Background. To carry out high-quality breeding work in the cattle populations, it is necessary to use next generation sequencing (NGS) methods, which allow large samples of the animals to be assessed for a significant number of the single nucleotide substitutions (SNPs) in a short period of time. Materials and methods. Genotyping of the Holstein black-and-white cattle bred in the Central Black Earth region of Russia (Belgorod region) was carried out using NGS sequencing (multilocus panel TruSeq® Bovine Parentage Kit, Illumina USA). Results. It was found that most of the animals studied are the inter-line hybrids. Correlation analysis of the "Reflection Sovereign", "Montvik Chieftain" and "Vis Back Ideal" lines showed no correlation dependency (r=0,165, p=0,106). Additionally, 10,4% of the cattle were carriers of the gene related to the Holstein red-and-white coat color. Furthermore, 61,5% of the cattle were carriers of the mutant allele G (_SNPchr8_108833985), which lowers immunity and contributes to the development of moraxellosis. Carriers of several unfavorable mutations were identified, such as Syndactyly (SY) – 8,3%, mutations leading to leukism (MITF gene) – 12,3%, and mutations in the melanocortin receptor gene (MC1R) – 10,4%. Carriers of the semi-lethal mutation (allele T) in the APAF1 gene accounted for 3,0% of the cattle, carriers of the lethal mutation (allele C) in the HH3 gene – 14,6%, carriers of the lethal mutation (allele C) in the HHB (BLAD) gene – 1,0%, carriers of the semi-lethal mutation (allele A) in the SLC35A3 gene – 2,0%. All this indicates errors in the breeding work. Against the backdrop of these unfavorable genetic factors, carriers of mutations with a positive effect on meat and milk productivity were observed, namely carriers of the MSTN gene mutation (allele T) – 50%, carriers of the ABCG2 gene mutation (allele C) – 4.2%, carriers of the DGAT1 gene mutation (allele A) – 28,1%, carrier of the CSN1S1 gene mutation (allele A) – 1,0%. Conclusion. We believe that adjusting the breeding work regarding these mutations, both with negative and positive effects, will allow farms to create core herds of high-productivity animals, which will contribute to increasing the quantity and quality of dairy products. EDN: RCSHPU

By sequencing ISSR DNA fragments of the Austrian snail (Caucasotachea vindobonensis), cloned into the pAL2-T vector, sequences of microsatellite loci (STR) and their flanking regions were obtained. For the nine identified STR loci with 2, 3 and 4 repeats, the design of primers adapted for fragment analysis was carried out. Subsequent genotyping of 100 individuals of the Austrian snail from various populations of Eastern Europe to estimate the volume allelic diversity of the species based on detected STR loci. All loci yielded unambiguous genotypes. Eight STR loci turn out to be polymorphic and have from 2 to 19 alleles.The data obtained can be used to assess the genetic diversity and phylogenetic analysis of populations of the Austrian snail, listed in the Red Book of the Belgorod region of Russia and the protection lists of Europe.

BACKGROUND: The aim of the study was to study the genetic structure of Caucasotachea vindobonensis populations in the south of the Central Russian Upland using SSR markers. MATERIALS AND METHODS Genetic structure of the populations was studied using eight microsatellite markers, first identified by the authors for the species under study. Fragment analysis of PCR products was carried out on an automatic capillary DNA sequencer Nanophore 05 (IAP RAS, Russia). A total of 498 individuals from 24 populations were studied. RESULTS: A total of 59 alleles were isolated in the eight studied STR loci, of which 21 (36%) turned out to be private. At the same time, private alleles were found in eleven studied populations, which is 46% of their total number. According to the data obtained, the effective number of alleles (Ae) averaged 1.6±0.06, the Chenon index (I) was 0.45±0.03, and the level of expected heterozygosity (He) was 0.27±0.021. A high level of spatial subdivision of population gene pools was noted (Fst=0.47±0.08) with a fairly low level of gene flow (Nm=1.15±0.91). PCA analysis showed that the populations of the central and eastern parts of the south of the Central Russian Upland form a closely related group, different from other populations of the region. Calculation of the effective abundance using the LD method showed that NeLd varies in different groups of snails from 0.6 to infinity. CONCLUSIONS: Populations of C. vindobonensis in the south of the Central Russian Upland live in conditions of significant isolation, as evidenced by the high degree of differentiation of the studied groups. The analysis of principal components based on genetic distances according to Nei and Wright's F-statistics indicate a pronounced division of populations in the study area into two clusters, which is probably associated with the historical features of landscape formation. Calculation of effective numbers indicates the high viability of the majority of the studied populations. At the same time, some groups of snails are clearly in a depressed state, which is reflected in low levels of genetic diversity and their effective size.

