K. I. Afanasiev

A search for null alleles at the microsatellite locus of chum salmon (Oncorhynchus keta Walbaum)

Population studies with the use of microsatellite markers face a problem of null alleles, i.e., the absence of a PCR product, caused by the mutations in the microsatellite flanking regions, which serve as the sites of primer hybridization. In this case, the microsatellite primer associated with such mutation is not amplified, leading to false homozygosity in heterozygous individuals. This, in turn, results in biased population genetic estimates, including the excess of homozygotes at microsatellite loci. Analysis of the population structure of a Pacific salmon species, chum salmon (Oncorhynchus keta Walbaum), revealed the presence of null alleles at the Oke3 microsatellite locus in the population samples, in which an excess of homozygotes was observed. The analysis was performed using different combinations of modified primers chosen to match the Oke3 locus. The use of these primers enabled identification of true heterozygotes among those individuals, which were previously diagnosed as homozygotes with the use of standard primers. Removal of null alleles eliminated the excess homozygotes in the chum salmon samples described. In addition to the exclusion of false homozygosity, the use of modified primers makes it possible to introduce polymorphic primer variants associated with certain microsatellite alleles into population studies.

Rapid expansion of an enhanced stock of chum salmon and its impacts on wild population components

A harvested stock of chum salmon homing to Kurilskiy Bay, Iturup Island, consists of two genetically distinct river populations that reproduce in two rivers that drain into the bay and are characterized by limited gene flow. One of these is small and can be regarded as wild, whereas the other is much larger and, until recently, was composed of naturally reproducing components spawning in the river’s mainstem and tributaries, with almost no hatchery reproduction during the past two decades. The only human impact on reproduction of the chum salmon stock was regulation of the escapement, with officially accepted limits to avoid ‘over-escapement’. Recently the hatchery began to release a large amount of chum salmon juveniles. As confirmed by data on variation in both age composition and microsatellite DNA, first-generation hatchery-origin fish that returned from the first large releases occupied spawning grounds and presumably competed directly with, and potentially displaced wild fish. The most dramatic example is a genetically distinct beach-spawning form of chum salmon that was swamped by much more numerous hatchery-origin fish of the river-spawning form. In order to restore and support naturally reproduced population components, careful estimation of the carrying capacity of natural spawning grounds is necessary with efforts to increase escapement to these habitats. We also recommend concerted efforts to restore and conserve a unique beach-spawning population of chum salmon. We further recommend development of a marking program for direct estimation of straying and evaluation of ecological and genetic impacts of hatchery fish on neighboring wild and natural populations.

Interregional differentiation of chum salmon from Sakhalin and South Kurils inferred from microsatellite markers

Variability at ten microsatellite loci was examined in wild and hatchery populations of chum salmon (Oncorhynchus keta Walbaum) from the Sakhalin Island and Southern Kuril Islands, Iturup and Kunashir. Substantial genetic differences between Sakhalin and South Kurils chum salmon (the differentiation reached 6.0%) were revealed. Statistically significant differences between chum salmon from Iturup and those from Kunashir were demonstrated, as well as between the chum salmon populations from different rivers within the islands. It was shown that in different types of population comparisons, different marker sets were most informative.

Differentiation of chum salmon Oncorhynchus keta Wallbaum populations as revealed with microsatellite and allozyme markers: A comparative study

The features and extent of population differentiation in chum salmon Oncorhynchus keta from Sakhalin and Iturup Islands were studied with 10 microsatellite and 12 allozyme markers. It was demonstrated with the example of allozyme polymorphism at the EstD locus that the effect of an individual locus with one major allele is capable of distorting the total picture of population differentiation. Multiallelic microsatellites were more efficient in revealing the genetic structure of chum salmon populations at the levels of differences between regional populations and between the stocks of individual rivers of the same region.

Population structure and variability of Pacific herring (Clupea pallasii) in the White Sea, Barents and Kara Seas revealed by microsatellite DNA analyses

Pacific herring, Clupea pallasii, have recently colonised the northeast Atlantic and Arctic Oceans in the early Holocene. In a relatively short evolutionary time, the herring formed a community with a complex population structure. Previous genetic studies based on morphological, allozyme and mitochondrial DNA data have supported the existence of two herring subspecies from the White Sea and eastern Barents and Kara Seas (C. p. marisalbi and C. p. suworowi, respectively). However, the population structure of the White Sea herring has long been debated and remains controversial. The analyses of morphological and allozyme data have previously identified local spawning groups of herring in the White Sea, whereas mtDNA markers have not revealed any differentiation. We conducted one of the first studies of microsatellite variation for the purpose of investigating the genetic structure and relationship of Pacific herring among ten localities in the White Sea, the Barents Sea and the Kara Sea. Using classical genetic variance-based methods (hierarchical AMOVA, overall and pairwise FST comparisons), as well as the Bayesian clustering, we infer considerable genetic diversity and population structure in herring at ten microsatellite loci. Genetic differentiation was the most pronounced between the White Sea (C. p. marisalbi) versus the Barents and Kara seas (Chesha–Pechora herring, C. p. suworowi). While microsatellite variation in all C. pallasii was considerable, genetic diversity was significantly lower in C. p. suworowi, than in C. p. marisalbi. Also, tests of genetic differentiation were indicating significant differentiation within the White Sea herring between sympatric summer- and spring-spawning groups, in comparison with genetic homogeneity of the Chesha–Pechora herring.

