation from Hardy-Weinberg equilibrium was observed at one locus, KSi221B. Such high variability indicates that these microsatellites should provide useful markers for studies of kinship and population genetics.     

Population genetic structure of wild and hatchery black rockfish Sebastes inermis in Korea, assessed using cross-species microsatellite markers

The population structure of the black rockfish, Sebastes inermis (Sebastidae), was estimated using 10 microsatellite loci developed for S. schlegeli on samples of 174 individuals collected from three wild and three hatchery populations in Korea. Reduced genetic variation was detected in hatchery strains [overall number of alleles (N(A)) = 8.07; allelic richness (A(R)) = 7.37; observed heterozygosity (H(O)) = 0.641] compared with the wild samples (overall N(A) = 8.43; A(R) = 7.83; H(O) = 0.670), but the difference was not significant. Genetic differentiation among the populations was significant (overall F(ST) = 0.0237, P < 0.05). Pairwise F(ST) tests, neighbor-joining tree, and principal component analyses showed significant genetic heterogeneity among the hatchery strains and between wild and hatchery strains, but not among the wild populations, indicating high levels of gene flow along the southern coast of Korea, even though the black rockfish is a benthic, non-migratory marine species. Genetic differentiation among the hatchery strains could reflect genetic drift due to intensive breeding practices. Thus, in the interests of optimal resource management, genetic variation should be monitored and inbreeding controlled within stocks in commercial breeding programs. Information on genetic population structure based on cross-species microsatellite markers can aid in the proper management of S. inermis populations.     

Genetic differentiation between collections of hatchery and wild masu salmon (<Emphasis Type="Italic">Oncorhynchus masou</Emphasis>) inferred from mitochondrial and microsatellite DNA analyses

There has been very little effort to understand genetic divergence between wild and hatchery populations of masu salmon (Oncorhynchus masou). In this study, we used mitochondrial (mt) NADH dehydrogenase subunit 5 gene (ND5) and six polymorphic nuclear microsatellite DNA loci to compare the genetic variability in three hatchery broodstocks of masu salmon with the variability in eight putative wild masu populations sampled in five rivers including one known source river for the hatchery broodstocks. Both ND5 and microsatellites showed no significant genetic divergence (based on F_ST estimates) between four annual collections from the source river population, suggesting no change in genetic diversity over this time period. The F_ST estimates, an analysis of molecular variance (AMOVA), and a neighbor-joining tree using both DNA markers suggested significant differentiation between the three hatchery and all eight putative wild populations. We conclude that genetic diversity of hatchery populations are low relative to putative wild populations of masu salmon, and we discuss the implications for conservation and fisheries management in Hokkaido.     

Microsatellite variability and differentiation of hatchery stocks of chum salmon Oncorhynchus keta Walbaum in Sakhalin

Variability at eight microsatellite loci was examined in five populations of chum salmon Oncorhynchus keta Walbaum from Sakhalin hatcheries. The population of Kalinino hatchery had the lowest heterozygosity and the lowest average number of alleles per locus. The populations examined exhibited significant differentiation, θ_ST = 0.026 on average per locus. The maximum genetic differences were found between the populations of the Kalinino and the Ado-Tymovo hatcheries; the latter differs from the remaining populations also by the highest number and high frequencies of specific alleles. The genetic features of the Taranai hatchery population, observed at microsatellite loci, reflect its “mixed” origin.     

Genetic Structure of Different Populations of Walking Catfish (Clarias batrachus L.) in Bangladesh

Information on genetic variation is essential for conservation and stock improvement programs. Seven dinucleotide microsatellite loci were analyzed to reveal genetic variability in three wild populations (Kella beel, Hakaluki haor, and Shobornokhali beel) and one hatchery population of the freshwater walking catfish, Clarias batrachus, in Bangladesh. Upon PCR amplification, the alleles were separated on polyacrylamide gel using a sequencing gel electrophoresis system and visualized by the silver-staining method. The loci were polymorphic (P95) in all the populations. Differences were observed in number and frequency of alleles as well as heterozygosity in the studied populations. Current gene diversity (He) was higher than expected under mutation-drift equilibrium, significantly in the Hakaluki haor and Shobornokhali beel populations, indicating a recent genetic bottleneck. Population differentiation (FST) values were significant (P<0.05) in all the population pairs. A relatively high level of gene flow and a low level of FST values were found between wild population pairs compared to hatchery-wild pairs. The unweighted pair group method with averages dendrogram based on genetic distance resulted in two major clusters: the hatchery population was alone in one cluster whereas the three wild populations made another cluster. The results reflect some degree of genetic variability in C. batrachus populations indicating potentialities for improving this species through a selective breeding program. The results revealed a recent bottleneck in some wild populations of C. batrachus. Protection of habitat may help increase the population size and lower the risk of vulnerability of the species in the future.     