Водный брюхоногий моллюск Lymnaea stagnalis характеризуется высокой изменчивостью конхиологических признаков. Для оценки степени вариаций размеров и формы раковины в популяциях данного вида, обитающих на юге Среднерусской возвышенности, был проведен сравнительный анализ с использованием методов классической и геометрической морфометрии. Попарное сравнение популяций с использованием Post-hoc теста для perMANOVA позволило показать, что при использовании геометрической морфометрии наблюдалось большее количество статистически значимых отличий между сравниваемыми группами улиток, чем при использовании классического конхиометрического анализа. Кроме того, картина распределения популяций по группам в ходе кластеризации, существенно различалась при использовании разных морфометрических методов. Полагаем, что методы геометрической морфометрии, позволяющие давать интегральную характеристику раковины, лучше подходят для понимания закономерностей микроэволюционных процессов в популяциях брюхоногих моллюсков, а также изучения экологических особенностей видов. Тем не менее, для предотвращения потери важных конхиометрических данных, которые могут быть использованы для видовой идентификации и оценки внутри- и межпопуляционной изменчивости, при использовании геометрической морфометрии также важно учитывать абсолютные размерные характеристики объекта исследования.

Using the PCR-RFLP method, we genotyped pigs for the genes GBP1 (mutation c. [10A>G; 11A>G]), FUT1 (mutation G307A) and MUC4(A243G) associated with resistance to infectious diseases. A total of 188 boars of four breeds (Duroc, Large White, Landrace and Yorkshire) from the Belgorod Region of Russian Federation were studied. The highest frequency of favorable genotype AG for the polymorphism GBP1E2, which is resistant to pig respiratory syndrome, was characterized by the Duroc breed (0.38). The highest frequency of Eischerichia coli F18-resistant AA genotype was observed in the Landrace population (0.19). And in terms of resistance to E. coli with fibrium type F4 (K 88), the highest frequency of favorable genotype GG is characterized by the breed Large White (0,23). In general, it can be noted that genetic resistance in the studied boars is observed predominantly only to one infectious disease.

AIM: The aim of the study was to carry out species identification of the hybridogenic complex of European water frogs (Pelophylax esculentus complex) in the conditions of transformed biotopes of the south of the Central Russian Upland based on molecular genetic markers MATERIALS AND METHODS: The study included 36 local populations (770 individuals) living in conditions of transformed biotopes of the south of the Central Russian Upland. Identification of cryptic forms was carried out by a Multiplex PCR. Two molecular genetic markers were used for amplification: intron 1 of the SAI-1 DNA serum albumin gene to determine hybrids and cryptic forms, and a fragment of the first subunit of the cytochrome oxidase COI mtDNA gene to determine maternal lines. RESULTS: According to the data obtained, pure R-type population systems predominate (58.33%) in the study region. Mixed RE-type population systems were identified in 14 localities, REL-type in the region is extremely rare and is noted only in one locality. No pure L-type, E-type, or mixed LE-type population systems have been identified. The study revealed a statistically significant (p 0.001) predominance of haplotypes of the Western form (Pelophylax ridibundus). CONCLUSIONS: The data obtained indicate active adaptive changes in the population structure of European water frogs in the study area. Degradation of water bodies caused by abiotic and anthropogenic factors forces amphibians to migrate to neighboring reservoirs in which hybridization of representatives of this complex occurs. Introgressive and hybrid forms of the marsh frog, as well as hybrid edible individuals with greater ecological plasticity and tolerance to anthropogenic pressure, displace populations of Pelophylax lessonae. Based on the above, we consider it necessary to include the pond frog (P. lessonae) in the Red Book of the Belgorod region.

The state of three populations of the adventitious species H. pomatia was studied on the basis of conchiometric, phenetic and genetic methods. According to the obtained data, the two newly described populations from the Belgorod and Lugansk regions differ both in metric and morphological indicators, as well as in the frequencies of alleles of isoenzyme loci. The reason for such disparity may be genetic-automatic processes, natural selection in newly developed territories, as well as chaotic invasion from various localities, either from native range, or from nearby biotopes.

