A rapid approach for isolation of microsatellites and other tandem repeated sequences in described. The method is based on hybridization capture of repetitive elements from digested genomic DNA using biotinylated oligonucleotide probes in solution and subsequent attachment to magnetic beads coated with streptavidin. Captured fragments are amplified by adapter polymerase chain reaction (PCR) and the PCR products enriched for microsatellites cloned directly into a T-vector for sequencing. The results presented here show that this approach is highly effective, allowing di- and trinucleotide repeats to be isolated and sequenced directly from fish and mammalian genomic DNA within four to five days. Assuming a density and relative abundance of repeats with AC/GT motifs corresponding to that found in the human genome, the protocol presented gives at least a 35-fold enrichment of AC/GT microsatellites using an (AC)10 oligo probe. In addition, four out of five sequences captured by a (CAG)9 oligo probe contained one or several CAG repeat arrays. The efficiency of this direct approach suggests that it can be used for extracting other types of tandem and interspersed repeated sequences (including transposons, rRNA and tRNA genes and proviruses) from vertebrate genomes.
To elucidate the colonization of freshwater fish into Norway following the last deglaciation of Europe 10,000 years ago, we have performed a survey using mitochondrial DNA variation (20 populations) and multilocus DNA fingerprinting (14 populations) of the widely distributed perch (Perca fluviatilis) from the Scandinavian peninsula and the Baltic Sea. Sequence analysis of a 378 bp segment of the perch mitochondrial control region (D-loop) revealed 12 different haplotypes. A nested clade analysis was performed with the aim of separating population structure and population history. This analysis revealed strong geographical structuring of the Scandinavian perch populations. In addition, the level of genetic diversity was shown to differ considerably among the various populations as measured by the bandsharing values (S-values) obtained from multilocus DNA fingerprinting, with intrapopulation S-values ranging from 0.19 in Sweden to 0.84 in the central part of Norway. Analysis of the intrapopulation S-values, with S-value as a function of lake surface area and region, showed that these differences were significant. The mitochondrial and DNA fingerprinting data both suggest that the perch colonized Norway via two routes: one from the south following the retreating glacier, and the other through Swedish river systems from the Baltic Sea area. Perch utilizing the southern route colonized the area surrounding Oslofjord and the lakes which shortly after deglaciation were close to the sea. Fish migrating from the Baltic Sea seem to have reached no further than the east side of Oslofjord, where they presumably mixed with perch which had entered via the southern route. It seems likely that the migration events leading to the current distribution of perch also apply to other species of freshwater fish showing a similar distribution pattern.
Genetic diversity within and between two wild and one farmed population of Atlantic salmon was estimated by 12 variable enzyme loci, three single locus‐ and one multilocus DNA minisatellite probe. The farmed salmon were fifth‐generation fish from one of the principal commercial strains in Norway and the wild salmon were from the rivers Numedalslågen and Tana, Norway. All three classes of markers detected significant differentiation between the populations, with the farmed population being genetically most divergent. The farmed fish showed less genetic variability than the wild populations, as all techniques revealed a lower (14%−45%) number of variable bands/alleles in this group. However, only multilocus DNA fingerprinting detected a significantly lower level of heterozygosity within the farmed population. Estimates of average heterozygosity within populations were almost identical for single locus and multilocus minisatellites (0.558 and 0.548, respectively), which were more than three times higher than for polymorphic allozymes (0.162). Estimates of interpopulation genetic differentiation, however, gave highly concordant results between the allozymes and minisatellites (Fstrange: 0.106‐0.135). Our results show that fifth generation farmed Atlantic salmon differ significantly from wild salmon in loci others than those chosen for selection in the breeding program. The study also suggests that the choice of genetic markers for studies of Atlantic salmon depends on the aim of the study (within‐versus between‐population variability).
The influence of sperm competition and individual mating behaviour in an externally fertilizing species of fish, the Atlantic salmon (Salmo salar), is estimated from video observations of multiple-male spawnings and subsequent paternity analyses. One male dominated the paternity during polygamous spawnings, fathering more than 80% of the progeny in a single nest. Behavioural analyses of the spawnings showed that the first-mating male had sperm precedence in 6 out of 10 cases. In three of the other spawnings, sperm limitation likely influenced individual success, as the first-mating male had participated in a large number of spawnings. In the final, nearly simultaneous spawning, male size was more important than the 0.6-s difference in spawning times. Thus, male fertilization success can be influenced by a variety of factors, including sperm precedence, male size, and spawning history.
UV-B tolerance and susceptibility of high Arctic morphotypes of tte Daphnia pulexl D. tenebrosa complex were assessed by in situ experiments at Ny-Alesund, Svalbard (79"N). Animals from local ponds were exposed to ambient light plus additional UV-B from lamps in a greenhouse facility. Taxonomic affinities did not appear as major determinants of UV susceptibility, but a major difference in UV-B tolerance was seen between morphotypes with pigmented carapaces and those without, the latter being far more susceptible. Assays on levels of carotene and the anti-oxidant enzymes catalase and superoxide dismutase did not reveal clear-cut differences between populations, and could not account for the higher tolerance in pigmented populations. Levels of glutathione transferase were higher in the transparent population, however. In the absence of blue light and UV, laboratory reared animals did not reconstitute their carapace melanization after moulting, indicating that short-wave light is the cue for melanin synthesis. Tests on melanized individuals and individuals of the same population reared indoors through 1-2 moults supported the major role of melanin for UV protection. Periods with high UV exposure during hatching of ephippia could induce shifts in morphotype or clonal dominance.
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