[Variability of Allozyme and cpSSR Markers in the Populations of Siberian Spruce].

The variability of 21 allozyme and three microsatellite loci of chloroplast DNA (cpDNA) was studied in the populations of Siberian spruce (Picea obovata Ledeb.) from Irkutsk oblast, Magadan oblast, Buryatia, and Mongolia. It was demonstrated that the highest level of genetic diversity among the examined populations at both allozyme and microsatellite loci was observed in the Tulyushka population from Irkutsk oblast. The lowest level of genetic diversity was observed in marginal isolated populations of Bogd Uul and Magadan. In the relict spruce population from Olkhon Island, differing from the other populations in the lowest allelic diversity of both types of markers, no expected decline of expected heterozygosity and haplotype diversity was observed. In this population, the variability parameters mentioned were close to the population mean. The obtained intrapopulation and intraspecific variability parameters of allozyme and microsatellite loci of chloroplast DNA and the data on the population differentiation at these loci indicate that the given markers can be used for the analysis of the population structure of Siberian spruce.

[Phylogeny of firs (genus Abies, Pinaceae) based on multilocus nuclear markers (AFLP)].

To study the phylogenetic relationships, evolutionary history, and molecular systematics of firs (genus Abies), the phylogenetic reconstruction, based on nuclear multilocus markers­amplified fragment length polymorphism (AFLP)­was conducted. Using seven combinations of selective primers, 84 samples of 39 taxa were genotyped for 553 polymorphic AFLP loci. A comparison with our earlier chloroplast and mitochondrial phylogenies of the genus (in 2014) shows that the nuclear phylogeny generally is more congruent to the chloroplast tree. Most of the clades resolved by the chloroplast phylogeny were supported also in the AFLP tree. Employing the nuclear DNA-based tree, we revealed the presence of new groups and the differences in the topology of several clades. AFLP confirmed the monophyly of Asian species of section Balsamea and their sister position in relation to the American group of species of this section. As shown by the tree of chloroplast DNA, Asian species of section Balsamea do not form a monophyletic group, but belong to the clade comprising the majority of Asian species. Phylogenetically mitochondrial DNA data to a large extent are not congruent to the nuclear and chloroplast DNA trees, and are more in line with geographical distribution of species. Conflicts between nuclear and cytoplasmic phylogeny were analyzed. Taking them into account, we consider the hypothesis of a hybrid origin of particular groups of firs, including ancient hybridization in section Balsamea. A comparison of molecular data with traditional taxonomy of the genus is discussed.

First data on genetic diversity of burbot (Lota lota L.) in the Western Siberian.

Analysis of genetic diversity of burbot (Lota lota Linneus, 1758) mitochondrial control region (mtCR) haplotypes from geographically distant localities in the Ob-Irtysh River basin in comparison with distribution of known burbot haplotypes was conducted. mtCR fragments from burbot samples, obtained in two localities (longitudinal part of the Irtysh near Tobolsk and the Sob River, a left-bank tributary of the Ob River), were sequenced.

[EXPERIENCE OF USING IMMUNE CHROMATOGRAPHY TEST FOR DIAGNOSTICS OF PNEUMOCOCCAL PNEUMONIA].

AIM: Justification of the use of immune chromatographic analysis for diagnostic of pneumococcal pneumonia. MATERIALS AND METHODS: Sensitivity and specificity of an immune chromatographic method (Binax Now test system) was studied for verification of pneumococcal pneumonia. Approbation of this method for etiologic deciphering of pneumonia in 260 patients hospitalized in infectious hospital was carried out. RESULTS: A high sensitivity (84.8%) and specificity (90.5%) of the chromatography test was established. Streptococcus pneumoniae antigen in urine of patients with community acquired pneumonia was determined 3.6 times more frequently when immune chromatographic test was used compared with bacteriological study of nasopharyngeal swab and 1.8 times more frequently compared with sputum bacteriological study. CONCLUSION: Application of immune chromatographic express test Binax Now could be recommended for timely verification of pneumococcal pneumonia along with bacteriological methods of study.

