[Long Sperm Storage in the Siberian Crane (Grus leucogeranus Pallas): Analysis of Paternity and Relatedness under Artificial Insemination].

Using ten microsatellite loci, paternity analysis has been conducted for 71 individuals of the Siberian crane (Grus leucogeranus Pallas) obtained under artificial insemination in Oka Crane Breeding Center in 2001­2014. The fathers of 39 chicks were the sires whose sperm was used for insemination directly before fertilized egg laying. Paternity of 23 fertilizations belonged to the sires whose sperm was used in the beginning or middle of insemination cycle. Nine cases of fertilization resulted from natural copulation of artificially inseminated females with their social partners. The terms of sperm storage in the female's reproductive ducts before fertilization were 0­6 days in the case of paternity of the last sperm donor and 2­15 days in the case of competing sperm by previous donors. Genetic relatedness by microsatellite loci between breeders of the captive Siberian crane population does not prevent fertilization and does not always lead to inbreeding depression.

[Microsatellite analysis of clonality and individual heterozygosity in natural populations of aspen Populus tremula L.: Identification of highly heterozygous clone].

Aspen Populus tremula L. (Salicaceae) is the fast-growing tree species of environmental and economic value. Aspen is capable of reproduction by both seeds and vegetative means, forming root sprouts. In an adult stand, identification of ramets of one clone among the trees of seed origin based on their morphology is difficult. A panel of 14 microsatellite loci developed for individual identification of aspen was applied for the clonal structure analysis in four natural aspen stands of the European part of Russia: Moscow and Voronezh oblasts, the Mari-El Republic, and the Republic of Tatarstan. In 52 trees from the Moscow sample, 41 multilocus genotypes were identified; in the Voronezh sample, among 30 individuals, 25 different genotypes were detected; and in the sample from Mari-El, 32 trees were represented by 13 genotypes. In the stand from Sabinsky Forestry, Tatarstan, all of the examined 29 trees were represented by a single genotype. The ancestral tree carrier of this genotype which was the most heterozygous (0.929) among all studied aspen individuals (sample mean, 0.598) obviously has spread over a large territory during several cutting and reproduction cycles, currently occupying the area of 2.2 ha. For aspen, usually suffering from Aspen trunk rot, such high viability is evidence of resistance to the main pathogens. The revealed superclone deserves further study with karyological methods and flow cytometry to determine ploidy level and analysis of the growth rate and the quality of wood for possible use in plantation forest production.

[Microsatellite data verify low genetic differentiation between western and eastern subspecies of the common crane Grusgrus L. (Gruidae, Aves)].

Using a set of 10 microsatellite loci (Gram-22, Gram-30, Gpa-12, Gpa-38, Gpa-39, Gj-M15, Gj-M34, Gj-4066, Gj-8077, Gj-2298) a high level of genetic variability (N(A) = 10.2, H(O) = = 0.684, H(E) = 0.728) and low genetic differentiation (F(ST) = 0.011) in the Common crane (Grus grus L.) was detected throughout its range. Genetic diversity in disputed western (G. g. grus) and eastern (G. g. lilfordi) sub- species was shown to be similar. Spatial distribution of multi-locus individual genotypes has not been revealed (R(XY) = 0.017). Despite low differentiation, subspecies and local populations of the Common crane should be considered as separate conservational units. Organization of programs for gene pool conservation and monitor- ing requires more detailed analysis based on combined analysis of various molecular markers.

[Offspring Sex Ratio in the Captive Population of Siberian Crane (Grus leucogeranus Pallas)].

The sex ratio of Siberian crane chicks (Grus leucogeranus Pallas) from the captive population ofthe Oka Crane Breeding Center was analyzed with the use of molecular sex marker EE0.6 in 2009-2014. We determined the sex of 84 birds bred from 12 dams by means of artificial insemination and natural breeding. The total primary sex ratio was 40:44, and the secondary sex ratio was 36:39 with a minor predominance of females. The mortality rate of embryos was the same for both sexes. The primary and secondary sex ratio among the first eggs in clutches showed the same trends as those observed in general analysis. The relatedness of parents by microsatellite DNA has no effect on offspring sex in both natural breeding and artificial insemination.

[Spatial Distribution of Intron 2 of nad1 Gene Haplotypes in Populations of Norway and Siberian Spruce (Picea abies-P. obovata) Species Complex].

