[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 frag- ment 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 pro- cessing 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 a constant differ- ence if the alleles are relatively small (not larger than 200-220 bp).

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.

[Population genetic differentiation of white-spotted char Salvelinus leucomaenis (Pallas) of Russian Far East].

The genetic structure of the populations in white-spotted char Salvelinus leucomaenis (family Salmonidae) was determined based on variations in ten microsatellite DNA loci in samples from different parts of the species range in the Russian Far East. In a number of samples, variations in 21 allozyme loci were examined, of which five loci were found to be polymorphic. The overall diversity level in the examined markers was comparable to that observed in a closely related char species, Dolly Varden. The estimates of interpopulation genetic differentiation were highly statistically significant in most of the pairwise comparisons amongthe samples. The overall evaluation of the spatial genetic differentiation in white-spotted char constituted F(ST) = 0.203 and R(ST) = 0.202. Cluster analysis and multidimensional scaling based on microsatellite allele frequencies indicated the possible subdivision of the examined samples into two main groups, i.e., northern (represented by the regions of the north of Khabarovsk krai, Kamchatka, Yama Bay) and southern (with regions including Sakhalin, Primorye, and the Kuril Islands). The allozome data demonstrated a similar pattern of differentiation. The level of intra- and interpopulation genetic diversity in the southern group was higher than in the northern group. The isolation-by-distance test did not identify a significant correlation between genetic and geographic distances among the samples. The data obtained enabled the suggestion that the genetic structure of the population of white-spotted char was shaped by the influence of historical geological climatic rearrangements of its range and the genetic drift, because of determined by relatively low population number and limited in the extent of the migration activity of its anadromous form.

[Genetic variations in Clupea pallasii herring from okhotsk sea based on microsatellite markers].

The genetic variations among spawning groups of herrings from different spawning grounds of the northwestern part of the Okhotsk Sea was assessed using ten microsatellite loci. All loci were polymorphic with the ex- pected heterozygosity estimates varying at different loci in the range of 0.7-95.0% (with a mean of 68.5%). The degree of genetic differentiation displayed by the herrings from the Okhotsk Sea was not statistically sig nificant (0 = 0.74%). The level of pairwise genetic differentiation F(ST) varied in the range of 0.002-0.014, no was it statistically significant in all comparison pairs between the herring samples.

[Microsatellite variation in white-spotted char Salvelinus leucomaenis from Sakhalin Oblast].

A panel of 12 polymorphic microsatellite markers was developed for the population genetic studies of white-spotted char Salvelinus leucomaenis. The four population samples examined consisted of 48 individuals each, and were collected in different geographical regions, including Sakhalin Island, Kunashir Island, and Iturup Island (two samples). The total number of different-sized alleles at different loci varied in the range of 2-31. In the population of white-spotted char subjected to strong anthropogenic pressure allelic diversity and expected heterozygosity indices were found to be lower than in wild populations of this species. The considerable genetic subdivision of different insular populations of white-spotted char observed was consistent with isolation by the distance model.

[Analysis of microsatellite loci variations in herring (Clupea pallasii marisalbi) from the White Sea].

The genetic diversity among spawning groups of herring from different parts of the White Sea was assessed using ten microsatellite loci. All loci were polymorphic with the expected heterozygosity estimates varying in the range of 12.7-94.1% (mean was 59.5%). The degree of genetic differentiation displayed by White Sea herring was statistically significant (theta = 2.03%). The level of pairwise genetic differentiation F(ST) was 0-0.085, and it was statistically significant in most of the comparison pairs between the herring samples. A hierarchical analysis of molecular variance (AMOVA) revealed the statistically significant differentiation of White Sea herring. 96.59% genetic variation was found within the samples and 3.41% variation was found among the populations. The main component of interpopulation diversity (1.85%) falls at the differences between two ecological forms of herring, spring- and summer-spawning. Within the spring-spawning form, the presence of local stocks in Kandalaksha Gulf, Onega Bay, and Dvina Bay was demonstrated.