[Development of New Mitochondrial DNA Markers in Scots Pine (Pinus sylvestris L.) for Population Genetic and Phylogeographic Studies].

Fragments of genomic DNA of Scots pine (Pinus sylvestris L.) homologous to the mitochondrial DNA (mtDNA) contigs of Norway spruce (Picea abies (L.) Karst.) and loblolly pine (Pinus taeda L.) were resequenced in a sample of the Scots pine trees of European, Siberian, Mongolian and Caucasian origin in order to develop mtDNA markers. Flanking non-coding regions of some mitochondrial genes were also investigated and resequenced. Five single nucleotide polymorphisms (SNPs) and a single minisatellite locus were identified. Caucasian samples differed from the rest by three SNPs. Two SNPs have been linked to an early described marker in.the first intron of the nad7 gene, and all together revealed three haplotypes in European populations. No variable SNPs were found in the Siberian and Mongolian populations. The minisatellite locus contained 41 alleles across European, Siberian and Mongolian populations, but, this locus demonstrated a weak population differentiation (F(ST) = 0.058), probably due to its high mutation rate.

[Molecular phylogenetic analysis of the genus Abies (Pinaceae) based on the nucleotide sequence of chloroplast DNA].

A phylogenetic study of firs (Abies Mill.) was conducted using nucleotide sequences of several chloroplast DNA regions with a total length of 5580 bp. The analysis included 37 taxa, which represented the main evolutionary lineages of the genus, and Keteleeria daviana. According to phylogenetic reconstruction the Abies species were subdivided into six main groups, generally corresponding to their geographic distribution. The phylogenetic tree had three basal clades. All of these clades contained American species, and only one of them contained Eurasian species. The divergence time calibrations, based on paleobotanical data and the chloroplast DNA mutation rate estimates in Pinaceae, produced similar results..The age of diversification among the clades of the present-day Abies was estimated as the end of the Oligocene-beginning of Miocene. The age of the separation of Mediterranean firs from the Asian-North American branch corresponds to the Miocene. The age of diversification within the young groups of Mediterranean, Asian, and boreal American firs (A. lasiocarpa, A. balsamea, A. fraseri) was estimated as the Pliocene-Pleistocene. Based on the phylogenetic reconstruction obtained, the most plausible biogeographic scenarios were suggested. It is noted that the existing systematic classification of the genus Abies strongly contradicts with phylogenetic reconstruction and requires revision.

[Mitochondrial DNA variation and reticulate evolution of the genus Abies].

The sequences of three regions of mitochondrial DNA (mtDNA) of a total length of 5226 bp were used to study the phylogeography of the genus Abies. The mtDNA haplotype network, comprising 36 studied Abies taxa, consisted of two branches: the first represented all American species plus two Asian, and the second included the remaining Eurasian species. Within these clusters, the haplotypes formed nine major groups, generally corresponding to the clades of the previously obtained phylogeny based on chloroplast DNA (cpDNA), but the relationships of these groups were significantly different: species assignment to the particular mtDNA haplotype group was more in line with its geographical distribution. In addition, the mtDNA haplotype network contains cycles indicating the recombination. It is assumed that the incongruence of cpDNA and mtDNA phylogenies is caused by the introgression capture of alien mtDNA during species hybridization and thus contains information about past migrations. The cases of incongruence of mitochondrial and chloroplast DNA suggesting a migration of Abies between Asia and North America are discussed.

[Microsatellite Loci polymorphism of chloroplast DNA of the pine tree (Pinus sylvestris L.) in Asia and Eastern Europe].