On the basis of molecular genetic analysis of the intron-1 of the nuclear serum albumin gene (SAI-1) were identified 177 individuals of Pelophylax esculentus complex of 9 localities Belgorod. Two types of population systems R and RE were identified. Pure populations of L-type, E-type and LE-type as well as P. lessonae individuals were not identified.

USING the PCR-RFLP method, pigs were genotyped for the leptin gene (the mutations T3266G and A1112G). The boars of four breeds (Duroc, Large White, Landrace and Yorkshire) from the Belgorod region of Russia were studied in the work. The LEP gene of pigs is located in the 18-th chromosome and serves as a promising candidate gene for pig growth and obesity evaluation. Purpose of the study: Evaluation of allele frequencies for the polymorphisms A1112G and T3266G among the boars of the Duroc, Large White, Landrace and Yorkshire breeds at the farms of the Belgorod region (Russia). Materials and methods: Genomic DNA was isolated from alcoholized earmarks using the DNA-Extran-2 reagent kit (SINTOL, Russia) according to the protocol. Pigs were genotyped by PCR-RFLP. PCR products were hydrolyzed with 4 units of restriction enzyme FokI or TaqI (SibEnzyme, Russia) for 16 hours. Gel blocks were stained with ethidium bromide (0.5 mu g/ml) and visualized on UV transilluminator to detect DNA fragments. The sequencing reaction was performed using the BigDye v. 3.1 Cycle Sequencing Kit. DNA sequencing was performed on the ABI 3500 genetic analyzer (Applied Biosystems, USA). Result: For both polymorphisms in all breeds of pigs they revealed a higher frequency of mutant alleles recorded in Yorkshires, as well as homozygous genotypes for them. At the same time, they showed a high degree of alleles and genotype frequencies dependence for two polymorphisms caused by linked inheritance. The allele frequencies of the studied breeds for the A1112G polymorphism correspond to the data of European selection. For T3266G polymorphism the Durocs revealed a significant deviation of genotype frequencies from the Hardy-Weinberg equilibrium, which may indicate an active breeding process in this breed. In addition to the studied polymorphisms relative to the reference sequence of the chromosome 18 (NC_010460.4), we identified 11 other mutations, including 8 SNPs and 3 deletions. Of these, 10 mutations were previously described during sequencing of the leptin gene (the sequences U66254.1 and AJ865080.1). For the first time, we described the G3297A mutation in the leptin gene intron among Duroc pigs, which can potentially act as a genetic marker of lipid metabolism and fat content in pigs. Conclusion: Based on the results of the pig LEP gene fragment sequencing, they confirmed the presence of polymorphisms T3266G and A1112G relative to the reference sequence U66254.1. They revealed a higher frequency of mutant alleles recorded in Yorkshires, as well as homozygous genotypes for both polymorphisms in all breeds of pigs. At the same time, they showed a high degree of dependence of alleles and genotype frequencies for two polymorphisms caused by linked inheritance. According to the T3266G polymorphism, a significant deviation of the genotype frequencies from the Hardy-Weinberg equilibrium was revealed among the Durocs, which may be the consequence of an active selection process in this breed. The Duroc breed showed a new SNP in the leptin gene G3297A.

In recent years, there has been an increasing amount of attention paid to the genetic health of domesticated animals and its relationship with the level of inbreeding and genetic diversity. At the same time, insufficient attention is still paid to the study of intrabreed genetic diversity and intrabreed stratification. The main goal of our work was to analyze the intra- and interbreed genetic diversity of commercial pig breeds on the basis of DNA microsatellite (MS-DNA) polymorphism. In total, the work used data for 3,308 pigs, which represented 11 herds. The animals belonged to four commercial pig breeds – Duroc (DR), Yorkshire (YR), Landrace (LN) and Large White (LW). 12 microsatellite loci recommended by ISAG-FAO and arranged in one multiplex panel (S0101, S0155, S0228, S0355, S0386, SW24, SW240, SW72, SW857, SW911, SW936, SW951) were used as DNA markers. When analyzing the intra- and interbreed variability of 11 herds, we found that all studied breeds significantly differed in terms of the proportion of both rare and the most common alleles. At the same time, the noted differences were determined, first of all, by the variability between individual herds within their breed. The location of herd centroids is random and is not consistent with their breed affiliation at all. When individuals belonging to the same breed are combined, the centroids of pig breeds in the space of first two axes from a Principal Coordinate Analysis form two clusters. The first one contains the only red pig breed (DR) used in the analysis, while the second one contains white pig breeds. In six pig herds the Ne estimates were below 50 inds., in two herds they were in the range of 50–100 inds., and finally in three herds the Ne estimates exceeded 100 inds. The analysis of the genetic variability of pigs of four commercial breeds showed that the high level of interbreed differences is caused, first of all, by the high variability among pig herds within each studied breed. Such intrabreed stratification can be formed due to the manifestation of many causes: different genetic basis of the founders of intrabreed genealogical groups, geographical isolation, different directions of selection within individual herds, exchange of animals between separate herds, the use of inbreeding in the practice of selection together with isolation, etc. Important consequences of intrabreed stratification are an increase in the level of interherd diversity (which is not lower than the level of interbreed diversity) against the background of a decrease in variability within individual herds, as well as a significant deficit of heterozygotes and an increase in the role of negative genetic and demographic processes. Thus, the existence of genetic heterogeneity within commercial pig breeds should be considered as an essential element in the history of their formation and breeding.

