Genetic variation within and among domesticated Atlantic salmon broodstocks in British Columbia, Canada.

Atlantic salmon have been reared in the British Columbia, Canada aquaculture industry since the early 1980s. No breeding programmes spanned the entire production period and pedigree records were not kept for broodstocks prior to or since importation. Of the three recognized industry strains, two are of European ancestry ('Mowi' from Norway and 'McConnell' from Scotland) and one is of North American heritage ('Cascade' from Gaspe, Quebec). We evaluated the amount and distribution of genetic variation within industry broodstocks by surveying microsatellite variation at 11 loci in 20 broodstock groups sampled from major production facilities. Allelic richness averaged 10.9 (range 5.8-13.8), compared with a value of 20.3 obtained for a North American wild population. Pairwise genetic distances (D(S)) between samples within strains were generally less than those between strains, with samples attributed to the same strain clustering together in a neighbour-joining dendrogram. Nevertheless, average distances between samples within the European strains were high (0.41 for Mowi; 0.71 for McConnell) but lower (0.06) for the Cascade strain. The reduced intra-sample and increased intra-strain genetic variation observed for the BC domesticated samples compared with wild populations was similar to observations for European domesticated Atlantic salmon. Evidence of introgression of the Cascade strain into European broodstocks was provided by the presence of large Ssa202 alleles (confined to North America in wild populations) in some Mowi and McConnell samples. Introgression likely also contributed to the decreased intercontinental genetic distance for the domesticated samples of this study compared with that observed for wild populations.

Geographic heterogeneity in natural selection on an MHC locus in sockeye salmon.

Balancing selection maintains high levels of polymorphism and heterozygosity in genes of the MHC (major histocompatibility complex) of vertebrate organisms, and promotes long evolutionary persistence of individual alleles and strongly differentiated allelic lineages. In this study, genetic variation at the MHC class II DAB-beta1 locus was examined in 31 populations of sockeye salmon (Oncorhynchus nerka) inhabiting the Fraser River drainage of British Columbia, Canada. Twenty-five percent of variation at the locus was partitioned among sockeye populations, as compared with 5% at neutral genetic markers. Geographic heterogeneity of balancing selection was detected among four regions in the Fraser River drainage and among lake systems within regions. High levels of beta1 allelic diversity and heterozygosity, as well as distributions of alleles and allelic lineages that were more even than expected for a neutral locus, indicated the presence of balancing selection in populations throughout much of the interior Fraser drainage. However, proximate populations in the upper Fraser region, and four of six populations from the lower Fraser drainage, exhibited much lower levels of genetic diversity and had beta1 allele frequency distributions in conformance with those expected for a neutral locus, or a locus under directional selection. Pair-wise FST values for beta1 averaged 0.19 and tended to exceed the corresponding values estimated for neutral loci at all levels of population structure, although they were lower among populations experiencing balancing selection than among other populations. The apparent heterogeneity in selection resulted in strong genetic differentiation between geographically proximate populations with and without detectable levels of balancing selection, in stark contrast to observations at neutral loci. The strong partitioning and complex structure of beta1 diversity within and among sockeye populations on a small geographic scale illustrates the value of incorporating adaptive variation into conservation planning for the species.

Denaturing gradient gel electrophoresis (DGGE): a rapid and sensitive technique to screen nucleotide sequence variation in populations.

We describe a rapid and sensitive method for the detection of nucleotide sequence variation that can be used for large-scale screening of population markers. Denaturing gradient gel electrophoresis (DGGE) detects sequence variants of amplified fragments by the differences in their melting behavior. DGGE detects most single-base substitutions when carried out on products amplified with a primer to which a GC clamp has been added. Although DGGE has been primarily used for the detection of limited numbers of single-base mutations in disease studies, it offers great potential for use in population analysis of genetic markers with greater levels of sequence variation. The methodology described was developed to identify the number and distribution of MHC class I alpha 1 alleles among chinook salmon (Oncorhynchus tshawytscha) populations. DGGE detects 28 of 31 identified alpha 1 sequences, which differ by between 1 and 16 nucleotides and a two-codon indel. By creating a network of control alleles, 22-23 of the MHC alleles can be resolved rapidly and accurately by a single gel run condition, and 27 alleles can be resolved by two gel run conditions. This techniques has been used in surveys scoring alleles from two MHC markers (class I alpha 1 and alpha 2) in 20,000 individuals of chinook and coho (O. kisutch) salmon. A single person in our laboratory now analyzes 160 salmon from one MHC locus per day with DGGE.

The salmonid class I MHC: limited diversity in a primitive teleost.