The variability of four microsatellite loci of chloroplast DNA was studied in 38 populations of Pinus sylvestris in the European part of Russia, Siberia, Kazakhstan, Transbaikalia and Mongolia. High variability was observed in all regions. In total, 152 haplotypes were identified. The level of population differentiation R(ST) was 2.1%. The differentiation of three geographical groups of populations (European, Siberian-Kazakhstan and Transbaikalian-Mongolian) was insignificant (R(CT) = 0.004%). At the same time, some rare haplotypes were found to be specific for a certain geographical region. Distribution of the rare haplotypes, which differentiated European populations from Asian populations and Mongolian and Transbaikalian populations from the Siberian, showed the independence of the history of these regions. This corresponds more to the hypothesis that the modern areal of Pinus sylvestris originated via settlement from many origins than to the hypothesized single center of the post-boulder-period of recolonization. The distribution of the pair differences between the individual specimens corresponded to the model of sudden population growth. The assessments of the age of this event for Pinus sylvestris (4.5-4.7 million years), which were obtained on the basis of this model, significantly exceeded the age of the quaternary period. Therefore, the revealed growth of populations is hardly due to the changes in flora related with the boulder-period, but rather mirrors the moment of the species formation.

[Genetic diversity of larch at the north of Primorskii Krai and distribution limits of Larix olgensis A. Henry].

The genetic diversity of four mtDNA fragments and five microsatellite loci of ctDNA was examined in six larch samples from the territory of the Sikhote-Alin Nature Reserve (northern Primor'e). In order to identify possible species-specific differences between the samples, the latter were collected at transects along the shore of the Sea of Japan and at different distances from the sea. Based on a number of morphological characters, some authors suggest that Olgan larch grows in the shoreline part of the reserve and, moving inland, it is replaced by Gmelin larch. According to the other data, the northern border of the Olgan larch range does not reach the reserve territory. The data obtained were compared to those obtained previously for three samples from the south of Primor'e, including those for the locus classicus of Olgan larch. In the examined larch accessions (186, for mtDNA and 200, for ctDNA), a total of five mitotypes and 52 chlorotypes were recovered. According to the results of the AMOVA test, the proportion of variations accounted for the differences among all Larix samples examined over both types of markers was higher (N(ST) = 0.435, for mtDNA and R(ST) = 0.041, for ctDNA) than that for the differences among the samples within the reserve (N(ST) = 0.079, for mtDNA and R(ST) = 0.005, for ctDNA). No differences were detected between the groups of shoreline and continental populations.

[Nucleotide diversity and linkage disequilibrium of adaptive significant genes in Larix (Pinaceae)].

Nucleotide polymorphism in genes potentially responsible for the adaptation to the latitudinal gradient of climate was investigated in the Siberian larch (Larix sibirica). The genes were selected among those previously studied in Douglas fir (Pseudotsuga menziesii), the variability of which indicates the influence of selection or shows the association with phenotypic traits that are important for adaptation to low temperatures and to geographically heterogeneous environment. Nucleotide sequences of eight genes, including coding and noncoding regions, were amplified and sequenced using newly developed primers specific to Larix. The sample size was 123 megagametophytes per locus in L. sibirica and 16.2 in L. occidentalis, which was taken as the out-group. The length of the sequenced fragments was 246-1700 bp. Nucleotide polymorphism π averaged 0.00536 (0.002 - 0.008), and haplotype diversity was H(d): 0.822 (0.625-0.948). Tajima's D was negative in all fragments and significant in three, while statistics D* and F* was significant in three and four segments, respectively, and F(s) was significant in three. This may indicate the presence of purifying selection on these genes or population growth. The HKA test revealed no significant deviations from the neutral model of evolution in all genes. The recombination parameter ρ/θ = 0.28 was close to the value obtained from P. menziesii. To investigate the association of polymorphic sites (factor) in these eight genes to the latitude of investigated individuals (trait), the generalized linear model (GLM) was used taking into account the population structure. After the False Discovery Rate correction no significant associations were found. The age of the split of American and Eurasian Larix lineages based on the nucleotide differences in the eight genes between L. sibirica and L. occidentalis is estimated to be 12 million years, which is much younger than the age of the most ancient Larix discoveries.