Population genetic structure in wild and hatchery populations of white cloud mountain minnow (Tanichthys albonubes): Recommendations for conservation

We used mitochondrial (mt) cytochrome b gene (cyt b) to compare the genetic variability in three hatchery broodstocks of white cloud mountain minnow with the variability in six wild populations sampled in two river drainages. A total of 43 haplotypes in 102 specimens were observed, with no haplotype shared between wild and hatchery populations. The nucleotide diversity of the wild samples (0.048) was significantly higher than that of the hatchery ones (0.007), but the haplotype diversity was almost similar between them. Two major phylogenetic haplotype groups were revealed and estimated to diverge about 6.531 myr (million years) ago. Significant genetic differentiation was revealed between wild and hatchery populations as well as among nine sampled populations, suggesting at chance effect during the founding process for the hatchery population and a subsequent genetic drift. According to the network, the connection between wild and hatchery populations indicates that present hatchery populations originated from single wild population. We suggested that two regions (Pearl River system and Lu River) identified by reciprocal mtDNA monophyly and SAMOVA should be regarded as three different ESUs and two different MUs in South China, respectively.     

Genetic structure of the Korean black scraper Thamnaconus modestus inferred from microsatellite marker analysis

The Korean black scraper, Thamnaconus modestus, is one of the most economically important maricultural fish species in Korea. However, the annual catch of this fish has been continuously declining over the past several decades. In this study, the genetic diversity and relationships among four wild populations and two hatchery stocks of Korean black scraper were assessed based on 16 microsatellite (MS) markers. A total of 319 different alleles were detected over all loci with an average of 19.94 alleles per locus. The hatchery stocks [mean number of alleles (N _A) = 12, allelic richness (A _R) = 12, expected heterozygosity (He) = 0.834] showed a slight reduction (P > 0.05) in genetic variability in comparison with wild populations (mean N _A = 13.86, A _R = 12.35, He = 0.844), suggesting a sufficient level of genetic variation in the hatchery populations. Similarly low levels of inbreeding and significant Hardy–Weinberg equilibrium deviations were detected in both wild and hatchery populations. The genetic subdivision among all six populations was low but significant (overall F _ST = 0.008, P < 0.01). Pairwise F _ST, a phylogenetic tree, and multidimensional scaling analysis suggested the existence of three geographically structured populations based on different sea basin origins, although the isolation-by-distance model was rejected. This result was corroborated by an analysis of molecular variance. This genetic differentiation may result from the co-effects of various factors, such as historical dispersal, local environment and ocean currents. These three geographical groups can be considered as independent management units. Our results show that MS markers may be suitable not only for the genetic monitoring of hatchery stocks but also for revealing the population structure of Korean black scraper populations. These results will provide critical information for breeding programs, the management of cultured stocks and the conservation of this species.     

Analysis of microsatellite DNA markers reveals no genetic differentiation between wild and hatchery populations of Pacific threadfin in Hawaii

Pacific threadfin, Polydactylus sexfilis, is popular fish in recreational fishing, as well as aquaculture in Hawaii. Its natural population has been continuously declining in the past several decades. Microsatellite DNA markers are useful DNA-based tool for monitoring Pacific threadfin populations. In this study, fifteen Microsatellite (MS) DNA markers were identified from a partial genomic Pacific threadfin DNA library enriched in CA repeats, and six highly-polymorphic microsatellite loci were employed to analyze genetic similarity and differences between the wild population and hatchery population in Oahu Island. A total of 37 alleles were detected at the six MS loci in the two populations. Statistical analysis of fixation index (F(ST)) and analysis of molecular variance (AMOVA) showed no genetic differentiation between the wild and hatchery populations (F(ST) = 0.001, CI(95%) = -0.01-0.021). Both high genetic diversity (H(o) = 0.664-0.674 and H(e) = 0.710-0.715) and Hardy-Weinberg equilibrium were observed in the wild and hatchery populations. Results of genetic bottleneck analysis indicated that the hatchery was founded with sufficient numbers of brooders as inbreeding coefficient is very low (F(IS) = 0.052-0.072) in both wild and hatchery populations. Further studies are needed for comprehensive determinations of genetic varieties of primary founder broodstocks and successive offspring of the hatchery and wild populations with increased number of Pacific threadfin sample collections.     

