Characterization and PCR multiplexing of novel highly variable tetranucleotide Atlantic salmon (Salmo salar L.) microsatellites

Seven novel and highly variable tetranucleotide microsatellite markers, and conditions for multiplexing and simultaneous genotyping six of these in a single run, are described for Atlantic salmon. These provide a highly informative and cost‐effective set of molecular markers for genetic studies on cultured and wild populations of the species. The primers sets showed cross‐species amplification of appropriately sized amplified products in a number of other salmonid species and suggests the primer sets may have wider application.     

Analysis of parentage determination in Atlantic salmon (Salmo salar) using microsatellites

Microsatellite genetic markers are becoming increasing important tools in the investigation of alternate reproductive strategies in wild plants and animals, and in the implementation of optimal breeding programs for endangered species, and managed cultured populations. Overall, little attention is paid to the frequency and impact of scoring errors and mutations on the resolution and accuracy of such analyses. Here, parentage of 792 Atlantic salmon ( Salmo salar ) reared communally were determined using di- and tetranucleotide microsatellites. Over 99·5% of the offspring could be unambiguously matched to one set of parents in the original 12 (1 × 1) experimental cross (each of 12 males uniquely crossed to one of 12 females) and in a simulated 36 (1 × 1) cross (involving additional parents), and over 80% in a 12 × 12 cross (all 12 males crossed to all 12 females). Mutations were rare (≈3·4 × 10⁻⁻⁴), though scoring errors were relatively common (2–3% per allele scored), with the rate of error varying among loci. Approximately 90% of scoring errors (or mutations) are expected to be detected in this analysis, and of those that are not, fewer than 0·5% should lead to a false or incorrect determinations of parentage. Based on several indices, we expect that greater than 99·7% of offspring assayed were matched to their true parents.     

Evidence for multiple sex-determining loci in Tasmanian Atlantic salmon (Salmo salar)

Phenotypic sex in salmonids is determined primarily by a genetic male heterogametic system; yet, sex reversal can be accomplished via hormonal treatment. In Tasmanian Atlantic salmon aquaculture, to overcome problems associated with early sexual maturation in males, sex-reversed females are crossed with normal females to produce all female stock. However, phenotypic distinction of sex-reversed females (neo-males) from true males is problematic. We set out to identify genetic markers that could make this distinction. Microsatellite markers from chromosome 2 (Ssa02), to which the sex-determining locus (SEX) has been mapped in two Scottish Atlantic salmon families, did not predict sex in a pilot study of seven families. A TaqMan 64 SNP genome-wide scan suggested SEX was on Ssa06 in these families, and this was confirmed by microsatellite markers. A survey of 58 families in total representing 38 male lineages in the SALTAS breeding program found that 34 of the families had SEX on Ssa02, in 22 of the families SEX was on Ssa06, and two of the families had a third SEX locus, on Ssa03. A PCR test using primers designed from the recently published sdY gene is consistent with Tasmanian Atlantic salmon having a single sex-determining gene that may be located on at least three linkage groups.     

Use of differential expression data for identification of novel immune relevant expressed sequence tag-linked microsatellite markers in Atlantic salmon (Salmo salar L.)

Despite the large number of genes contributing to the immune response, wildlife immunogenetic studies have tended to focus mostly on the major histocompatibility complex-related genes. Here, we utilized previously published microarray and competitive RNA hybridization information to identify 3750 immune relevant Atlantic salmon (Salmo salar) expressed sequence tags. We then identified those expressed sequence tags containing microsatellites and subsequently designed 48 primer pairs and tested them for polymorphism in Atlantic salmon. Altogether, 16 polymorphic markers were characterized, with allele numbers ranging from two to 18, and these 16 loci were further tested in five other salmonid species.     

Genetic protein variation and natural selection in Atlantic salmon (Salmo salav, L.) parr

The principal focus of this study was an examination of the relationship between diallelic variation at the MEP-2 * locus and growth and survival in Atlantic salmon parr. In addition, patterns of growth and survival among genotypes at the AAT-I *, IDDH-I * and ZDHP-2 * loci were also examined. Significant differences in growth were found among MEP-2 * genotypes, with * 100/ 100 homozygotes largest in one of two independent comparisons and the * 125/I25 homozygotes largest in the other. No growth differences were observed among genotypes at any other locus. Although no significant differences were found in survival among genotypes at any locus, results of joint analysis of the patterns of growth and survival among MEP-2 * genotypes were consistent with a model of positive size-selective mortality. The demonstration of growth differences among MEP-2 * genotypes in parr and smolts may indicate an association between selection on this locus in fresh water and previously reported differences in sea-age at maturity among MEP-2 * genotypes in Atlantic salmon.     