Three MHC class I genes have been characterized in salmonids: A, B, and UA. Levels of polymorphism vary among the genes, but they all share one common feature: a lack of sequence diversity. Although individual species can carry over 30 alleles at a given locus (A), intraspecific diversity is generally less than 5% in Pacific salmon (genus Oncorhynchus), and less than 10% in Atlantic salmon (genus Salmo). These levels of diversity suggest that few ancient allelic lineages have persisted within species, and that most of the allelic radiation has occurred during or since speciation. Also apparent is the greater retention of allelic lineages in Atlantic salmon than Pacific salmon, which reflects historic differences of the two genera. Comparison of the salmonid class I sequences with those of other teleosts reveals two well supported groups: one containing the Cypriniformes and the salmonid UA, and the other containing the neoteleosts and the salmonid A and B. There is no homology between known Cypriniformes and neoteleostean sequences. If this relationship is borne out, it offers strong support for the hypothesis that the higher teleosts diverged more recently from the Salmoniformes than the Cypriniformes. The salmonid MHC may provide a snapshot of the neoteleostean MHC prior to the extensive class I duplication that has taken place in at least some of the more advanced species.

Molecular evolution at Mhc genes in two populations of chinook salmon Oncorhynchus tshawytscha.

The DNA sequences of four exons of the MHC (major histocompatibility complex) were examined in chinook salmon (Oncorhynchus tshawytscha) from an interior (Nechako River) and a coastal (Harrison River) population in the Fraser River drainage of British Columbia. Mhc class I A1, A2 and A3 sequences and a class II B1 sequence were obtained by PCR from each of 16-20 salmon from each population. The class I A1 and a pair of linked A2-A3 exons were derived from two different classical salmonid class I genes, Sasa-A and Onmy-UA, respectively. Allelic variation for B1, A1 and A2 was characterized by the high levels of nonsynonymous substitution indicative of the effects of natural selection on Mhc domains that contain peptide binding regions. The number of alleles detected at each of the four exons ranged from three (B1) to 22 (A1), but levels of nucleotide sequence divergence at all four exons were low relative to classical mammalian Mhc genes. The nucleotide similarity among alleles ranged between 89 and 99% over all exons, and all four domains possessed only two major sequence motifs. Allelic distributions at B1, A1 and A3 confirmed the genetic distinctiveness of the Harrison and Nechako chinook salmon populations revealed in previous studies. The two major allelic motifs of B1 and A1 segregated strongly between the populations. In spite of evidence that allelic diversity at these chinook salmon Mhc exons has been generated by selection, the level and distribution of diversity in the two salmon populations strongly reflected the demographic history of the species, which has been characterized by repeated bottlenecks and isolation-by-distance in glacial refugia.

Mhc diversity in Pacific salmon: population structure and trans-species allelism.

Geographic variation at an Mhc class I A1 exon was surveyed in 14 populations of coho salmon (Oncorhynchus kisutch) and 15 populations of chinook salmon (O. tshawytscha) inhabiting rivers of British Columbia, Canada. A total of 2,504 fish were sampled using denaturing gradient gel electrophoresis (DGGE), which distinguished 17 alleles in coho salmon and 20 alleles in chinook salmon. Heterozygosity at the A1 locus was moderately high for both coho (0.7) and chinook (0.6) salmon, but sequence divergence was low, with mean inter- and intraspecific nucleotide similarities of approximately 0.96. In a maximum parsimony tree, all of the observed alleles clustered into two trans-specific lineages. Within each lineage, coho and chinook alleles tended to fall into species-specific subclusters. Much of the intraspecific allelic variation within each lineage could be accounted for by nonsynonymous point mutation, indicative of balancing selection. The FST values for both coho (0.11) and chinook (0.13) salmon indicated that much of the allelic diversity was partitioned among populations. Neighbor-joining analyses of A1 allelic frequencies among coho and chinook salmon populations showed strong patterns of geographic differentiation similar to those based on neutral genetic markers such as microsatellite loci. Both natural selection and the salmonid zoogeographic history of frequent population bottlenecks have shaped the patterns of diversity observed at this and other Mhc exons in Pacific salmonids.

Sequence analysis of a polymorphic Mhc class II gene in Pacific salmon.