[Systematics and genegeography of Juniperus communis inferred from isoenzyme data].

Using isoenzyme analysis, 35 populations of Juniperus communis L. from various parts of the Russian species range and by one population from Sweden and Alaska were studied. The total sample size was 1200 plants. As a result, the existence ofJ. communis var. oblonga in North Caucasus and J. communis var. depressa in North America was confirmed, but genetic differences between J. communis var. communis and J. communis var. saxatilis were not detected in the main part of the Russian species range (European part of Russia, Ural, Siberia). These populations proved to be genetically uniform with the same predominant allelic frequencies, which may evidence recent settling of this species from one of Central or East European refugium. J. communis var. saxatilis from northeastern Russia inhabiting the region behind Verkhoyansk mountain and Russian Far East showed considerable differentiation in frequencies of alleles at three loci and geographical subdivision. These populations also exhibit high intrapopulation variation. This can be connected with the refugium in this territory. The origin of this group is probably connected with migrations from Central Asia (Tibet) in the direction to northeastern Russia along mountains connecting Central and North Asia. It is also assumed that migrations of this species previously proceeded across the Beringian land bridge.

[Spread of mitochondrial DNA haplotypes in population of Scots pine (Pinus sylvestris L.) in northern European Russia].

The variability of the first intron of the nad7 gene of Scots pine mitochondrial DNA was investigated in 15 populations in northeast of European Russia and in three populations in Belarus, Sweden, and the Voronezh region. Restriction Fragments Length Polymorphism of the PCR product (PCR-RFLP) and sequencing were used. The investigated samples were compared with the populations studied previously. The haplotype, which is absolutely dominant in the eastern part of the Scots pine range, was fixed in the Kirov, Arkhangelsk, and Kostroma regions; Komi; and Chuvashia. The extreme northeastern discovery of an alternative haplotype that is present in most European populations and occurs the most frequently in eastern Scandinavia was made in the Vologda region. These results support the hypothesis that the population of Scots pine in northeast Russia and Fennoscandia originated from different glacial refugia.

[Genetic variability of Siberian fir (Abies sibirica Ledeb.) inferred from AFLP markers].

Genetic variability of AFLP markers was studied in 20 populations of Siberian fir (Abies sibirica (Pinaceae) and in two populations of Far-Eastern Manchurian fir A. nephrolepis and Sakhalin fir A. sachalinensis each. Four pairs of selective primers were used. In total, 168 samples from three fir species were genotyped for 117 polymorphic loci. According to the AMOVA results, the variability proportion characterizing the differences between three Abies species was several times higher (F(CT) = 0.53) than that accounting for among-population differences within the species (F(SC) = 0.125). Differentiation of the A. sibirica populations based on AFLP markers exceeded 14% (F(ST) = 0.141). Significant correlation between the genetic distances calculated from the AFLP data and the geographic distances between populations was found. The results of AFLP variability analysis supported and supplemented the conclusions inferred previously from allozyme and cpSSR data: several genetically similar geographic groups of Siberian fir were identified. These groups differ both in allele frequencies and in the levels of genetic variation.

[Genetic variation of some varieties of common juniper Juniperus communis L. inferred from analysis of allozyme loci].

Using the method of allozyme analysis, genetic variation, diversity, and population structure of Juniperus communis L. var. communis and J. communis L. var. saxatilis Pall. (= J. sibirica Burgsd. = J. nana Wild), growing on the territory of Russia, J. c. var. communis from Sweden, and J. c. var. depressa Pursh from Northern America (Alaska), was investigated. The total level of genetic variation of these varieties was found to be higher than the values obtained for the other conifers. The population samples of J. c. var. depressa from Alaska and J. c. var. saxatilis from Sakhalin were noticeably different from all other populations examined. Between the other samples, no substantial genetic differences were observed. These populations were characterized by weak interpopulation differentiation along with the absence of expressed geographical pattern of the allele frequency spatial distribution. The only exception was the procumbent form of common juniper from the high mountain populations of South and North Ural, which was somewhat different from the others.