Genetic diversity and differentiation of the Korean starry flounder (Platichthys stellatus) between and within cultured stocks and wild populations inferred from microsatellite DNA analysis

The Korean starry flounder, Platichthys stellatus, is economically valuable coastal resident fish species. However, the annual catch of this fish has fluctuated and suffered major declines in Korea. We examined the genetic diversity and population structure for four wild populations and three hatchery stocks of Korean starry flounder to protect its genetic integrity using nine microsatellites. A group of 339 genotypes belonging to seven populations were screened. High degrees of polymorphism at the microsatellite loci were observed within both the wild and hatchery populations. Compared to the wild populations, genetic changes, including reduced genetic diversity and highly significant differentiation, have occurred in cultured stocks. Significant population differentiation was also observed in wild starry flounder populations. Similar degrees of inbreeding and significant Hardy–Weinberg equilibrium deviations were detected in both the wild and the hatchery populations. The genetic connectivity pattern identified four distinct metapopulations of starry flounder in Korea by clustering in the phylogenetic tree, Bayesian analyses, molecular variance analysis, PCA and multidimensional scaling analysis. A pattern of isolation-by-distance was not significant. This genetic differentiation may be the result of the co-effects of various factors, such as historic dispersal, local environment or anthropogenic activities. These results provide useful information for the genetic monitoring of P. stellatus hatchery stocks, for the genetic improvement of this species by selective breeding and for designing suitable management guidelines for the conservation of this species.     

Analysis of gene associated tandem repeat markers in Atlantic salmon (<Emphasis Type="Italic">Salmo salar L.</Emphasis>) populations: implications for restoration and conservation in the Baltic Sea

Patterns of genetic diversity and differentiation among five wild and four hatchery populations of Atlantic salmon in the Baltic Sea were assessed based on eight assumedly neutral microsatellite loci and six gene-associated markers, including four expressed sequence tag (EST) linked and two major histocompatibility complex (MHC) linked tandem repeat markers (micro- and mini-satellites). The coalescent simulations based on the method of Beaumont and Nichols (1996, Proc. R. Soc. Lond. Ser. B – Biol. Sci., 263, 1619–1626) indicated that two loci (MHCIIα and Ssa171, with the lowest and highest overall F_ST estimates, respectively) exhibited significant departures (P<0.05) from the neutral expectations. Another coalescent-based test for selective neutrality (Vitalis et al. 2001, Genetics, 158, 1811–1823) further supported the outlier status of the Ssa171 microsatellite locus but not of the MHCIIα linked minisatellite. In addition, actin related protein linked microsatellite locus was identified with this test as an outlier in six pairwise population comparisons. All genetic diversity estimates revealed more genetic variation in hatchery stocks than in the small wild salmon populations from the Gulf of Finland. However, the wild populations possessed alleles at gene-associated markers (e.g. MHCI and IGF) not found in the hatchery stocks, which together with moderate genetic differentiation and distinctive environmental conditions justifies the special conservation measures for the last remaining native salmon populations in the Gulf of Finland.     

Genetic differences between broodstock and offspring of seven-band grouper in a hatchery

The seven-band grouper (Epinephelus septemfasciatus) is an important fishery resource of a target for prospective aquaculture diversification and maintenance of stock quality is thus important. To explore the sustainability of fry production, genetic variations in 83 seven-band groupers from two broodstock and offspring populations of a hatchery strain were analyzed using 13 polymorphic nuclear microsatellite DNA loci; 133 alleles were identified. Allelic variability ranged from 4 to 18 in the broodstock and from 3 to 11 in the offspring. The average observed and expected heterozygosities were 0.669 and 0.734 in broodstock and 0.674 and 0.649 in offspring, respectively. Although no statistically significant reductions in heterozygosity or allelic diversity were evident in offspring, considerable loss of rare alleles was apparent. The broodstock and offspring populations exhibited significant genetic differences (F _ST = 0.033, P < 0.001) indicating that genetic drift has likely promoted differentiation between the two populations, which may have negative effects on sustainable fry production. Therefore, genetic variations between broodstock and offspring should be monitored, and inbreeding should be controlled, to ensure the success of commercial breeding programs. Our data provide a useful genetic basis for future planning of sustainable culture and management of E. septemfasciatus in fisheries.     