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

Variation of microsatellite loci Cpa 10, Cpa 113, Cpa4, and Cpa7 was for the first time examined in Pacific-type herring Clupea pallasii from the White Sea (CI. pallasii marisalbi), the Kara Sea (CI. pallasii suworowi), the Sea of Okhotsk, and Lake Nerpich'e, Kamchatka Bay, northwestern Pacific (CI. pallasiipallasii). 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.

[Extension of a set of microsatellite markers for more precise identification of chum salmon (Oncorhynchus keta Walbaum)].

A set often microsatellite loci enabling fairly accurate identification of the chum salmon individuals from geographically distant groups was designed at the Laboratory of Genetic Identification, Vavilov Institute of General Genetics, Russian Academy of Sciences. However, identification of the individuals from closely located basins performed using these loci was not sufficiently precise. The present study was focused on the improvement of the resolution of the method through increasing the number microsatellite loci used. In this study, typing of additional microsatellite loci of chum salmon and evaluation of the change of the degree of identification with the increase of the number ofmicrosatellite loci used is described. It was shown that the identification accuracy permanently increased with the increase of the number of microsatellite markers used.

[Analysis of microsatellite variation in the rainbow trout (Parasalmo (Oncorhynchus) mykiss) from Kamchatka].

Genetic variation of Kamchatka rainbow trout Parasalmo (O.) mykiss was examined using 10 microsatellite DNA loci, and phylogeographic comparison with other representatives of the species across the distribution range was performed. It was demonstrated that Kamchatka populations differed from other geographic groups of rainbow trout in a number of microsatellite loci. These populations also displayed distinct clustering and were characterized by lower genetic diversity. Analysis of a set of 26 different microsatellite loci (personal and literature data) demonstrated that most of the populations within the Kamchatka region were separated from one another, characterized by marked geographic differentiation, and affiliation to certain river basins. In Kamchatka rainbow trout, with high degree of probability, three geographic clusters (northwestern, southwestern, and eastern) were identified. In general, analysis of microsatellite DNA supported the data on low genetic diversity of the Kamchatka group Parasalmo (O.) mykiss, based on the variation estimates for a number of genes of nuclear and mitochondrial DNA, and allozyme loci.

[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.

[Analysis of microsatellite DNA of rainbow trout (Parasalmo (Oncorhynchus) mykiss) of Kamchatka: selection of loci and optimization of the method].

The variation of a sample of rainbow trout (Parasalmo (Oncorhynchus) mykiss) from natural populations of several rivers of the Kamchatka Peninsula with respect to 43 microsatellite DNA loci has been studied. These loci were earlier used for analysis of Asian populations of closely related salmonids. Ten of them may be regarded as markers and seen promising for further studies on intraspecific relationships of rainbow trout of Kamchatka. Their use in studies on more numerous samples from different localities and populations of Parasalmo (O.) mykiss in the Asian part of the species range will ensure efficient population genetic analysis of the Kamchatka population group of this species.

[Interregional differentiation of chum salmon from Sakhalin and South Kurils infered from microsatellite markers].

Variability at ten microsatellite loci was examined in wild and hatchery populations of chum salmon from the Sakhalin Island and Southern Kuril Islands, Iturup and Kunashir. Substantial genetic differences between Sakhalin and South Kurils chum salmon (the differentiation theta reached 6.0%) were revealed. Statistically significant differences between chum salmon from Iturup and that 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, required different marker sets most informative were.

[Genetic differentiation of pink salmon oncorhynchus gorbuscha Walbaum in the Asian part of the range].

