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.     

Genetic variation of wild and hatchery populations of the catla Indian major carp (Catla catla Hamilton 1822: Cypriniformes, Cyprinidae) revealed by RAPD markers

Genetic variation is a key component for improving a stock through selective breeding programs. Randomly amplified polymorphic DNA (RAPD) markers were used to assess genetic variation in three wild population of the catla carp (Catla catla Hamilton 1822) in the Halda, Jamuna and Padma rivers and one hatchery population in Bangladesh. Five decamer random primers were used to amplify RAPD markers from 30 fish from each population. Thirty of the 55 scorable bands were polymorphic, indicating some degree of genetic variation in all the populations. The proportion of polymorphic loci and gene diversity values reflected a relatively higher level of genetic variation in the Halda population. Sixteen of the 30 polymorphic loci showed a significant (p < 0.05, p < 0.01, p < 0.001) departure from homogeneity and the F(ST) values in the different populations indicated some degree of genetic differentiation in the population pairs. Estimated genetic distances between populations were directly correlated with geographical distances. The unweighted pair group method with averages (UPGMA) dendrogram showed two clusters, the Halda population forming one cluster and the other populations the second cluster. Genetic variation of C. catla is a useful trait for developing a good management strategy for maintaining genetic quality of the species.     

Genetic diversity of two Portuguese populations of the pullet carpet shell Venerupis senegalensis, based on RAPD markers: contribution to a sustainable restocking program

The pullet carpet shell Venerupis senegalensis (=V. pullastra) is a commercially important species in Portugal, Spain, France, and Italy. In Portugal, this species was once abundant in the Ria Formosa (southern Portugal). However, in the early 1980s, its abundance declined dramatically due to overfishing. In order to reverse this negative trend, the genetic sustainable management of the wild stocks of V. senegalensis should be performed by promoting successful restocking actions and the development of an aquaculture commercial production program of this species. In order to find the best broodstock for aquaculture purposes and therefore minimize the deleterious effects of hatchery practices, we analyzed the genetic diversity of the natural population to be restocked (Ria Formosa) but also of another potential genetically close population (Ria de Aveiro) by RAPD. Similar and substantive percentage of polymorphic loci, effective number of alleles, Nei’s gene diversity, and Shannon’s diversity index was found within both populations. This high genetic variability within populations suggests that they might have a gene pool with sufficient genetic plasticity to support changes in the environmental conditions. Analyses of population genetic structure also revealed a small genetic differentiation between the two populations. The high genetic variability of the natural population to be restocked makes it the preferential broodstock for aquaculture purposes. However, the Ria de Aveiro population could also be a viable alternative, due to its genetic plasticity and the genetic similarity of both populations. The results of this study can be useful to the sustainable management of wild stocks as well as in promoting successful restocking actions based on aquaculture production.     

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.     

Partitioning kin groups of broodstock in the critically endangered Chinese sturgeon,Acipenser sinensis Gray 1835

Pedigrees of broodstock with unknown relationship of the critically endangered Chinese sturgeon, Acipenser sinensis, was evaluated using microsatellite markers to facilitate genetic management in restocking programs with small broodstock size. We characterized the distributions of relatedness values to reconstruct kin groups in four hatchery families with known pedigrees using microsatellites. The distributions of relatedness values for kin classes were used for partitioning full sibling groups of wild A. sinensis broodstock kept in two hatcheries, resulted in 13 full sibling clusters, four of which containing 62% of all the wild individuals. This indicates high probability of choosing close related breeder pairs in random mating, thus selective breeding is necessary to minimize inbreeding and maintain genetic diversity. This study provides a useful tool for genetic management in conservation programs of A. sinensis in aim of preserving self‐sustained wild populations.     

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.     

Randomly amplified polymorphic DNA analysis of four different populations of the Indian major carp, Labeo rohita (Hamilton)

The level of genetic variation provides the raw material for selective improvement of a stock. Random amplified polymorphic DNA (RAPD) assay was used to assess the genetic variation in three rivers: the Halda, the Jamuna and the Padma as well as in one hatchery population of the commercially important Indian major carp, Labeo rohita. RAPD markers were amplified from DNA samples of 35 fish from each of the four populations using six decamer random primers. The polymorphic loci proportions were 0.33, 0.28, 0.28 and 0.26 and Nei's gene diversity values were 0.06, 0.07, 0.06 and 0.05 for the Halda, the Jamuna, the Padma and the hatchery populations, respectively. The pairwise population differentiation (F_ST) values indicated a low level of genetic differentiation between the population pairs. From the unweighted pair group method of arithmetic mean (UPGMA) dendrogram based on Nei's genetic distances a correlation between genetic affinities and geographical area was found. The populations were segregated into two groups: the Halda in one group and the Jamuna, the Padma and the hatchery in another group. Overall, the RAPD technique can be introduced as a tool in the population genetics of the rohu fish to provide information on their genetic stock structure.     