[Rubella in Russia: variability of the infectious agent during the period of vaccine prophylaxis].

AIM: To assess influence of vaccination against rubella on the genetic diversity of rubella virus. MATERIALS AND METHODS: Vaccine strains of rubella virus Wistar 27/3 and Orlov-B as well as sera from patients with rubella obtained in Perm region during 1999 - 2005 period and standard serologic, molecular, epidemiologic and statistical methods were used. The study was performed according to the WHO recommendations on the genotyping of wild rubellavirus strains. RESULTS: Strains of rubella virus isolated in Perm region, vaccine strain Orlov-B (Saint Petersburg), and 4 Russian strains isolated in 1967-1997 before vaccine introduction belong to the same genetic group with high degree of homology - genetic divergence do not exceed 0 - 1%. This group was identified as genotype 2c which, according to WHO's data, circulates only in Russia. Periods of epidemic peaks of rubella incidence and its falls as well as selective immunization of girls and women of childbearing age did not influence on the genetic stability of the virus (divergence did not exceed 0.6 - 2.0%). On the contrary, mass immunization of children aged 1 - 2 years during 4 years resulted in statistically significant changes of rubella virus subtype inside the genotype 2c. CONCLUSION: Ten-year experience of rubella vaccination in Perm region demonstrates necessity of inclusion of monitoring for rubella virus variability in the system of epidemiological surveillance and control for rubella infection during period of its prevention by vaccine.

[Mitochondrial DNA variation pattern in larches of Eastern Siberia and the Far East].

Genetic variation has been studied in 32 Eastern Siberian and Far Eastern populations of Larix Mill. with the use of three mitochondrial markers based on polymerase chain reaction. Eight multilocus haplotypes with a heterogeneous spatial distribution (G(ST) = 0.788, N(ST) = 0.829) have been found, which indicates limited gene flows between populations. Several geographic regions with specific larch haplotype sets have been determined: (1) Japan, (2) southern Sakhalin and the Kuril Islands, (3) Primor'e and Korea, (4) Kamchatka, and (5) Eastern Siberia and the northern Far East. The haplotype fixed in the Kamchatka is absent in the Magadan oblast or Chukotka but is present in southern Primor'e and Sakhalin Island. This may be explained by either the postglacial recolonization of Kamchatka by larch that spread from Primor'e through Sakhalin and the Kuril Islands or its survival through the last glacial maximum in the Kamchatka Peninsula. The biogeography of larch and other woody plants indicate that boreal species have a common history of the colonization of Kamchatka.

[The diversity of chloroplast microsatellite loci in Siberian fir (Abies sibirica Ledeb.) and two Far East fir species A. nephrolepis (Trautv.) Maxim. and A. sachalinensis Fr. Schmidt].

The genetic variability in 29 populations of Abies sibirica, three of A. nephrolepis, and seven of A. sachalinensis was studied using SSR markers of chloroplast DNA. Stable amplification and polymorphic products were obtained using primer pairs Pt71936 and Pt30204 (with nine and forteen alleles, respectively) of 10 pairs. Totally, 70 haplotypes were found, 43 in A. sibirica, 49 in A. sachalinensis, and 31 in A. nephrolepis. The highest values of genetic diversity parameters were observed in A. sachalinensis, and the lowest in A. nephrolepis. The Siberian fir differs from Far East species by the uneven multimodal frequency distributions of allele length in both cpSSR loci, which is explained by the presence of few separated from each other dominating haplotypes. This fact indicates that A. sibirica and the Far East species have different demographic histories. In A. sibirica, the proportion of diversity between populations in the total genetic diversity, calculated taking into account the differences between haplotypes (R(ST)) was 8.34% and 4.42% without accounting for haplotypes differences (R(ST) > G(ST), P= 0.01). The pairwise G(ST) correlate significantly with geographic distances between the populations A. sibirica and with genetic distances D calculated from allozyme data. No such correlations were found with the R(ST) parameter. The results of cpSSR variability analysis strongly support the conclusions inferred from allozyme data: several geographic groups of comparatively genetically close populations are identified, which may be explained by the invasion of colonization of the present-day Siberian fir range.