Genetic variation at 19 allozyme (including 11 polymorphic) and 10 microsatellite loci was examined in the population samples of odd- and even-broodline pink salmon from the southern part of Sakhalin Island, Southern Kuril Islands, and the northern coast of the Sea of Okhotsk. The estimates of relative interpopulation component of genetic variation over the allozyme loci, per broodline, were on average 0.43% (GST), while over the microsatellite loci it was 0.26% (the theta(ST) coefficient, F-statistics based on the allele frequency variance), and 0.90% (the rho(ST) coefficient, R-statistics based on the allele size variance). The values of interlinear component constituted 2.34, 0.31, and 1.05% of the total variation, respectively. Using the allozyme loci, statistically significant intralinear heterogeneity was demonstrated among the regions, as well as among the populations of Southern Sakhalin Island. Multivariate scaling based on the allozyme data demonstrated regional clustering of the sample groups, representing certain populations during the spawning run or in different years. Most of the microsatellite loci examined were found to be highly polymorphic (mean heterozygosity > 0.880). The estimates of interlinear, interregional, and interpopulation variation over these loci in terms of theta(ST) values were substantially lower than in terms of rho(ST) values. Regional genetic differentiation, mostly expressed at the allozyme loci among the populations from the northern and southern parts of the Sea of Okhotsk (i.e., between the Sakhalin and Kuril populations), was less expressed at the microsatellite loci. The differentiation between these regions observed can be considered as the evidence in favor of a large-scale isolation by distance characterizing Asian pink salmon. It is suggested that in pink salmon, low genetic differentiation at neutral microsatellite loci can be explained by extremely high heterozygosity,of the loci themselves, as well as by the migration gene exchange among the populations (the estimate of the genetic migration coefficient inferred from the "private" allele data constituted 2.6 to 3.4%), specifically, by the ancient migration exchange, which occurred during postglacial colonization and colonization of the range.

[Selective effect of the duration of the critical temperature period on some allozyme loci of Atlantic salmon Salmo salar L. (Salmonidae)].

Genetic structure of juvenile fish from the populations of the Atlantic salmon Salmo salar inhabiting the rivers of Murmansk oblast, Arkhangelsk oblast, and Karelia, as well as of juveniles from hatcheries was examined at five allozymic loci: aspartate aminotransferase (AAT-4*), isocitrate dehydrogenase (IDHP-3*), iditol dehydrogenase (IDDH-2*), esterase D (ESTD*), and malic enzyme (MEP-2*). High genetic differentiation of both natural and "hatchery" juvenile fish was revealed. It was demonstrated that the gene pool of juveniles at three of the five loci tested was to a considerable degree formed by natural selection. In this case, the role of limiting factor was played by the duration of critical temperature regime in the rivers. The differentiation factors for juveniles from nature were not established, however, their clusterization pattern pointed to a possible role of natural selection in this process.

[A comparison of the spawning migration and genetic and biological parameters of stellate sturgeon from the Volga population in 1985 and 1996].

The dynamics of changes in several genetic and biological parameters of the Volga stellate sturgeon during the spawning runs of 1985 and 1996 were studied by electrophoresis and morphometry and compared. The dynamics of changes in genetic diversity of the spawners running spawners is repeated in many respects every 11 years, suggesting that the intrapopulation diversity is maintained in time. The among-sample diversity of late spring stellate sturgeon, whose spawners spawners are used in hatchery, decreased twofold. Along with decreases in body length and weight and mean age, the females displayed increased heterozygosities and genotype diversity index at LDH3 and PGM1 loci, while the males displayed a statistically significant decrease in body length. During the period in question, the heterozygosity at EST2 locus significantly decreased. The changes observed are connected with the artificial reproduction and non-rational fishing. Preservation of genetic diversity in stellate sturgeon requires that the specific features of the structure and ecology of the species reproduced are taken into account.

[Variation in morphometric and genetic characteristics of stellate sturgeon juveniles raised at different densities].

Genetic and size/weight characteristics of stellate sturgeon juveniles were examined under standard and twice lower than standard densities. Viability, growth rate, and variability of juveniles were higher at standard density. Individuals with different genotypes exhibited higher viability and faster growth at different densities, which was particularly pronounced in case of the PGM1 locus. Despite the high phenotypic variability of sturgeon juveniles under standard conditions, its genetic component was higher at low density. It was concluded that low density conditions, which are closer to the natural ones, mediate maintenance of genetic variability.

[Microsatellite variability and differentiation of hatchery stocks of chum salmon Oncorhynchus keta Walbaum in Sakhalin].

Variability at eight microsatellite loci was examined in five populations of chum salmon Oncorhynchus keta Walbaum from Sakhalin hatcheries. The population of Kalinino hatchery had the lowest heterozygosity and the lowest average number of alleles per locus. The populations examined exhibited significant differentiation, theta ST = 0.026 on average per locus. The maximum genetic differences were found between the populations of the Kalinino and the Ado-Tymovo hatcheries; the latter differs from the remaining populations also by the highest number and high frequencies of specific alleles. The genetic features of the Taranai hatchery population, observed at microsatellite loci, reflect its "mixed" origin.