The length and sequence variations among intron 2 haplotypes of the mitochondrial DNA nad1 gene have been studied in the Norway and Siberian spruce (Picea abies (L.) H. Karst.-P. obovata Ledeb.) species complex. Twenty-two native populations and 15 provenances were analyzed. The distribution of the northern European haplogroup (haplotypes 721, 755, 789, 823, 857, 891, and 925) is delimited in the west by the Ural region inclusively. Haplotype 712 is widespread in populations of Siberia, in the Far East and in northeastern Russia. A novel variant of the Siberian haplogroup (780) containing three copies of the first minisatellite motif (34 bp) was found for the first time. The absence of an admixture of the northern European and Siberian haplotypes in the zone of spruce species introgression previously marked by morphological traits and nuclear allozyme loci was demonstrated. This may be evidence of the existence of a sharper geographic boundary between the two haplogroups, as compared to a boundary based on phenotypic and allozyme data. A high proportion of the interpopulation component of variation (65%) estimated by AMOVA indicates a substantial genetic subdivision of European and Siberian populations of the Palearctic spruce complex by mtDNA, which can be putatively explained by natural barriers to gene flow with seeds related, for instance, to the woodless regions of the western Siberian Plain in the Pleistocene and the probable floodplains of large rivers.

[Distribution of the genetic diversity of the Siberian stone pine, Pinus sibirica Du Tour, along the latitudinal and longitudinal profiles].

The Siberian stone pine (Pinus sibirica Du Tour) is one of the main forest-forming coniferous species of the boreal ecosystems of Western Siberia. We used the isozyme method to analyze 11 ecotypes representing the latitudinal and longitudinal profiles within the species range, including samples from the geographic boundaries of the distribution. The genetic structure of the ecotypes is described on the basis of the variability for 26 isozyme loci encoding for 16 enzyme systems. The greatest genetic diversity was observed in the taiga ecotypes in the central part of the studied area, while the ecotypes along the species range boundaries were shown to be genetically depauperized. Approximately 8.1% of the observed genetic diversity is attributed to differences between the studied ecotypes. We detected high levels of genetic diversity for the Fest_2, Pgm_1, Sod_4, and a few otherloci, as well as a correlation between allele frequencies and geographical locations of the populations. The results of multivariate analysis of allelic frequencies as well as cluster analysis allowed us to discriminate three major groups of ecotypes: north-eastern, central and south-western. In view of our results, we compare two hypotheses: one which attributes the spatial distribution of genetic variations to the selectivity for some of the polymorphic allozyme loci, and the other based in the history of the formation of the range of the Siberian stone pine.

[Genetic variability of sable Martes zibellina L., pine marten M. martes L., and their hybrids in Western Siberia: protein and DNA polymorphism].

Using four types of markers, the genetic variability of sable and pine marten inhabiting Western Siberia was examined. Izoenzyme and restriction endonuclease analysis of the mtDNA cytochrome b gene fragment, as well as the ISSR-PCR and analysis of microsatellite variation, revealed a low differentiation level of sable and pine marten and confirmed the hybrid origin of atypical representatives of these species. The hybrids were characterized by an increased heterozygosity level and were genetically closer to sable than to pine marten. In atypical martens, the presence of mtDNA haplotypes of eastern sable was identified. This could be the consequence of the reintroduction of the Barguzin sable in the 20th century. In Western Siberia, the introgression of genes between sable and pine marten was massive and symmetrical. It apparently occurred in the past and continues in the present.

[Genetic passportization and identification of Siberian cranes (Grus leucogeranus Pallas) in captivity].

The genetic diversity of the founders of an artificial population of the Siberian crane Grus leucogeranus Pallas (rare species of cranes) was characterized using 10 microsatellite loci. It was established that the allelic diversity (on average, 5.9 alleles per locus) and genic (H(o) = 0.739) diversity of the Siberian crane is rather high and comparable with the estimations for natural populations of different crane species. Genetic passportization of the birds (119 individuals) from the register of the Siberian crane International Studbook was carried out at the initial stage. The efficiency of genetic passportization for individual identification, identification of the origin, paternity analysis, and exclusion of inbreeding was demonstrated in Siberian cranes under natural mating and artificial insemination. Cases of natural reproduction in pairs of Siberian cranes imprinted to the human and continuous storage of spermatozoa in the female reproductive ducts were registered.

[Genetic diversity and relatedness in different generations of the Siberian crane (Grus leucogeranus Pallas) captive population].