Do hatchery-reared sea urchins pose a threat to genetic diversity in wild populations?

In salmonids, the release of hatchery-reared fish has been shown to cause irreversible genetic impacts on wild populations. However, although responsible practices for producing and releasing genetically diverse, hatchery-reared juveniles have been published widely, they are rarely implemented. Here, we investigated genetic differences between wild and early-generation hatchery-reared populations of the purple sea urchin Paracentrotus lividus (a commercially important species in Europe) to assess whether hatcheries were able to maintain natural levels of genetic diversity. To test the hypothesis that hatchery rearing would cause bottleneck effects (that is, a substantial reduction in genetic diversity and differentiation from wild populations), we compared the levels and patterns of genetic variation between two hatcheries and four nearby wild populations, using samples from both Spain and Ireland. We found that hatchery-reared populations were less diverse and had diverged significantly from the wild populations, with a very small effective population size and a high degree of relatedness between individuals. These results raise a number of concerns about the genetic impacts of their release into wild populations, particularly when such a degree of differentiation can occur in a single generation of hatchery rearing. Consequently, we suggest that caution should be taken when using hatchery-reared individuals to augment fisheries, even for marine species with high dispersal capacity, and we provide some recommendations to improve hatchery rearing and release practices. Our results further highlight the need to consider the genetic risks of releasing hatchery-reared juveniles into the wild during the establishment of restocking, stock enhancement and sea ranching programs.     

Genetic monitoring of the Amazonian fish matrinchã (Brycon cephalus) using RAPD markers: insights into supportive breeding and conservation programmes

The importance of genetic evaluations in aquaculture programmes has been increased significantly not only to improve effectiveness of hatchery production but also to maintain genetic diversity. In the present study, wild and captive populations of a commercially important neotropical freshwater fish, Brycon cephalus (Amazonian matrinchã), were analyzed in order to evaluate the levels of genetic diversity in a breeding programme at a Brazilian research institute of tropical fish. Random Amplified Polymorphic DNA fingerprinting was used to access the genetic variability of a wild stock from the Amazon River and of three captive stocks that correspond to consecutive generations from the fishery culture. Although farmed stocks showed considerably lower genetic variation than the wild population, a significantly higher level of polymorphism was detected in the third hatchery generation. The results seem to reflect a common breeding practice on several hatchery fish programmes that use a small number of parents as broodstocks, obtaining reproductive success with few non‐identified mating couples. The obtained data were useful for discussing suitable strategies for the genetic management and biodiversity conservation of this species.     

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.     

A tool for tracking genetic contributions of wild Penaeus (Melicertus) plebejus broodstock to hatchery populations

Stock enhancement, restocking and sea ranching are being increasingly applied in both fisheries and conservation. The contribution of hatchery stock to fishery harvest and the maintenance of the genetic structure of stocked populations are both important considerations when releasing captive‐bred organisms into natural systems. Use of wild‐caught broodstock generally overcomes some of the genetic problems associated with domesticated hatchery populations, but there is still a need to ensure that a sufficient proportion of the natural population contribute to production of the stocked cohort to realise the genetic benefits of using wild‐caught broodstock. Releases of Penaeus (Melicertus) plebejus are under investigation as a means of increasing prawn production in recruitment‐limited areas. We used the highly variable mitochondrial control region (mtCR) to assign post‐larvae to maternal lineages in the hatchery and also to investigate the reproductive performance of female broodstock in terms of contribution to the production of the cohorts of post‐larvae in the hatchery. Our data showed that mtCR can be a useful tool for tracking lineages and provided genetic evidence that unequal contribution and underproducing females can occur even in wild‐caught broodstock. This work therefore highlights the importance of monitoring the genetic composition of pre‐release hatchery stocks.     