To address the decline in biodiversity, international cooperation in monitoring of threatened species is needed. Citizen science can play a crucial role in achieving this challenging goal, but most citizen science projects have been established at national or regional scales. Here we report on the establishment and initial findings of the European Stag Beetle Monitoring Network (ESBMN), an international network of stag beetle (Lucanus cervus) monitoring schemes using the same protocol. The network, started in 2016, currently includes 14 countries (see results) but with a strong variation in output regarding the number of transects (148 successful transects in total) and transect walks (1735). We found differences across European regions in the number of stag beetles recorded, related to phenology and temperature, but not for time of transect start. Furthermore, the initial experiences of the ESBMN regarding international cooperation, citizen science approach, and drop-out of volunteers is discussed. An international standardised protocol that allows some local variation is essential for international collaboration and data management, and analysis is best performed at the international level, whereas recruiting, training, and maintaining volunteers is best organised locally. In conclusion, we appeal for more joint international citizen science-based monitoring initiatives assisting international red-listing and conservation actions.

Anthropogenic pollution of water bodies is a serious problem for the sustainability of aquatic ecosystems. The main sources of pollution of water bodies are wastewater, washouts from agricultural fertilizers, pesticides and roads. Many of highly persistent pollutants are dangerous mutagens and carcinogens. The disruption they cause to the DNA and RNA structure and properties of organisms can have long-term consequences and can affect the livelihood of future generations. Among existing DNA damage estimation methods, DNA comet assay is the most widespread due to its accuracy and reliability. In our study, the marsh frog (Pelophylax ridibundus Pallas, 1771) was chosen as an object of study. This species is adapted to life under anthropogenic influence and actively inhabits transformed biotopes. The aim of our work was to estimate the level of DNA damage by DNA comet assay of P. ridibundus inhabiting Belgorod city and its suburbs. We studied a total of 6 marsh frog populations, with collection sites differing in the degree of anthropogenic pressure (See Table 1 and Fig. 1), and we selected 5 individuals of P. ridibundus from each locality. The experimental animals were handled in accordance with international ethical standards. Animal liver was used as the material for the study and analysis was performed on the day of collection. Tissue was homogenized in phosphate-salt buffer (pH 7.5) containing 20 mMEDTA-Na2 and 10% dimethyl sulfoxide (DMSO) at +4 degrees C. To precipitate large fragments, tubes were incubated for 5 mM at room temperature before transferring 1.5 ml of the top layer to microcentrifuge tubes and centrifuged in a 5415 R Eppendorf microcentrifuge (Germany) at 1000 g for 10 mM at +4 degrees C. The supernatant was removed and the cell precipitate was diluted in 1 ml of phosphate salt buffer cooled to +4 degrees C. To obtain microarrays, 60 mu l of cell suspension was mixed with 240 mu l of 1% fusible agarose solution (t<42 degrees C). The lysis was performed for at least one hour at +4 degrees C (lysis buffer: 10mM Tris-HCl (pH 10) 2.5M NaCl, 100 mM EDTA-Na2 and 1% Triton X-100 with 10% DMSO). Electrophoresis was performed in a darkened room in Tris-EDTA-borate electrode buffer (TBE) for 20 mM at a voltage of 1V/cm. At the end of electrophoresis, the preparations were fixed with 70% ethanol for 15 mM. The preparations were stained with SYBR Creen I dye, with subsequent analysis using a luminescent trinocular microscope MIKMED-2 of Lomo (Russia), with at least 100 cells analyzed from each preparation, which were divided into 5 types; cells in the state