Eight variable microsatellite loci were analyzed in terms of studying the genetic structure of different generations of a captive population of a rare endemic Russian species, the Siberian crane (Grus leucogeranus Pallas). It was shown that the founding population of natural origin (15 birds) is characterized by high genetic diversity (N(A) = 6.625, H(O) = 0.767, H(E) = 0.731) and a lack of relatedness (R = -0.079). In the total sample of descendents of the founders (122 individuals from generations F1, F1/F2, F,/F3, F2, F2/F3), this characteristic level of genetic variation was retained; however, we observed a decrease in allelic richness in some generations (F1/F2, F1/F3, F2). We found a low level of relatedness inthe sample of descendents of the founders (F1, F1/F2, F1/F3), while the relatedness was maximal (R = 0.302) in the first-generation descendents of the breeders. A small sample of breeders related to each other of generations F1 and F2 (eight birds) does not represent the entire gene pool of the founders of the Siberian Crane captive population. In view of this, we discuss the need to form a new genetically heterogeneous generation of breeders that would also include Siberian Cranes from the virtually extinct Western Siberian population.

[Sex determination in ten crane species by DNA marker EE0.6].

Using a unique DNA sequence of W-chromosome EE0.6, we carried out molecular sex determination in 383 individuals often species of cranes (Grusgrus L., G. leucogeranus Pallas, G. japonensis Muller, G. vipio Pallas, G. Canadensis L., G. antigone L., G. monacha Temminck, Anthropoides virgo L., Balearica regulorum Bennett, and B. pavonia L.) kept in zoos and other centers of captive propagation. In 211 birds, sex was determined or verified for the first time. The efficiency of using the sex marker EE0.6 for chicks and immature and adult cranes of different species, as well as for interspecific hybrids was shown.

[Non-invasive method of sex identification of crane chicks by the DNA from capillary vessels of alantois].

The non-invasive method of determining the sex of chicks after hatching based on the DNA isolated from capillary vessels of allantois of the egg-shell membranes was demonstrated on four crane species (Gruinae, Aves), which were bred in the Crane Breeding Centre of the Oka Nature Reserve in 2009-2012. Using the EE0.6 molecular marker of sex, the gender of 26 Siberian (Grus leucogeranus), 15 Red-crowned (G. japonensis), 4 Common (G. grus) and 1 Demoiselle (Anthropoides virgo) crane chicks was identified. This method can be recommended for determining the sex of chicks and the ratio of sexes in cranes that reproduce both in captivity and natural populations.

[Testing of microsatellite primers with different populations of Eurasian spruces Picea abies (L.) Karst. and Picea obovata Ledeb].

From a clone library containing microsatellite DNA fragments of Norwegian spruce, seven pairs of primers were selected. These primers were tested to be the markers in the genetic structure analysis of nine populations of Eurasian spruce species Picea abies (L.) Karst. and Picea obovata Ledeb. Five pairs of these primers identified polymorphic loci with the allele numbers from 6 to 15. In the populations examined, the observed and expected heterozygosity values assessed at five loci varied from 0.1778 to 0.6556 and from 0.7800 to 0.900, respectively. In the populations examined, the values of F(st) index varied from 0.0691 to 0.2551 with the mean value of 0.1318. On the dendrogram based on Nei genetic distances, the populations formed three groups: Pskov-Ciscarpathia, Komi-Tatarstan-Arkhangelsk, Kazakhstan-Karelia(natural)-Karelia(culture)-Krasnoyarsk. Five of the primer pairs tested proved useful for analysis of the population genetic structure in Eurasian spruce species.

[Mitochondrial DNA genetic differentiation of the muksun Coregonus muksun (Pallas) and related Siberian species of Coregonus (Coredonidae, Salmoniformes)].

Restriction enzyme analysis of the mitochondrial DNA (mtDNA) fragment encoding subunit 1 of the NADH dehydrogenase complex (ND-1) amplified via polymerase chain reaction (PCR) has been used to obtain data on genetic differentiation of muksun Coregonus muksun (Pallas) populations. Population polymorphism with respect to the restriction sites of 18 endonucleases has been described. It has been demonstrated that the muksun is genetically related to the pidschian C. pidschian (Gmelin), its sympatric species in Siberian waters. Analysis of the median network of mtDNA haplotypes has shown that haplotypes of muksun from various Siberian basins form a common group with haplotypes of pidschian of the Arctic Ocean basin, some frequent haplotypes been found in both forms. This raises the question as to the validity of the muksun as a species. Differences within this group of haplotypes are much smaller than those typical of species of the genus Coregonus. The possibility of a hybrid origin of the muksun from a pidschian-like ancestor and species of the cisco-peled (C. sardinella-C. peled) complex is discussed.

[Dynamics of allozyme heterozygosity in Siberian dwarf pine Pinus pumila (Pall.) Regel populations of the Russian Far East: comparison of embryos and maternal plants].