Molecular Evidence for Introgression and Loss of Genetic Variability in Salmo (trutta) macrostigma as a Result of Massive Restocking of Apennine Populations (Northern and Central Italy)

We assessed structural gene variation (allozymes and mtDNA) of brown trout to evaluate the genetic variability of Apennine stream populations (Northern and Central Italy) and the possibility of introgression by alien genomes after massive restocking with hatchery strains (Atlantic stocks). Genetic variability within and between Apennine populations was extremely low in our samples. Only two allozyme loci were polymorphic and mean hetero-zygosity was also reduced compared to other brown trout populations. Allelic frequencies determined for both loci were similar to the ones detected in the corresponding hatchery spawners. The reduction or total absence of the Mediterranean nuclear (LDH-5) and mitochondrial (16S rDNA) diagnostic markers suggests the domestic origin of most populations, and the introgression effects carried out by non-native genomes. From a taxonomic point of view, a clear differentiation emerges among basins placed on opposite sides of the Apennine chain (Tyrrhenian and Adriatic regions). In particular, the presence of Mediterranean genotypes and haplotypes characterizing Salmo (trutta) macrostigma is sporadic along the eastern Apennine side, adding additional doubts on the original presence and wide distribution of this salmonid along the Adriatic side of the mountain chain. In spite of conservation programs devoted to preservation of local genetic characteristics of S. t. macrostigma, massive restocking practices with hatchery strains obtained by a few spawners is the major cause of significant `founder effect' and `inbreeding depression' even in Apennine regions.     

Conservation of population structure and genetic diversity under captive breeding of remnant coaster brook trout (<Emphasis Type="Italic">Salvelinus fontinalis</Emphasis>) populations

Rebuilding wild populations often involves captive broodstocks derived from small, remnant populations. We measured a hatchery program’s ability to conserve genetic diversity when founding captive broodstocks from such populations. Migratory coaster brook trout were extirpated from most of their historic range in US waters of Lake Superior and were proposed for listing under the Endangered Species Act. Two captive broodstocks, one with 19 founders and another with 99 founders, were established to rebuild US populations. We used microsatellite markers to examine genetic variation in source populations and early hatchery generations. Broodstocks retained the strong differentiation found between source populations; however, one founder, with a low probability of belonging to either source population, sired 5.7% of F₁ progeny. We found small changes in within-population genetic variation across successive wild and hatchery generations of broodstocks. Evaluation of stage-specific survivorship indicated that equalizing family sizes of embryos produced modest gains in the effective number of breeders, and that survival in the hatchery was nearly random across families. Our study demonstrates the value of genetic monitoring during initial stages of hatchery programs for small and declining populations.     

Genetic structure and management of the Neotropical migratory fish Megaleporinus obtusidens on a highly impacted river basin

Habitat fragmentation caused by hydroelectric dams has depleted fish populations worldwide. Restocking actions are usually adopted to recover those populations, but hatchery management protocols rarely guarantee the maintenance of genetic diversity and a balanced contribution among captive breeders each generation. Here, a set of 10 microsatellite markers was used to assess the genetic diversity (average allelic richness A_R?=?10.87 and expected heterozygosity H_E?=?0.742, respectively) and structuring of Megaleporinus obtusidens, a migratory freshwater fish inhabiting over 2500 km of the São Francisco River in Brazil. Three main genetic clusters were identified in this species across the river basin that could be related to the sharply different climatic and hydrologic regimes from the Upper to the Lower course. A significant reduction (> 50%) in genetic diversity was observed in the broodstock when compared to their wild conspecifics, especially in the allelic richness. The information here presented will aid for management of genetic resources of this species in the São Francisco River taking as reference the genetic clusters identified. Furthermore, the results indicated that restocking is not necessary unless signals of population depletion occurs and, if so, hatchery reproductive protocols should rely on artificial fertilization rather than mass spawning.

     

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.     

Population structure of the olive flounder (Paralichthys olivaceus) in Korea inferred from microsatellite marker analysis