An analysis of the distribution of juvenile Atlantic salmon (Salmo salar) in nature as a function of relatedness using microsatellites

The major objective of this study was to test the hypothesis that juvenile Atlantic salmon kin occupy adjacent territories in their natural habitat in order to profit from the benefits associated with kin-biased behaviours, as has been observed under controlled laboratory conditions. Microsatellites were used to establish the relatedness of salmon fry (in their first summer of life) and parr (in their second and third summer of life) captured in adjacent territories. We did not observe a relationship between the proximity and the relatedness of either parr of the same cohort or fry in their natural habitat. Although many pairs of fry were identified as being related when sampled immediately after emergence, most family groups did not occupy adjacent territories. The high dispersal potential in rivers, the low occupation rate of the habitat and the incidence of half-sibs in nature most probably reduce the opportunity and advantage of kin-biased behaviour, in contrast to laboratory studies conducted in artificial, high-density conditions.     

Comparative analysis of population structure across environments and geographical scales at major histocompatibility complex and microsatellite loci in Atlantic salmon (Salmo salar)

Evidence of selection acting on major histocompatibility complex (MHC) genes has been illustrated with the analysis of their nucleotide sequences and allele frequency distribution. Comparing the patterns of population differentiation at neutral markers and MHC genes in the wild may provide further insights about the relative role of selection and neutrality in shaping their diversity. In this study, we combine both methods to assess the role of selection on a MHC gene in Atlantic salmon. We compare variation at a MHC class II B locus and microsatellites among 14 samples from seven different rivers and seven subpopulations within a single river system covering a variety of habitats and different geographical scales. We show that diversifying selection is acting on the sites involved in antigen presentation and that balancing selection maintains a high level of polymorphism within populations. Despite important differences in habitat type, the comparison of the population structure at MHC and microsatellites on large geographical scales reveals a correlation between patterns of differentiation, indicating that drift and migration have been more important than selection in shaping population differentiation at the MHC locus. In contrast, strong discrepancies between patterns of population differentiation at the two types of markers provides support for the role of selection in shaping population structure within rivers. Together, these results confirm that natural selection is influencing MHC gene diversity in wild Atlantic salmon although neutral forces may also be important in their evolution.     

Spawning success in Atlantic salmon (Salmo salar L.): a long-term DNA profiling-based study conducted in a natural stream

Spawning success of Atlantic salmon (Salmo salar L.) was investigated, under near-natural conditions, in the Girnock Burn, an 8-km long tributary of the River Dee in Scotland. Employing minisatellite-based DNA profiling, mating outcomes were resolved over three spawning seasons by assigning parentage to progeny samples removed from spawning nests ('redds'). While individual spawning patterns differed markedly, consistent trends were present over the 3 years studied. Multiple spawning was found to be prevalent. More than 50% of anadromous spawners of both sexes contributed to more than one redd. Up to six redds for a single female and seven for a single male were detected. Both sexes ranged extensively. Distance between redds involving the same parent varied from a few metres to > 5 km. Distances > 1 km were common. Both males and females ranged to a similar extent. Range limit was not correlated to fish size. Pairs were not monogamous, both males and females mating with different partners at different sites. Size assortative mating was apparent among 1991 spawners but was not detected for 1992 or 1995. Redd superimposition was found to be common (17-22% of redds over the 3 years), although it was not correlated to the number of anadromous spawners present. High levels of nonanadromous mature parr mating success (40-50% of total progeny sampled) were recorded, and these likely contribute greatly to the effective population size. The relevance of these findings at the individual and population level is discussed, with particular reference to management implications.     

Quantitative genetics of disease resistance in vaccinated and unvaccinated Atlantic salmon (Salmo salar L.)

Furunculosis (Aeromonoas salmonicida) is an important disease in Atlantic salmon (Salmo salar) farming. Vaccination and selective breeding for increased resistance to the disease on the basis of challenge tests of unvaccinated fish are used as complementary prophylactic methods. An important issue is whether genetic predisposition to infection is consistent across vaccinated and unvaccinated fish. Hence, the main objective of this study was to determine the magnitude of the genetic associations (correlations) between resistance to furunculosis in vaccinated and unvaccinated fish, and to estimate the magnitude of the correlation of resistance to furunculosis with resistance to the viral diseases infectious pancreatic necrosis (IPN) and infectious salmon anaemia (ISA). Sub-samples of unvaccinated and vaccinated salmon from 150 full-sib families were subjected to separate cohabitation challenge tests. Substantial genetic variation was found in resistance to furunculosis in both the unvaccinated (heritabilities of 0.51 ± 0.05) and vaccinated (0.39 ± 0.06) fish. However, the genetic correlation between resistance to furunculosis in the two groups was low (0.32 ± 0.13), indicating a weak genetic association between resistance in the two groups. Hence, the current selection strategy on the basis of challenge tests of unvaccinated fish is likely to produce low genetic improvement in resistance to furunculosis under field conditions, where fish are vaccinated with an effective vaccine. Evidence was found of significantly favourable genetic associations of resistance to furunculosis in unvaccinated (but less so for vaccinated) fish with resistance to both IPN and ISA (unvaccinated fish), indicating that vaccination 'mask' genetic associations between resistance to different diseases.     