of apoptosis were counted separately (Durnev, et al., 2006). The degree of DNA damage was estimated using the Kruskel-Wallis criterion ("DNA comet index") (Struwe et al., 2007; Filippov, 2014). To determine the statistical significance of differences between the studied populations we used one-way analysis of variance (ANOVA); statistically significant differences were considered at p < 0.05. According to the data obtained, in all studied populations, the mean value of the DNA damage index (DDI) did not exceed the first threshold of DNA degradation (See Table 2). This may indicate, on the one hand, the absence of high concentrations of genotoxic pollutants here (which is highly doubtful) and, on the other hand, the efficient operation of the reparative system of marsh frog. Nevertheless, the calculated DDI values made it possible to construct a gradational scale of DNA destruction level in the studied groups of frogs depending on the degree of pollution and proximity of residential and industrial buildings (See Table 2-4). Various chemical compounds from industrial production, particularly nitrogen compounds, are known to have genotoxic effects. Due to the lack of possibility to perform chemical analysis of water to identify concentrations of genotoxic pollutants, we analysed data on surface water pollution in some of the investigated locations from open literature sources. As a result of this analysis, we revealed the dynamics of surface water pollution. All investigated points by the mean value of the statistical DDI surpassed reliably the least polluted site "Sevrukovo", which is located in the green zone and at a considerable distance from urbanized areas (See Table 2-4). High values of DDI were fixed in a zone of industrial and multi-storey buildings, localities "Seversky Donets" (DDI = 0.454) and "Veselka" (DDI = 0.416). Exceeding maximum permissible concentration of nitrogen compounds is observed in the above mentioned rivers. Noteworthy is the fact that the highest value of the DDI (0.521) was recorded in the estuary of the Razumnaya River remote from the industrial part of the city ("Razumnoye"). It should be noted that in this locality there is the greatest number of damaged cells, including apoptosis (See Table 2, Fig. 2 and 3). The highest DDI value is due to the high level of pollutants, since the predominant part of the river is occupied by "non-sewered" private low-rise buildings, as well as arable land. At the same time in the upper part of the river ("Sevryukovo") due to the lack of obvious pollutants and the intensive current contributing to self-purification of the river, we recorded the minimum value of the DDI (0.057) and the absence of cells in the state of apoptosis. Thus, the present study revealed an increase in the degree of DNA destruction in populations of marsh frogs in the impacted areas of the city of Belgorod and its surroundings. At the same time, increased levels of toxic substances may contribute to a decrease in the viability of individuals of both analyzed species and other hydrobiont species, which will negatively affect the biodiversity of the region as a whole.

Pelophylax ridibundus БелгородАннотация: Известно, что в городских ландшафтах часто происходят резкие изменения природных условий, которые могут негативно сказываться на жизнеспособности обитающих здесь популяций.Это в свою очередь вызывает сокращение биоразнообразия в целом.Одним из эффективных методов анализа степени деградации экосистемы является изучение трофических связей животных.Особый интерес в этом отношении вызывают бесхвостые земноводные, в частности зеленые лягушки, которые являются толерантными к антропогенному прессу и часто используются в качестве биоиндикаторов.Одним из таких видов является озерная лягушка (Pelophylax ridibundus).Данный вид обладает высокой экологической пластичностью и часто заселяет как антропогенно измененные, так естественные ландшафты.В работе представлены данные о пищевых предпочтениях озерной лягушки в условиях урбанизированных территорий г.Белгорода