Microsatellite analysis of two captive populations of sable (Martes zibellina L.)

The high value of sable (Martes zibellina L.) fur and stable demand for it over the centuries have led to suboptimal hunting patterns and, as a result, considerable fluctuations in the sizes of natural populations of this species. To maintain the traditional export of sable fur, efforts towards commercial domestication of sable have been made in Russia. The first farm population of sable consisted of animal from eight natural populations was founded in 1929. After the problems related to breeding in captivity were solved, directional selection began. Eighty years of breeding have resulted in sable herds with homogeneous quantitative characters. Prospects for further breeding depend on the current level of genetic diversity in the captive populations of sables formed during the first stages of domestication. The sable populations of the Pushkinsky and Saltykovsky fur farms located in Moscow oblast, which were the objects of this study, are the progenitors of the existing captive populations. The first estimation of genetic variation of this species by means of a panel of micro-satellite markers was developed for this study. Two captive sable populations were analyzed using ten micro-satellite loci; a total of 75 alleles were found in both populations. Population-specific alleles were identified (6 and 13 in the Pushkinsky and Saltykovsky populations, respectively). The populations studied were found to be differentiated with respect to four microsatellite loci.

Efficiency of the inbreeding coefficient f and other estimators in detecting null alleles, as revealed by empirical data of locus oke3 across 65 populations of chum salmon Oncorhynchus keta

A survey of 65 populations of chum salmon Oncorhynchus keta across the species range revealed homozygote excess (947 homozygotes in 2954 fish) at a polymerase chain reaction (PCR)-based simple sequence repeat (SSR) locus oke3 with multiple alleles, whereas re-designed PCR primers indicated that 328 of these homozygotes were actually heterozygotes. Statistically significant high positive values of inbreeding coefficients, f, in multiple populations appeared to be a reliable predictor of null alleles. Based on these data, three methods were checked for their ability to estimate null-allele frequencies.

Data on variation of microsatellite loci in Kildin cod Gadus morhua kildinensis (Gadidae)

Variation of microsatellite loci Gmo8, Gmo-G12, Gmo-G18, Gmo19, PGmo32, Gmo34, and Gmo35 is investigated in Kildin cod Gadus morhua kildinensis. The investigated loci are characterized by a low level of variation: five loci are represented by two-allele systems and three loci are monomorphic. Mean value of heterozygosity calculated by all investigated loci is lower in Kildin cod than in Atlantic Gadus morhua—0.2854 vs. 0.5667.

A detection of allelic variants at microsatellite markers by using capillary and traditional electrophoresis

Microsatellite alleles are detected by PCR (polymerase chain reaction) that provides a manifold increase in the number of copies (amplification) of a given DNA fragment. The fragment visualization can be reached by two different methods. These are fragment analysis by capillary electrophoresis in denaturing gel and fragment separation in non-denaturing gel with subsequent gel staining. The first method is more accurate and automated, but expensive. The second method is much cheaper but less convenient. It requires manual processing and is presumably less accurate. In this study, we present the results of comparison of the allele typing at nine microsatellite loci using these two methods for one of the species of Pacific salmon, sockeye salmon Oncorhynchus nerka Walbaum. In most cases, both methods give identical fragment sizes or with a constant difference if the alleles are relatively small (not larger than 200–220 bp).

Preliminary data on variation of four microsatellite loci in Pacific herring Clupea pallasii

Variation of microsatellite loci Cpa110, Cpa113, Cpa4, and Cpa7 was for the first time examined in Pacific-type herring Clupea pallasii from the White Sea (Cl. pallasii marisalbi), the Kara Sea (Cl. pallasii suworowi), the Sea of Okhotsk, and Lake Nerpich’e, Kamchatka Bay, northwestern Pacific (Cl. pallasii pallasii). All loci exhibitedhigh genetic diversity. The estimates of expected heterozygosity varied from 41.5 to 95.6% (mean, 82%). The level of pairwise genetic differentiation Fst at all microsatellite loci varied from 0.005 to 0.076 (0.019, on average) and t was statistically significant (p < 0.05) in most of the pairs of herring samples. Estimates of genetic differentiation among the herring of one subspecies were lower than between the groups belonging to different subspecies.