[Show abstract][Hide abstract] ABSTRACT: Microsatellite genotyping is a common DNA characterization technique in population, ecological and evolutionary genetics research. Since different alleles are sized relative to internal size-standards, different laboratories must calibrate and standardize allelic designations when exchanging data. This interchange of microsatellite data can often prove problematic. Here, 16 microsatellite loci were calibrated and standardized for the Atlantic salmon, Salmo salar, across 12 laboratories. Although inconsistencies were observed, particularly due to differences between migration of DNA fragments and actual allelic size ('size shifts'), inter-laboratory calibration was successful. Standardization also allowed an assessment of the degree and partitioning of genotyping error. Notably, the global allelic error rate was reduced from 0.05 ± 0.01 prior to calibration to 0.01 ± 0.002 post-calibration. Most errors were found to occur during analysis (i.e. when size-calling alleles; the mean proportion of all errors that were analytical errors across loci was 0.58 after calibration). No evidence was found of an association between the degree of error and allelic size range of a locus, number of alleles, nor repeat type, nor was there evidence that genotyping errors were more prevalent when a laboratory analyzed samples outside of the usual geographic area they encounter. The microsatellite calibration between laboratories presented here will be especially important for genetic assignment of marine-caught Atlantic salmon, enabling analysis of marine mortality, a major factor in the observed declines of this highly valued species.
[Show abstract][Hide abstract] ABSTRACT: Approximately half of the mitochondrial genome inherent within 546 individual Atlantic salmon (Salmo salar) derived from across the species' North Atlantic range, was selectively amplified with a novel combination of standard PCR and pyro-sequencing in a single run using 454 Titanium FLX technology (Roche, 454 Life Sciences). A unique combination of barcoded primers and a partitioned sequencing plate was employed to designate each sequence read to its original sample. The sequence reads were aligned according to the S. salar mitochondrial reference sequence (NC_001960.1), with the objective of identifying single nucleotide polymorphisms (SNPs). They were validated if they met with the following three stringent criteria: (i) sequence reads were produced from both DNA strands; (ii) SNPs were confirmed in a minimum of 90% of replicate sequence reads; and (iii) SNPs occurred in more than one individual.
Pyrosequencing generated a total of 179,826,884 bp of data, and 10,765 of the total 10,920 S. salar sequences (98.6%) were assigned back to their original samples. The approach taken resulted in a total of 216 SNPs and 2 indels, which were validated and mapped onto the S. salar mitochondrial genome, including 107 SNPs and one indel not previously reported. An average of 27.3 sequence reads with a standard deviation of 11.7 supported each SNP per individual.
The study generated a mitochondrial SNP panel from a large sample group across a broad geographical area, reducing the potential for ascertainment bias, which has hampered previous studies. The SNPs identified here validate those identified in previous studies, and also contribute additional potentially informative loci for the future study of phylogeography and evolution in the Atlantic salmon. The overall success experienced with this novel application of HT sequencing of targeted regions suggests that the same approach could be successfully applied for SNP mining in other species.
[Show abstract][Hide abstract] ABSTRACT: Three ecologically and morphologically distinct forms of Arctic charr (Salvelinus alpinus L.) have been identified in Loch Rannoch, Scotland, whose evolutionary status and origins are incompletely understood. A
study was made of restriction fragment length polymorphism (RFLPs) detected variation in the D-loop, ND1 and cytochrome b regions of the mitochondrial genome, encompassing >3500bp. Eight RFLP haplotypes were identified that clustered into three
distinct clans based on restriction differences and into four clans based on sequence differences. Significant differences
in RFLP frequencies were found among all morph groups. The pelagic morph was highly divergent from the two benthic forms,
with the benthic forms having variants from only one genetic clan while the pelagic was dominated by a single variant from
another clan. The relative divergence observed among benthic and pelagic forms is ~10 fold greater when nucleotide divergence
among the haplotypes, as well as haplotype frequency differences, is taken into account. Sequence divergence between haplotypes
in the two main clans is of a similar order to that between haplotypes in these clans and a charr from North America. In contrast,
divergence among the two benthic morphs relates entirely to differences in haplotype frequencies. The study confirms the genetic
distinctiveness of the pelagic and benthic forms as well as of the two benthic forms. It strongly supports previous evidence
that the genetic divergence between the pelagic and benthic populations is allopatric in origin. Additionally, the results
strongly suggest that the two benthic populations have undergone peripatric divergence through the sequential colonisation
of the two basins by one lineage, followed by their spatial separation and reproductive isolation.