USING the method PCR-RFLP, the pigs were genotyped for the H-FABP gene (T314C mutation). The study examined 188 boars of four breeds (Large White, Landrace, Duroc, and Yorkshire) from the Belgorod region of Russia. H-FABP3 plays a key role in fatty acid metabolism and fat deposition and is considered a candidate gene for the assessment of pig fatness and intramuscular fat (IMF) levels. Purpose of the study: Evaluation of alleles and genotype occurrence frequency for the HinfI polymorphism in the H-FABP gene among the Large White, Duroc, Landrace and Yorkshire breeds at the farms of the Belgorod region of Russia. Materials and methods: Genomic DNA was isolated from alcoholized earmarks using the DNA-Extran-2 reagent kit (SINTOL, Russia) according to the protocol. Pig genotyping for the T103C mutation was performed by PCR-RFLP method. The PCR product in the amount of 10 mu l was hydrolyzed with 4 units of the HinfI restriction enzyme (SibEnzyme, Russia) for 16 hours. The hydrolysis products were separated by horizontal electrophoresis in 2% agarose gel (Mini-Sub Cell GT, BioRad, USA). Gel blocks were stained with ethidium bromide (0.5 mu g/ml) and visualized by UV transilluminator to detect DNA fragments. Result: The highest frequency of the economically valuable allele H and the genotype HH was noted for the Yorkshire breed (0.968 and 0.94, respectively), and the smallest frequencies - for the Large White breed (0.628 and 0.36, respectively). Based on the.2 test, all examined breeds are in a status of genetic equilibrium based on Hardy-Weinberg. They revealed the dependence of H-FABP alleles and genotype frequencies on the T3469C polymorphism of the leptin gene (LEP). In particular, they revealed the tendency to C allele frequency increase for T3469C (LEP) with H allele frequency decrease of the H-FABP gene (r = -0.81 +/- 0.41). Besides, a positive correlation was found between the frequencies of homozygous genotypes TT (LEP) and HH (H-FABP) (r = 0.87 +/- 0.35), as well as heterozygous genotypes CT (LEP) and Hh (H-FABP) (r = 0.93 +/- 0.26). Conclusion: The highest frequency of the economically valuable H allele and the HH genotype were noted for the Yorkshire breed, and the lowest - for the Large White breed. They determined the originality of the gene pool of the main breeds of pigs in the Belgorod region for the polymorphism H-FABP T314C, caused by the peculiarities of local selection and crossing systems thorough the producing meat products. They revealed the dependence of alleles and genotype frequencies in the FABP and LEP genes.

Cepaea vindobonensis (Gastropoda, Pulmonata, Helicidae) is a terrestrial mollusk, protected in the Belgorod Region (European Russia). In the Bekaryukovsky Bor Natural Monument, its peripheral population is known considering to be one of the most abundant in the north-eastern part of its range. In this paper, changes in the morphogenetic structure of C. vindobonensis population and its relationships with microclimatic environmental factors were analysed for the last 20 years. The results of the Kruskal-Wallis test of conchiometric features showed statistically significant (p < 0.05) differences between the studied years for all measurements. We found a statistically significant correlation between the parameters of the adult shell and some climatic characteristics, including both the average values for a four-year period and values recorded during the shell formation at the juvenile life cycle stage. This correlation apparently indicates the epigenetic nature of this population adaptation to the environment. The calculation of the average number of phenotypes (identified according to Zhivotovsky) showed the lowest level in 2020 (mu = 1.44 +/- 0.20), and the highest level in 2002 (mu = 2.19 +/- 0.21). To determine the population vitality degree, we analysed the genetic structure of the C. vindobonensis population on the basis of esterase izozyme loci. The highest changes in allele frequencies were observed in EST8 locus, where there was a statistically significant (p < 0.05) decrease in the EST82 allele frequency in 2006-2011, while being absent in the population in 2012-2020. At the same time, in 2020 the studied population became monomorphic for the EST8-3 allele. It is assumed that the transition to the homozygous state in this locus was caused not only by the genetically automatic process, but also by changes in habitats. In addition, there was a considerable lack of heterozygotes (inbreeding coefficient F = 0.517 +/- 0.395) in 2020. The effective population size (Ne), calculated using the temporal method, was equal to 13.52. The forecast of the lifetime of the.. vindobonensis population based on genetic data demonstrated the value of about 100 years. The obtained results show a low vitality degree of the studied peripheral C. vindobonensis population. This may lead to its further extinction, especially in the case of changes in environmental parameters. However, the maintaining of nature conservation measures in the study area may allow avoiding this event.