Looking for a needle in a haystack: Discovery of indigenous Atlantic salmon (Salmo salar L.) in stocked populations

Microsatellite analysis of Atlantic salmon fromfive Danish rivers was performed to determinethe stocked or indigenous status ofindividuals. Genetic variation at six highlypolymorphic microsatellite loci was assayed andused for individual based analyses (assignmenttests). Contemporary samples of adult returningspawners and fry were compared to baseline datafrom: 1) historical DNA samples (from oldscales) representing the indigenouspopulations, 2) samples from another Danishpopulation (Skjern River) used for stocking,and 3) five exogenous populations used forstocking. Assignment power was high. Thepercent of stocked salmon correctly assigned topopulation of origin ranged from 83% to 99%and the percent of indigenous salmon correctlyassigned to population of origin ranged from83% to 90%. For two of the riverssignificantly more individuals were assigned tothe indigenous populations than expected frommisclassification alone, suggesting that someremains of the indigenous populations hadpersisted. Still, many fish were of exogenousorigin. Simulated hybrids among releasedexogenous salmon and between exogenous andreleased Danish salmon (Skjern River) revealedthat natural hybridisation among released fishwas not likely to be the source of the fryclassified as indigenous, however, thepossibility of hybridisation among indigenousand released fish could not be dismissed.Several full sib groups were found amongindigenous natural fry ruling out one or a fewmatings as the source of the indigenous fry.These results show that some native populationsmay persist even after years of introductionand environmental perturbation; geneticinformation can be used to identify thesepopulations and identify individualsrepresenting these populations for use inrestoration programs.     

Microsatellite DNA markers for the study of Atlantic salmon (Salmo salar) kinship,population structure,and mixed‐fishery analyses

Eleven microsatellite DNA loci were identified and characterized for Atlantic salmon (Salmo salar) collected from the Penobscot River, Maine, USA and the River Nith, Scotland, UK. The markers revealed high levels of genetic diversity (seven to 48 alleles per locus), heterozygosity (to 100%), and allelic heterogeneity (all comparisons). Considerable differentiation was observed as the genetic distance (chord) between the two collections was 0.680 and the pairwise F_ST, 0.12, was highly significant. These findings are consistent with patterns of continental‐level differentiation observed previously using an alternate suite of microsatellite loci. Locus‐by‐locus analyses of molecular variance suggested that most markers were suitable for delineating kinships and population genetic structure.     

Genetic markers for Atlantic salmon (Salmo salar L.): single locus inheritance and joint segregation analyses of minisatellite (VNTR) DNA loci

Relatively few genetic markers are available for detailed studies of Atlantic salmon. The detection of 12 distinct minisatellite DNA loci in this species (by 10 Atlantic salmon and brown trout derived probes) and subsequent inheritance analyses in two half-sib families are reported here. Disomic Mendelian inheritance was confirmed at all loci. Only a single aberrant progeny genotype (at Ssa -A60) was observed among 138 progeny screened. None of the loci was sex-linked. The tight linkage association Str -A22/1 with Str -A22/2, previously reported for brown trout, was found to be conserved in the Atlantic salmon genome. An additional male-specific linkage group, Ssa -A34 with Str -A9/2, was also noted. These highly polymorphic loci should find widespread use as chromosomal, individual, familial and, probably, population markers.     

The use of genetic marking to assess the reproductive success of mature male Atlantic salmon parr (Salmo salar, L.) under natural spawning conditions

The reproductive success of mature male Atlantic salmon parr ( Salmo salar ) under natural spawning conditions was estimated using the polymorphism at the MEP - 2 * locus as a genetic marker. The percentage of eggs per redd fertilized by parr varied considerably over the five redds examined (0·9–27.7%, mean 10·8%), but a gametic contribution from mature parr was detected in each case. Parr reproductive success has important implications for the population structure and evolution of the Atlantic salmon through its effect on gene flow.     