[Genetic divergence of chars of the genus Salvelinus from Kronotsky Lake (Kamchatka Peninsula)].

Chars from the genus Salvelinus, inhabiting lakes and lake-river systems, belong to morphologically and ecologically different forms whose taxonomic status is under dispute. In the present work, we have examined genetic variation and divergence in various chars from the Kronotsky lake basin: the lacustrine chars (white, nose, and long-head) and Dolly Varden char Salvelinus malma. The study was conducted using analysis of allozyme and microsatellite loci, myogens, RAPD, and restriction analysis of two mtDNA segments. The estimates of heterozygoisty at allozyme and microsatellite loci were similar to the corresponding parameters in populations of northern Dolly Varden and Arctic char. Heterozygote deficit was recorded in both samples of individual forms, and in the combined sample of all chars from Kronotsky Lake. For both markers, appreciable genetic differentiation among the samples of different char forms was found, which was comparable to that among the spatially isolated populations of northern Dolly Varden. This result indicates reproductive isolation among the char forms examined. However, this isolation is not complete, because no fixed differences between the forms by any of the genetic systems analyzed was found. The genetic differentiation among different forms of lacustrine chars, which corresponds to the interpopulation rather than interspecies level, is thought to be explained by their comparatively recent divergence.

[Far Eastern mullet Mugil soiuy Basilewsky (Mugilidae, Mugiliformes): the genetic structure of populations and its change under acclimatization]. / Dal'nevostochnyi pilengas Mugil soiuy Basilewsky (Mugilidae, Mugiliformes): geneticheskaia struktura populiatsii i ee izmeneniia pri akklimatizatsii.

The introduction of Far Eastern mullet (pilengas) in the Azov Sea in the 1970s-1980s has resulted in the formation of a self-reproducing commercial population. We have carried out a comparative population-genetic analysis of the mullet from the native (Primorye, the Sea of Japan basin) and the new (The Azov Sea basin) ranges. Genetic characteristics of three Primorye and three Azov local samples were studied using electrophoretic analysis of 15 enzymes encoded by 21 gene loci. In the Azov mullet, the initial heterozygosity characteristic of the donor population was preserved while the genotype and the allele compositions changed; the changes included a 1.9-fold reduction in the percentage of polymorphic loci and 1.5-fold reduction in the mean number of alleles per locus. The genetic differences between the Azov and the Primorye sample groups were highly significant. In the native range, no genetic differentiation among the mullet samples from different areas was found (Gst = 0.42%), whereas in the Azov Sea basin, the samples from spatially isolated populations (ecological groups) exhibited genetic differences (Gst = 1.38). The genetic divergence of the subpopulations and the excess of heterozygotes at some loci in the Azov mullet suggest selection processes that formed genetically divergent groups associated with the areas of different salinity in the new range. The salinity level is assumed to be the most probable factor of local differentiating selection during fast adaptation and naturalization of the introduced mullet.

Genetic markers in the Tuvan population of Todja, Siberia.

Todja is a secluded region of northern Tuva-situated in the Sayany Mountains, Siberia. The aboriginal population of Todja is Tuvan. A total of 128 healthy Tuvans living in Todja were typed for HLA-A, -B and -C antigens and several plasma and erythrocyte protein polymorphisms (Hp, Tf, Gc, ESD, ACP, PGM1, PGD and ADA). The observed frequencies of all 8 blood protein and HLA genotypes were in agreement with Hardy-Weinberg expectations. The most frequent HLA antigens in Todjans are A2 (0.36), A3 (0.24), A9 (0.50), B15 (0.34) and B40 (0.50). HLA haplotypes A2B5, A2B40, A9B15 and A9B40 are most common in this population. The observed frequencies of protein polymorphisms and HLA antigens and haplotypes in Todjans are similar to those of other Mongoloid populations. A comparison of HLA frequencies currently observed in Todjans with those obtained 20 years ago at the same locality showed minor changes attributable to the effect of migration.