BACKGROUND: The water frog (Pelophylax esculentus complex) is hybrid in composition. In view of the fact that a large number of data on the species composition of the water frog and very scarce material on the genetic structure of populations are available in the literature, we aimed to analyze the genetic structure of populations of the water frog in the southern part of the Middle Russian upland, which was one of the refugia for many species during the glacial epoch and the center of dispersion in the postglacial time, based on DNA microsatellite markers. MATERIALS AND METHODS: The study involved 36 local populations. DNA variability was analyzed by multiplex SSR-PCR. Seven loci (Res 14, Res 15, Res 17, Res 22, Rrid059A, Rrid082A, and Rrid171A) were used for amplification. Fragment analysis of PCR products was performed on an ABI PRISM 3500 automated capillary DNA sequencer (Applied Biosystems, USA). RESULTS: The total number of alleles detected ranged from 13 to 41. The effective number of alleles (Ae) averaged 4.569 0.219, the Chenon index (I) 1.567 0.04, level of expected heterozygosity (Не) 0.68 0.01. According to Wrights model, the greatest contribution to genetic variability is made by the heterogeneity of individuals within populations, some of which are of a hybrid nature (Fis = 0.281 0.069, Fit = 0.413 0.053, Fst = 0.180 0.017). The average indicator of the intensity of gene exchange between populations (Nm) was 1.212 0.142 individuals per generation. The calculation of the effective abundance using the LD method indicates a high level of viability of the studied groups of the frogs. CONCLUSION: The results demonstrated a high level of genetic diversity and viability of most of the studied groups, which, due to the intense gene exchange between them, can represent a single panmictic population. The data of the genetic analysis support the active adaptation of P. esculentus complex to living in an urbanized environment.

Background. Morphological features are one of the most important markers for bioindication. Among them, the polymorphism of phenotypic traits occupies a special place. In tailless amphibians, dorsal colouration variability is widespread: the presence or absence of a light dorsomedial stripe or dark spots. The phenotypic composition is known to change in the impacted areas. The purpose of the study was to analyze the phenotypic structure of Pelophylax esculentus complex populations in the urbanized landscapes in the South Central Russian Upland. This study is a part of the comprehensive analysis of the population structure of green frogs in the South Central Russian Upland. Materials and methods. A total of 748 individuals were sampled from 35 localities in the South Central Russian Upland. Collection points differed in the degree of anthropogenic pressure. Dorsal coloration variations were analyzed according to the generally accepted grading. Analysis of phenotypes diversity was performed according to the indicators proposed by L.A. Zhivotovsky. Results. Four main phenotypes “Maculata” (M), “Striata” (S), “Punctata” (P), “Burnsi” (B), and their combinations SM, SP and SMP were registered in the study area. The highest phenotypic diversity was observed in natural or sparsely disturbed biotopes “Sazon” (6.815 ± 0.251), “Aidar” (6.527 ± 0.306), “Goluboi Dunai” (6.43 ± 0.418), while under anthropogenic pressure an increase in the proportion of rare phenotypes was observed. The principal component analysis has demonstrated the phenotypic similarity of most of the populations. At the same time, unique groups were found in both impact and natural habitats, which may indicate an effective compensatory response of the green frog populations in the study area to anthropogenic pressures. Conclusions. The results obtained confirm the data of the previous studies on the phenotypic polymorphism of green frogs, according to which the phenotypic diversity declines and the proportion of rare phenotypes increases under conditions of anthropogenic pressure. In addition, in a number of populations, both in urbanized and relatively clean biotopes, unique combinations of phenotypic frequencies are observed, indicating active adaptation of green frogs to changing environmental conditions.

USING the method PCR-RFLP, they performed genotyping of cattle for the CSN2 gene (A1/A2 polymorphism). The work studied the cows of six main dairy breeds: Holstein, Simmental, Ayrshire, Brown Swiss, Jersey, Red-and -White from nine different farms of the Central Black Earth Region of Russia. The gene for beta-casein (CSN2) is located in the chromosome 6 and may be an important marker of milk yield and milk quality. Purpose of the study: to assess the frequencies of the A2 variant and the A2A2 genotype in various breeds of dairy cattle at the breeding farms of the Central Chernozem region of Russia. Materials and Methods: Genomic DNA was isolated from whole blood. 106 b.p DNA fragment was amplified using the primers from the CSN2 gene containing the target mutation. The PCR product was hydrolyzed with the restriction enzyme BstDEI. Gel blocks were stained with ethidium bromide and visualized on UV transilluminator to detect DNA fragments. The samples with the genotype A1A1 had the DNA fragments of 106 b.p., A1A2 -the fragments of 106, 74, 32 b.p., and A2A2 -74, 32 b.p. Result: The highest frequency of the variant A2 was found in the livestock of the Jersey (0.94) breed, and the lowest in the Simmental breed (0.38). At the same time, there is a significant scatter of A2 frequencies in different cattle of the same breed: in the Holstein breed -from 0.53 to 0.70, in the Ayrshire breed -from 0.48 to 0.91. In general, the frequencies of the A2 CSN2 variant in most of the studied breeds take values higher than the average as compared with the data on the same breeds from other regions and countries. In some herds, they revealed a significant deviation of genotype frequencies from the Hardy-Weinberg equilibrium, which indicates the breeding work related to milk quality.