[Show abstract][Hide abstract] ABSTRACT: PCR primers were successfully designed to amplify small ND1 gene fragments for RFLP genotyping of degraded Atlantic salmon Salmo salar mtDNA. Analysis of archival scales with these primers, when existing primer sets failed, show Atlantic salmon from the George River, Quebec, to include European haplotypes and those from the Kapisidlit River, West Greenland, to be fixed for a European haplotype characteristic of Baltic populations.
Journal of Fish Biology 04/2005; 60(1):266 - 270. · 1.83 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Small populations may be expected to harbour less genetic variation than large populations, but the relation between census
size (N), effective population size (N
e), and genetic diversity is not well understood. We compared microsatellite variation in four small peripheral Atlantic salmon
populations from the Iberian peninsula and three larger populations from Scotland to test whether genetic diversity was related
to population size. We also examined the historical decline of one Iberian population over a 50-year period using archival
scales in order to test whether a marked reduction in abundance was accompanied by a decrease in genetic diversity. Estimates
of effective population size (N
e) calculated by three temporal methods were consistently low in Iberian populations, ranging from 12 to 31 individuals per
generation considering migration, and from 38 to 175 individuals per generation if they were regarded as closed populations.
e/N ratios varied from 0.02 to 0.04 assuming migration (mean=0.03) and from 0.04 to 0.18 (mean=0.10) assuming closed populations.
Population bottlenecks, inferred from the excess of heterozygosity in relation to allelic diversity, were detected in all
four Iberian populations, particularly in those year classes derived from a smaller number of returning adults. However, despite
their small size and declining status, Iberian populations continue to display relatively high levels of heterozygosity and
allelic richness, similar to those found in larger Scottish populations. Furthermore, in the R. Asón no evidence was found
for a historical loss of genetic diversity despite a marked decline in abundance during the last five decades. Thus, our results
point to two familiar paradigms in salmonid conservation: (1)␣endangered populations can maintain relatively high levels of
genetic variation despite their small size, and (2) marked population declines may not necessarily result in a significant
loss of genetic diversity. Although there are several explanations for such results, microsatellite data and physical tagging
suggest that high levels of dispersal and asymmetric gene flow have probably helped to maintain genetic diversity in these
peripheral populations, and thus to avoid the negative consequences of inbreeding.
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: Matrilineal phylogenetic divergence among Atlantic salmon stocks of the Bay of Fundy in south eastern Canada is investigated. Sequence variation in two regions of the mitochondrial ND1 gene, encompassing 710 base pairs, is described for 168 salmon from 11 rivers. Mean overall haplotype and nucleotide diversity (h and pi) observed are 0.5014 and 0.00095, respectively. Nested clade analysis (NCA) and molecular analysis of variance (AMOVA) both point to highly restricted gene flow among rivers and show the haplotype distribution to be geographically structured. Variation among predefined regions of the Bay (16%) is greater than among populations within these regions (14%) The main regional differentiation occurs between rivers of the geographically isolated inner Minas Basin and those elsewhere in the Bay. Differentiation most probably reflects the pattern and nature of the historical processes associated with post-glacial colonisation of the area by salmon following the last Pleistocene glacial maximum c. 180,00 yrs BP.
[Show abstract][Hide abstract] ABSTRACT: Current understanding of the postglacial colonization of Nearctic and Palearctic species relies heavily on inferences drawn from the phylogeographic analysis of contemporary generic variants. Modern postglacial populations are supposed to be representative of their Pleistocene ancestors, and their current distribution is assumed to reflect the different colonization success and dispersal patterns of refugial lineages. Yet, testing of phylogeographic models against ancestral genomes from glacial refugia has rarely been possible. Here we compare ND1 mitochondrial DNA variation in late Pleistocene (16,000-40,000 years before present), historical and contemporary Atlantic salmon (Salmo salar) populations from northern Spain and other regions of western Europe. Our study demonstrates the presence of Atlantic salmon in the Iberian glacial refugium during the last 40,000 years and points to the Iberian Peninsula as the likely source of the most common haplotype within the Atlantic lineage in Europe. However, our findings also suggest that there may have been significant changes in the genetic structure of the Iberian refugial stock since the last ice age, and question whether modern populations in refugial areas are representative of ice age populations. A common haplotype that persisted in the Iberian Peninsula during the Pleistocene last glacial maximum is now extremely rare or absent from European rivers, highlighting the need for caution when making phylogeographic inferences about the origin and distribution of modern genetic types.