Polymorphism of the nucleotide sequences encoding 149 amino acids of linked major histocompatibility complex (Mhc) class II B1 and B2 peptides, and of the intervening intron (548-773 base pairs), was examined within and among seven Pacific salmon (Oncorhynchus) species. Levels of nucleotide diversity were higher for the B1 sequence than for B2 or the intron in comparisons both within and between species. For the codons of the peptide binding region of the B1 sequence, the level of nonsynonymous nucleotide substitution (dN) exceeded the level of synonymous substitution (dS) by a factor of ten for within-species comparisons, and by a factor of four for between-species comparisons. The excess of dN indicates that balancing selection maintains diversity at this salmonid Mhc class II locus, as is common for Mhc loci in other vertebrates. Levels of nucleotide diversity for both the exon and intron sequences were greater among than within species, and there were numerous species-specific nucleotides present in both the coding and noncoding regions. Thus, neighbor-joining analysis of both the intron and exon regions provided phylogenies in which the sequences clustered strongly by species. There was little evidence of shared ancestral (trans-species) polymorphism in the exon phylogeny, and the intron phylogeny depicted standard relationships among the Pacific salmon species. The lack of shared allelic B1 lineages in these closely related species may result from severe bottlenecks that occurred during speciation or during the ice ages that glaciated the rim of the north Pacific Ocean approximately every 100000 years in the Pleistocene.

Genetic variation in survival of chinook salmon (Oncorhynchus tshawytscha) alevins exposed to an unidentified agent of mortality.

Mortality of an unknown etiology occurs after hatching and before emergence among Harrison River chinook salmon (Oncorhynchus tshawytscha) alevins incubated in the Chehalis River Hatchery, British Columbia. Inter- and intra-stock genetic variation for alevin survival and time to death was investigated at Chehalis Hatchery in factorial crosses among chinook salmon from the Harrison and Capilano rivers. Alevin survival by family ranged from 0 to 100%, with a mean value of 35.2%. The mean family survival of pure Harrison alevins (13.0%) was significantly lower than that of Capilano alevins (64.1%). For the Harrison stock, estimates of the heritability of survival were 1.05 +/- 0.62 (sire component) and 0.03 +/- 0.07 (dam component). For the Capilano stock, the corresponding estimates were 0.79 +/- 0.53 and 0.80 +/- 0.54. Family means of time to death ranged from 7.5 to 48 days after exposure to mortality-inducing agents. The mean times to death for pure Harrison (15.3 days) and Capilano (21.8 days) families were not significantly different. Sire and dam component heritability estimates for time to death were high for the Harrison stock (1.39 +/- 0.87 and 0.71 +/- 0.46) but low for the Capilano stock (0.06 +/- 0.11 and 0.17 +/- 0.18). Values of survival and time to death for the reciprocal interstock hybrid alevins generally fell between those of the parental stocks. Neither survival nor time to death differed significantly between the reciprocal hybrids, but both traits were more strongly influenced by sire than by dam. The possibility of asynchronous paternal and maternal allele activation during embryonic development was proposed as an explanation for the strong paternal effects observed in this study.

Electrophoretic polymorphism and sexual dimorphism in the freshwater and anadromous threespine sticklebacks (Gasterosteus aculeatus) of the Little Campbell River, British Columbia.

We used single-family crosses to confirm the Mendelian interpretation of allozyme variation and to examine linkage relationships at five polymorphic loci in freshwater and anadromous threespine sticklebacks (Gasterosteus aculeatus) from the Little Campbell River, British Columbia. The electrophoretic pattern of a sixth locus, mitochondrial NADP-dependent isocitrate dehydrogenase (IDH), was found to be sexually dimorphic but otherwise invariant in sticklebacks. This raised the possibility of a sex-linked Idh locus. No differences in IDH quantity or substrate affinities (apparent Km values) were detected between male and female sticklebacks. The electrophoretic pattern of IDH expression was not changed in sticklebacks in which sexual development was altered by hormonal treatment.

Heterozygosity and morphological variability of chum salmon (Oncorhynchus keta) in southern British Columbia.

We compared variability in two meristic and six morphometric characters with heterozygosity within and among 27 populations of chum salmon (Oncorhynchus keta) in southern British Columbia. Among individuals, there was no relationship between levels of heterozygosity at 10 electrophoretic loci and degree of meristic or morphometric variation. Decreased morphological variance was not associated with increased heterozygosity. Morphological variance and heterozygosity did not change with age for chum salmon maturing at three to five years of age. Among populations of chum salmon, increased levels of average heterozygosity were not associated with decreased variance of morphometric or meristic characters. Our results do not support the hypothesis that more heterozygous individuals show less phenotypic variability than more homozygous ones due to a canalization of morphology during development. Genetic distances between pairs of chum salmon populations were significantly correlated with pairwise Mahalanobis distances derived from meristic, but not from morphometric characters. Chum salmon are morphometrically adapted to the natal stream environment, whereas biochemical and meristic characters in these populations may be less affected by local selective forces.