The results of studies of the genetic structure of the Central European wild boar (Sus scrofa scrofa) population and four breeds of domestic pigs (Duroc, Yorkshire, Large White and Landrace) bred in the Central Black Earth region of Russia are presented in this work. Based on 12 microsatellite loci, a significant ( p <0.05) decrease in the level of genetic variability in bred breeds was shown. The expected heterozygosity and Shannon index were as follows: in the wild boar, Ho = 0.763 ± 0.026, I = 1.717 ± 0.091; in the Duroc breed, Ho = 0.569 ± 0.068, I = 1.191 ± 0.157; in the Landrace, Ho = 0.618 ± 0.062, I = 1.201 ± 0.147; in the Large White, Ho = 0.680 ± 0.029, I = 1.362 ± 0.074; and in the Yorkshire, Ho = 0.642 ± 0.065, I = 1.287 ± 0.156. The results of checking genotypic Hardy–Weinberg equilibrium based on the G-test of maximum likelihood demonstrated that the overwhelming majority of loci in the wild boar population were in the state of said equilibrium. By contrast, in pig breed populations, some loci demonstrated a significant deviation from the indicated equilibrium. In addition, the Yorkshire, Large White, and Landrace populations had loci, for which the hypothesis of neutrality was reliably rejected based on the results of the Ewens–Watterson test. The revealed private alleles, characteristic of the wild boar and breeds, can later be used to identify them. The ordination of the centroids of different herds in the space of the first two principal coordinates based on the matrix of pairwise estimates of Nei’s genetic distances showed that the most distant populations are the Duroc and Boar breeds, and the most genetically close are the Yorkshire and Landrace breeds. The closest to the wild boar population was the Large White breed. The assessment of the effective size, carried out using the method based on the linkage disequilibrium and the molecular coancestry method, showed that in all studied groups, including the wild boar population, the effective size was less than 100 individuals. The low effective size of the wild boar population (Ne = 21.8, Neb = 4.0) is probably caused by the death and shooting of animals due to Pestis africana suum.

The pigs were genotyped for the dystrophin gene (DMD) using the ACRS-PCR method. The study examined 215 boars of four breeds (Duroc, Large White, Landrace and Yorkshire) from the Belgorod region of Russia. The mutation in the DMD gene leads to dystrophin expression decrease in the muscles of the lumbar region, longissimus dorsi, and also in the diaphragm. The animals with the mutant allele T had the average daily gain decrease in live weight, the fat thickness of the muscle layer depth reduction. To assess the frequency of alleles and genotype occurrence for the DMD gene among the Landrace, Duroc, Large White and Yorkshire breeds that are bred on the farms of the Belgorod region of Russia. Genomic DNA was isolated from alcoholized earmarks. Pig genotyping was performed using ACRS-PCR (Amplification Created Restriction Sites). Using primers, 151 b.p. DNA fragment was amplified from the forty-first exon of the dystrophin gene containing the target mutation. Primers were designed using the Primer3 software. 10 mu l of the PCR product were hydrolyzed with 4 units of the Mox20I restriction enzyme (SibEnzyme, Russia) for 16 hours. The hydrolysis products were separated by horizontal electrophoresis in 4% agarose gel. Then, based on the results obtained, the pigs were genotyped by TaqMan PCR. The mutant allele T is observed only in the Landrace livestock with the frequency of 0.405. This is significantly higher than in the livestock of this breed from other regions and can be caused by the peculiarities of local selection. Based on the verified genotypes, they developed the method of pig genotyping for the DMD gene using real-time PCR technology based on TaqMan fluorescent probes. The high efficiency and reliability of the results of the TaqMan PCR technique and the possibility of its use in routine practice have been shown. The methods of pig genotyping for DMD gene mutations have been developed using ACRS-PCR and real-time PCR using TaqMan technology. The analysis of allele frequencies and genotypes of boars from the Duroc, Large White, Landrace and Yorkshire breeds at the farms of the Belgorod region of Russia was carried out according to the polymorphism of the dystrophin gene. As compared to previously obtained data from other regions of Russia, higher frequencies of the mutant allele T were revealed for the Landrace breed, which may be associated with the peculiarities of local selection for productive traits.