Genetic differences between wild and hatchery populations ofDiplodus sargus andD. vulgaris inferred from RAPD markers: implications for production and restocking programs design

Restocking and stock enhancement programs are now recognized as an important tool for the management of fishery resources. It is important, however, to have an adequate knowledge on the genetic population structure of both the released stock and the wild population before carrying out such programs. In this study, random amplified polymorphic DNA (RAPD) markers were applied to assess genetic diversity and population structure of wild and hatchery populations of the white seabreamDiplodus sargus and the common two-banded seabreamD. vulgaris (Sparidae). The estimated values for intrapopulation genetic variation, measured using the percentage of polymorphic loci (%P), Shannon indexH’, and Nei’s gene diversity (h), showed high values for all populations. The percentage of genetic variation withinD. sargus andD. vulgaris populations, based on coefficient of gene differentiation, reached 82.5% and 90% of the total genetic variation, respectively. An undeniable decrease in genetic variation was found in both hatchery populations, particularly inD. sargus, compared to the wild ones. However, the high values of variation within all populations and the low levels of genetic variation among populations did not indicate inbreeding or depression effects, thus indicating a fairly proper hatchery management. Nevertheless, the results of this study highlight the importance of monitoring the genetic variation of hatchery populations, particularly those to be used in restocking programs. The creation of a genetic baseline database will contribute to a more efficient conservation management and to the design of genetically sustainable restocking programs.     

Genetic Differences Between Hatchery Stocks and Natural Populations in Pacific Abalone (<Emphasis Type="Italic">Haliotis discus</Emphasis>) Estimated Using Microsatellite DNA Markers

Genetic variations within and between nine hatchery stocks and seven natural populations of abalone including Ezo-abalone (Haliotis discus hannai) and Kuro-abalone (H. d. discus) were assayed with nine microsatellite markers. Marked reductions of genetic variability in the hatchery stocks were recognized in the allelic diversity and mean heterozygosity compared with the natural populations. Thirteen of 16 significant HWE deviations in hatchery stocks revealed heterozygotes excess, while all natural populations did not show such a tendency. Highly significant F _ST values were observed for all cases between the hatchery stocks, and between the hatchery stocks and natural populations. Genetic distance (D _A) between each hatchery stock and the geographically proximal population (mean ± SD, 0.108 ± 0.035) were similar to those estimated for between the natural Ezo-abalone and Kuro-abalone (0.101 ± 0.021). The self-assignment test, which allocated individuals to their own stock with a high success rate, provided evidence of solid genetic differences among the nine hatchery stocks. These results suggests that the allelic composition and diversity in the natural populations was not necessarily reflected in the hatchery stocks owing to population bottleneck and genetic drift through seedling process, and thus the seedling and stocking practice of these hatchery stocks should take much notice of the results to conserve the genetic diversity of natural populations.     

New polymorphic microsatellite markers for the masu salmon (Oncorhynchus masou masou) from Korea and their application to wild and hatchery populations

Masu salmon, Oncorhynchus masou masou, is an economically important fish species in the Far East and occurs in two life history forms: sea-run migratory (anadromous) and freshwater resident (non-anadromous). The non-anadromous form has recently become a popular freshwater food and game fish during a well-known Korean winter festival. However, the genetic background of this species remains largely unknown, partly due to a lack of molecular genetic markers. In this study, we developed new polymorphic microsatellite markers for masu salmon using next-generation sequencing technology. From 40 primer sets, 11 primer sets (27.5% of the primer sets selected) were successfully amplified with 106 alleles (range 2–9) in 64 individuals from different populations: two wild and one hatchery. Observed and expected heterozygosities ranged from 0.304 to 0.947 and 0.278 to 0.865, respectively. Significant departures from the Hardy–Weinberg equilibrium were detected for four markers (OMM11, OMM17, OMM28, and OMM33) in a single population. All pair-wise F_ST values were highly significant between the wild and hatchery populations (range 0.084–0.183, P < 0.0001). We identified a set of robust microsatellite markers that worked well even in formalin-fixed samples, which will be suitable for biogeographical and population structure analyses of the masu salmon.