Siberian dwarf pine, or Japanese stone pine, Pinus pumila (Pall.) Regel is widespread in eastern Siberia and the Russian Far East; the species is bird-dispersed and has a unique spreading or shrub living form. A mixed mating system (predominant outcrossing with self-pollination and matings of close relatives) leads to the formation of partly inbred progenies in P. pumila, as in the majority of other conifers. The question arises as to whether inbred individuals persist in the reproductive part of a population, which can have negative genetic consequences. The ADH, FDH, FEST, GDH, GOT, IDH, LAP, MNR, MDH, PEPCA, 6-PGD, PGI, PGM, SKDH, and SOD isozyme systems were analyzed to study the dynamics of heterozygosity in four P. pumila natural populations from the Pacific region, optimal for the species. Samples were collected in northern Koryakia, southern Kamchatka (two samples), and the Kunashir Island (Kurils). Wright's fixation index was used to estimate the level of inbreeding in embryos from dormant seeds resulting from free pollination and in maternal plants. A substantial level of inbreeding (F(IS) = 0.124-0.342) was observed in the embryo samples but not in three out of the four adult samples. The inbreeding level at the reproductive age was high only in the sample from Koryakia, which was explained by a relatively young age of plants in the population frequently affected by fires. A general increase in heterozygosity, characteristic of other conifers as well, was attributed to elimination of inbred progenies and by balancing selection for heterozygotes, which is a key factor maintaining allozyme polymorphism in populations.

[Allozyme polymorphism of Swiss stone pine Pinus cembra L. in mountainous populations of the Alps and the Eastern Carpathians].

Swiss stone pine Pinus cembra L. is a species with fragmented range, occurring in the Alpine-East Carpathian mountain system. Seeds of P. cembra are dispersed by nutcrackers, which offers potential possibilities for gene exchange among populations. Using isozyme analysis, we have examined five samples from two parts of the Swiss stone pine range: the Alps (Switzerland and Austria) and the Carpathians (two samples from the northern macroslope of the Gorgany Ridge, Eastern Carpathians, Ivano-Frankovsk oblast and one sample from Trans-Carpathian oblast of Ukraine). The allele frequencies of 30 isozyme loci, coding for enzymes ADH, FDH, FEST, GDH, GOT, IDH, LAP, MNR, MDH, PEPCA, 6PGD, PGI, PGM, SDH, SKDH, SOD, were analyzed using cluster analysis and methods of principal components. Two clusters, corresponding to the isolated Alpine and Carpathian parts of the range, were found. The main contribution to these differences were made by loci Adh-1, Adh-2, Fest-2, Lap-3, Mdh-4, and Sod-4. The interpopulation differentiation proved to be somewhat higher than that typical for pines (F(ST) = 7.4%), but within the limits characteristic for taxonomically close species. Thus, isolation of the populations did not lead to their marked differentiation, which may be explained by gene flow and balancing selection, which equalizes gene frequencies across the fragmented species area. Interlocus F(ST) heterogeneity (from 0.003 to 0.173) suggests adaptive significance of some of the allozyme polymorphisms or linkage of some loci with adaptive genes. The Carpathian populations were shown to have higher gene diversity than the Alpine ones (expected heterozygosities 0.095-0.114 and 0.060-0.080, respectively). A deficit of heterozygotes (as compared to the Hardy-Weinberg proportions), observed in the embryo sample, was probably explained by inbreeding. The reduction in the area of Carpathian pine forests in Holocene, caused by the global climatic changes and the anthropogenic impact, is hazardous for the gene pool of the species. The maintenance of genetic uniqueness of both Carpathian populations of P. cembra in general, and individual stands in particular, requires special measures for protection of Swiss stone pine in the Eastern Carpathians.

Allozyme evidence for polyzygotic polyembryony in Siberian stone pine (Pinus sibirica Du Tour).

Approximately 4000 mature seeds from 350 trees in nine populations (12-75 trees per population) of Siberian stone pine were investigated for multiple embryos (polyembryony). Haploid megagametophytes and embryos were genotyped for eight allozyme loci. Eight-yone seeds (2.11%) had more than 1 embryo. Of these, 71 seeds had 2 embryos (1.85%), 6 seeds had 3 embryos (0.16%), 3 seeds had 4 embryos (0.08%) and 1 seed had 6 embryos (0.026%). Allozyme comparison of megagametophytes and embryos could distinquish two types of polyembryony in 56 of the 81 seeds. In 28 seeds (50%) the polyembryony was polyzygotic (independent fertilizations of more than one egg cell in the ovule); 25 seeds (45%) had most likely monozygotic polyembryony (genetically identical embryos resulting from the cleavage of a single proembryo) and 3 seeds had both genetically different and genetically identical embryos. To the best of our knowledge, this is the first genetic evidence for the form of polyembryony in conifer seeds.