The population structure of olive flounder Paralichthys olivaceus was estimated using nine polymorphic microsatellite (MS) loci in 459 individuals collected from eight populations, including five wild and three hatchery populations in Korea. Genetic variation in hatchery (mean number of alleles per locus, A = 10·2–12·1; allelic richness, A_R = 9·3–10·1; observed heterozygosity, H_O = 0·766–0·805) and wild (mean number of alleles per locus, A = 11·8–19·6; allelic richness, A_R = 10·9–16·1; observed heterozygosity, H_O = 0·820–0·888) samples did not differ significantly, suggesting a sufficient level of genetic variation in these well‐managed hatchery populations, which have not lost a substantial amount of genetic diversity. Neighbour‐joining tree and principal component analyses showed that genetic separation between eastern and pooled western and southern wild populations in Korea was probably influenced by restricted gene flow between regional populations due to the barrier effects of sea currents. The pooled western and southern populations are genetically close, perhaps because larval dispersal may depend on warm currents. One wild population (sample from Wando) was genetically divergent from the main distribution, but it was genetically close to hatchery populations, indicating that the genetic composition of the studied populations may be affected by hydrographic conditions and the release of fish stocks. The estimated genetic population structure and potential applications of MS markers may aid in the proper management of P. olivaceus populations.     

Mitochondrial DNA variation and population genetic structure in the small yellow croaker at the coast of Yellow Sea and East China Sea

Small yellow croaker is one of the most important fishery species in China. The mass–scale artificial propagation of this fish species was initially developed in 2015 with the aim of facilitating the fish production stock enhancement and aquaculture programs in the future. In the present study, the wild broodfish and its corresponding progeny along with three other wild populations were sampled and subjected to sequence analysis of the mitochondrial cytochrome c oxidase subunit I gene. The genetic diversity and population genetic structure were evaluated with a total sample size of 141 individuals representing the populations of the Yellow Sea (Qingdao and Lvsi populations) and the East China Sea (Xiangshan and Ningde populations). The wild populations were characterized by high haplotype diversity (0.925–0.976) and low nucleotide diversity (0.376%–0.560%). The hierarchical analysis of molecular variance (AMOVA) analysis and the values of pairwise Ф-statistics (Ф_ST) indicated non-significant genetic differentiation among the four wild populations. The hatchery stock XSH exhibited lower indices of genetic diversity compared with the wild populations that could be attributed to the small effective population size. The findings of the present study have valuable insight to the sustainable management and utilization of this resource.     

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 analysis of two Portuguese populations of <Emphasis Type="Italic">Ruditapes decussatus</Emphasis> by RAPD profiling

The clam Ruditapes decussatus is commercially important in the south of Portugal. The random amplified polymorphic DNA (RAPD) technique was applied to assess the genetic diversity and population structure of two Portuguese populations occurring in the Ria Formosa (Faro) and the Ria de Alvor, respectively. Twenty-five individuals of each population were investigated by RAPD profiles. Genetic diversity within populations, measured by the percentage of polymorphic loci (%P), varied between 68.57% (Alvor) and 73.88% (Faro). Shannon’s information index (H) and Nei’s gene diversity (h) were 0.281 and 0.176, respectively, for the Alvor population and 0.356 and 0.234 for the Faro population. Overall, genetic variation within R. decussatus populations was high. The total genetic diversity (H _T) was explained by a low variation between populations (G _ST = 0.145), which is consistent with high gene flow (N _m = 2.9). The analysis of molecular variance (AMOVA) showed that 65% of variability is within populations and 35% between populations (Φ_PT = 0.345; P ≥ 0.001). The value of Nei’s genetic distance was 0.0881, showing a low degree of population genetic distance, despite the different geographic origin. This is the first study on the population genetics of R. decussatus by RAPD technique. The results may be useful for restocking programs and aquaculture.     

Comparative genetic diversity of wild and hachery populations of Korean threadsail filefish Stephanolepis cirrhifer using cross-species microsatellite markers

The threadsail filefish Stephanolepis cirrhifer is a highly commercial fisheries resource in Korea that suffers intensive anthropogenic pressure across much of its range. For basic information about its current genetic status in relation to stock enhancement, the level and distribution of genetic variation between a wild and a hatchery-bred population were investigated using 10 microsatellite markers developed for Thamnaconus modestus. High levels of polymorphism were observed between the two populations. A total of 95 different alleles were found at all loci, with some alleles being unique. The allelic variability ranged from six to 13 in the wild population and from five to 13 in the hatchery one. The average observed and expected heterozygosities were estimated to be 0.72 and 0.80 in the wild sample and 0.70 and 0.79 in the hatchery one, respectively. These results showed similar 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 = 0.016, P < 0.05). Genetic drift in the intensive breeding practices for stock enhancement has probably promoted differentiation between populations. Significant deviations from Hardy-Weinberg equilibrium were detected in both populations. Our results indicate that further studies using species-specific microsatellite markers will be necessary for a more reliable assessment of genetic diversity of the species.