Yu. S. Belokon

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 crawling 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 open pollination and in maternal plants. A substantial level of inbreeding (FIS = 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 can be explained by a relatively young age of plants in the population frequently affected by fires. A general increase in heterozygosity in the course of ontogeny, characteristic of other conifers as well, was attributed to elimination of inbred progenies and by balancing selection in favor of heterozygotes, which is a key factor maintaining allozyme polymorphism in populations.

Interspecific genetic variability of the oyster mushroom Pleurotus ostreatus as revealed by allozyme gene analysis

Allozyme variation in natural populations of basidiomycete fungus Pleurotus ostreatus (88 individuals) from three regions of central Russia was studied. The species was shown to have 92.86% of polymorphic allozyme loci and expected heterozygosity He = 0.49. The mean number of alleles per locus was 3.5. The genetic differences among populations were supported by F-statistics (FST = 0.750). The low level of inbreeding (FIS = 0.018) suggests that the P. ostreatus populations are panmictic, and the main reproduction mode involves basidiospores dispersing at long distances. Using cluster analysis, geographically isolated populations and intersterile groups were differentiated within the complex P. ostreatus species.

Microsatellite loci variation and investigation of gene flow between two karyoforms of Cricetulus barabensis sensu lato (Rodentia, Cricetidae)

We examine the diversity of six microsatellite loci and partial RAG1 exon of “barabensis” and “pseudogriseus” karyoforms in Cricetulus barabensis sensu lato species complex. A total of 435 specimens from 68 localities ranging from Altai to the Far East are investigated. The results of the population structure analysis (factor analysis and NJ tree based on Nei genetic distances) support subdivision into two well-differentiated clusters corresponding to the two karyoforms. These karyoforms are also well differentiated by the level of microsatellite variability. In several “barabensis” specimens, we found microsatellite alleles that are common in “pseudogriseus” populations but are otherwise absent in “barabensis.” Most of these specimens originate from a single population in one of the zones of potential contact between karyoforms, Kharkhorin in Central Mongolia. These molecular results are consistent with previously published karyological data in suggesting that rare hybridization events between the two chromosomal races occur in nature.

[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 other loci, 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.

Gene pool state and degree of infestation by bark beetle ( Ips tipographus L.) of Norway spruce ( Picea abies L. Karst.) natural populations and planted stands in Moscow region

A comparative analysis of the gene pool state in natural populations and planted stands of Norway spruce and the degree of their infestation by the bark beetle in the Moscow region was conducted taking into account the dynamic state of communities (4 populations, 148 samples, 24 isoenzyme loci). The degree of infestation by the bark beetle of conditionally native communities is 0%; for planted stands, it is 90–100%; and for a short-term community, it is 15–20%. The comparison of “healthy” populations and those infested with bark beetle by average values of observed heterozygosity (HO) detected no significant differences. However, the test on allelic frequency heterogeneity demonstrated the difference of planted stands from conditionally native populations both by three loci (Fe-2, Idh-1, Mdh-3) and by the totality of 18 polymorphic isoenzyme loci; the short-term population differs from conditionally native population only by two loci. The value of the inbreeding coefficient by the Idh-1 locus is significantly higher in both populations infested with the bark beetle than in “healthy” populations. The results of conducted studies demonstrate the necessity of continuation of the study on the gene pool state in Norway spruce populations owing to the degree of their infestation by the bark beetle along with the study on the dynamic state of the communities; this can provide a key to solving the problem of the forest preservation from pests.

Development of microsatellite genetic markers in Siberian stone pine ( Pinus sibirica Du Tour) based on the de novo whole genome sequencing

Siberian stone pine, Pinus sibirica Du Tour is one of the most economically and environmentally important forest-forming species of conifers in Russia. To study these forests a large number of highly polymorphic molecular genetic markers, such as microsatellite loci, are required. Prior to the new high-throughput next generation sequencing (NGS) methods, discovery of microsatellite loci and development of micro-satellite markers were very time consuming and laborious. The recently developed draft assembly of the Siberian stone pine genome, sequenced using the NGS methods, allowed us to identify a large number of microsatellite loci in the Siberian stone pine genome and to develop species-specific PCR primers for amplification and genotyping of 70 microsatellite loci. The primers were designed using contigs containing short simple sequence tandem repeats from the Siberian stone pine whole genome draft assembly. Based on the testing of primers for 70 microsatellite loci with tri-, tetra- or pentanucleotide repeats, 18 most promising, reliable and polymorphic loci were selected that can be used further as molecular genetic markers in population genetic studies of Siberian stone pine.

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.

Genetic diversity and differentiation of Siberian stone pine populations at the southern range margin in the West Siberia lowland area

Based on the analysis of the variability of 25 allozyme loci, genetic variation within and differentiation between populations of Siberian Stone Pine growing at the trailing-edge range margin in the southern taiga up to the forest-steppe transition zone in Western Siberia have been estimated. At the current stage, climate change and anthropogenic impact have not caused a substantial reduction in genetic diversity within and increase in differentiation between isolated small stands at the southern edge of their distribution in the lowland areas of the Siberian Stone Pine range.

Comparative analysis of Pleurotus ostreatus natural isolates

A comparative analysis was performed of the polymorphism of the oyster mushroom Pleurotus ostreatus (Fr.) Kumm naturally occurring strains isolated from the natural substrates from two geographically remote Russian natural preserves, the Central Forest Biosphere Tver State Preserve and the Moscow State University Zvenigorod Biological Station (Moscow oblast), and within the city of Moscow. The results of the frequency analysis for the allozyme loci alleles and for the sexual and somatic incompatibility groups are presented; the genetic structure and the interpopulation relations among 58 Pleurotus ostreatus dikaryotic strains are estimated. The natural samples from the Moscow and Tver oblasts are shown to have a high degree of polymorphism with a genetic differentiation of 0.743; in spite of their territorial remoteness, they are, however, actively exchanging genetic material. The natural fungal isolates form two reproductively isolated groups.

Differentiation between Commercial Strains of Oyster and Button Mushrooms Using Molecular Markers

Analysis of commercial strains of two edible mushrooms, Pleurotus ostreatus and Agaricus bisporus, using PCR and isozyme electrophoresis techniques allowed us to differentiate groups of genetically similar and distant strains. Among the commercial strains of P. ostreatus, the level of genetic variation was higher suggesting a broader genetic basis employed in breeding of this mushroom. The cultivars and hybrids of A. bisporusshowed a higher level of homology. The isozyme markers (nonspecific esterase, leucinaminopeptidase, and phosphoglucoisomerase) are recommended for identification of the commercial strains of edible mushrooms.