Variation in the timing of river entry of Atlantic salmon (Salmo salar L.) in the Baltic

The timing of river entry in the Atlantic salmon is known to depend on genetic,demographic and environmental factors,but little is known about the relative magnitude of among population and among year variation and covariation in this respect in natural state Atlantic salmon rives.To investigate this,variability in the timing of river entry in three historical Finnish Atlantic salmon populations were analyzed using salmon trap data collected during 1870-1902.The analyses reveled that 1)the timing of river e...     

Comparision of a serological potency assay for furunculosis vaccines (Aeromonas salmonicida subsp. salmonicida) to intraperitoneal challenge in Atlantic salmon (Salmo salar L.)

Batch potency testing of salmonid vaccines is mainly performed by in vivo challenge, which requires a lot of animals and causes severe pain. Due to the animal welfare concerns associated with in vivo immunization challenge tests, methods which could refine, reduce or replace (3Rs) these tests are needed.The aim of this study was to assess the use of serological assay (immunization & antibody estimation with an enzyme-linked immunosorbent assay (ELISA) for batch potency testing of oil adjuvanted, inactivated commercial furunculosis vaccines. In total ten vaccines were included in the study: two commercial multi-component vaccines and two experimental single-component furunculosis vaccines with 5% and 20% antigen content (relative to the commercial vaccine), from two manufacturers. In addition two experimental single component vaccines based on A-layer positive and A-layer negative Aeromonas salmonicida respectively were included. Challenge and blood sampling were conducted 9 weeks post vaccination.There was a correlation between antibody response against A. salmonicida as measured by ELISA and protection in i.p. challenge.This study shows that the ELISA assay can be used for testing different vaccine formulations and can potentially replace in vivo challenge tests for batch potency testing of furunculosis vaccines.     

Heart rate as an indicator of stress during the critical swimming speed test of farmed Atlantic salmon (Salmo salar L.)

A swim tunnel is to fish as a treadmill is to humans, and is a device used for indirect measuring of the metabolic rate. This study aims to explore the fish stress (if any) during the critical swimming test routines (fish handling, confinement, and swimming) using heart rate (f_H, heartbeat per minute) bio-loggers in farmed Atlantic salmon (Salmo salar L.). In addition, the recovery dynamics of exercised fish using f_H were explored for 48 h post swim tests. Continuous f_H data were acquired following the surgical implantation and throughout the trials, such as during fish handling, swim tests (critical swimming speed, U_crit), and 48 h post swim tests. After 3 weeks of surgical recovery, f_H stabilized at 46.20 ± 1.26 beats min⁻¹, equalizing a ~38% reduction in f_H recorded post-surgical tachycardia (74.13 ± 1.44 beats min⁻¹). Interestingly, f_H was elevated by ~200% compared to baseline levels not only due to the U_crit (92.04 ± 0.23 beats min⁻¹) but also due to fish handling and confinement in the swim tunnel, which was 66% above the baseline levels (77.48 ± 0.34 beats min⁻¹), suggesting fish stress. Moreover, significantly higher plasma cortisol levels (199.56 ± 77.17 ng mL⁻¹) corresponding to a ~300% increase compared to baseline levels (47.92 ± 27.70 ng mL⁻¹) were identified after U_crit, predicting post-swim test stress (physiological exhaustion). These findings reinforce the importance of fish acclimation in the swim tunnel prior to the swimming tests. However, f_H dropped over the course of the 48-h post-swim test, but remained comparatively higher than the basal levels, suggesting fish should be given at least 48 h to recover from handling stress for better fish welfare. This study further explored the influence of fish tagging on U_crit, which resulted in reduced swimming capabilities of tagged fish (1.95 ± 0.37 body lengths s⁻¹) compared to untagged fish (2.54 ± 0.42 body length s⁻¹), although this was not significant (p = 0.06), and therefore future tagging studies are warranted.     

An evaluation of the quality of fall-fingerling Atlantic salmon (Salmo salar L.) released to natural lacustrine nursery areas in Newfoundland,Canada

Results of experiments on controlled releases of fall-fingerling Atlantic salmon to lacustrine habitats in Newfoundland are reviewed in terms of survival during freshwater residence and smolt yield per unit of standing water habitat. Average survival from parr to smolt in three consecutive annual releases was 12.9% and we conclude that the quality of fall-fingerling salmon, produced from our semi-natural rearing techniques, was satisfactory. Year-class survival decreased progressively with consecutive releases. Rate of increase in juvenile salmon biomass remained stable over the duration of the study, suggesting that the average annual biomass of 1.6 kg ha^–1 of juvenile salmon released did not exceed habitat carrying capacity. Precocity did not appear to represent a significant deterrent to fall-fingerling survival. Annual smolt yield (by year-class) from stocking ranged from 53 to 130 ha^–1 (mean = 77 smolts ha^–1).