[Genetic variation and population differentiation in Siberian fir Abies sibirica lebed. Inferred from allozyme markers].

Genetic variation in 24 populations of Siberian fir Abies sibirica Lebed. from the Urals, West Siberia, East Siberia, South Siberia, and the Baikal region were examined using allozyme markers. Three out of fifteen allozyme loci proved to be polymorphic. Heterozygosity He was 6.6-9.6%, which is substantially lower than that in other widely spread boreal conifers. Our results suggest that the Siberian fir populations are subdivided into four geographic groups: (1) the Baikal Lake group, (2) the Sayan and the Altai group, (3) the Middle and Southern Urals group, and (4) Subpolar and Northern Urals group. This pattern of geographic differentiation may be explained by the preservation of the Siberian fir during the last glacial maximum (18 000-22000 years B.P.) in isolated refugia with subsequent recolonization of the present area. FST in the populations examined was 10.16%, which is comparable to the estimate for Larix sibirica (7.9%), a conifer species having a similar range and pattern of geographic population differentiation.

Absence of geographical structure of chloroplast DNA variation in sallow, Salix caprea L.

In the present study, we have used PCR-RFLP markers to investigate the chloroplast DNA variation in 24 European populations of Salix caprea L. A subset of these populations has also been analysed with chloroplast microsatellites. The main feature of both markers is the absence of a clear geographic structure (G(ST(PCR-RFLP))=0.090, G(ST(microsatellites))=-0.017) and high levels of variation within populations. This lack of phylogeographic structure in S. caprea is suggested to be the consequence of the joint action of several factors: (i) presence of intermediate latitude refugia with large population sizes during the last glacial maximum, (ii) a high speed of recolonisation and dispersal ability, (iii) a high mutation rate and (iv) extensive hybridisation with other willow species. In addition to the S. caprea samples, a limited number of individuals from several other Salix species were also analysed with PCR-RFLP: S. cinerea, S. aurita, S. purpurea, S. atrocinerea, S. appendiculata, S. elaeagnos, S. fragilis and S. alba. Many of the haplotypes found in Salix caprea were also detected in S. cinerea, S. aurita, S. purpurea, S. atrocinerea and/ or S. appendiculata but not in S. alba, S. elaeagnos or S. fragilis. Our data suggest that hybridisation and gene flow have occurred within these two groups but not between them.

Genetic mapping of sex-linked markers in Salix viminalis L.

A total of 88 selective primer combinations were screened using bulked males and females sampled from four families of Salix viminalis. A total of more than 1000 polymorphic fragments was obtained, of which only four cosegregated with sex. These four sex-linked markers were subsequently scored in individuals that were used for bulked sample preparation in additional individuals of the same families, and in individuals in other families. A pair of primers that amplified the sex-linked fragments was constructed from one of the sex-linked amplified fragment length polymorphism (AFLP) fragments. In hybridization of Southern blot filters with the sex-linked DNA fragments, the band was present in females and absent in males, but the opposite pattern of band segregation (a band found in males and no band in females) was never observed in either the AFLP or RFLP experiments. Two of the sex-linked markers were placed on a linkage map. They both map at the same location in a linkage group comprising other markers not segregating with sex. Our data suggest that a single locus governs the sex determination and that nonrecombining sex chromosomes are absent in S. viminalis. A close association was found between skewed sex ratio and segregation distortion at this locus.