Random transgene integration is a powerful tool for developing new genome-wide screening approaches. These techniques have already been used for functional gene annotation by transposon-insertion sequencing, for identification of transcription factor binding sites and regulatory sequences, and for dissecting chromatin position effects. Precise localization of transgenes and accurate artifact filtration are essential for this type of method. To date, many mapping assays have been developed, including Inverse-PCR, TLA, LAM-PCR, and splinkerette PCR. However, none of them is able to ensure localization of both transgene’s flanking regions simultaneously, which would be necessary for some applications. Here we proposed a cheap and simple NGS-based approach that overcomes this limitation. The developed assay requires using intentionally designed vectors that lack recognition sites of one or a set of restriction enzymes used for DNA fragmentation. By looping and sequencing these DNA fragments, we obtain special data that allows us to link the two flanking regions of the transposon. This can be useful for precise insertion mapping and for screening approaches in the field of chromosome engineering, where chromosomal recombination events between transgenes occur in a cell population. To demonstrate the method’s feasibility, we applied it for mapping SB transposon integration in the human HAP1 cell line. Our technique allowed us to efficiently localize genomic transposon integrations, which was confirmed via PCR analysis. For practical application of this approach, we proposed a set of recommendations and a normalization strategy. The developed method can be used for multiplex transgene localization and detection of rearrangements between them.

The aim of the present study was to identify the influence of extra- (EOV) and intraovarian vitrification (IOV) on mitochondrial activity (MA) and chromatin state in porcine oocytes during maturation in vitro. During EOV porcine oocytes were exposed in cryoprotective solutions (CPS): CPS-1 – 0.7 M dimethyl sulfoxide (DMSO)+0.9 M ethylene glycol (EG); CPS-2 – 1.4 M DMSO+1.8 M EG; CPS-3 – 2.8 M DMSO+3.6 M EG+0.65 M trehalose. At IOV the ovarian fragments were exposed in CPS-1 – 7.5 % EG+7.5 % DMSO, then in CPS-2 – 15 % EG, 15 % DMSO and 0.5 M sucrose. Straws with oocytes and ovarian fragments were plunged into LN2 and stored. For devitrification, the EOV oocytes were washed in solutions of 0.25, 0.19 and 0.125 M of trehalose, the IOV – in 0.5 and 0.25 М trehalose. Oocytes were cultured in NCSU-23 medium with 10 % fluid of follicles, follicular walls, hormones. 0.001 % of highly dispersed silica nanoparticles (ICP named after A.A. Chuyko of the NAS of Ukraine) were added to all media. The methods of fertilization and embryo culture are presented in the guidelines developed by us. MA and chromatin state were measured by MitoTracker Orange CMTMRos and the cytogenetic method. Significant differences in the level of oocytes with high-expanded cumulus between control and experimental vitrified groups (81 % versus 59 % and 52 %, respectively, p ≤ 0.001) were observed. The percentage of pyknotic cells in native oocytes was 19 %, EOV or IOV oocytes were 39 % and 49 %, respectively. After culture, the level of matured native oocytes was 86 %, 48 % EOV and 33 % IOV cells finished the maturation (p ≤ 0.001). Differences were also observed in the level of MA between groups treated by EOV and IOV (89.4±7.5 µA and 149.2±11.3 µA, respectively, p ≤ 0.05). For the first time, pre-implantation embryos were obtained from oocytes treated by IOV.

Narrow-leaved lupine (Lupinus angustifolius L.) is a cultivated multipurpose species with a very short history of domestication. It is used as a green manure, and for feed and food. This crop shows good prospects for use in pharmacology and as a source of fish feeds in aquaculture. However, its genetic potential for the development of productive and adaptable cultivars is far from being realized. For crop species, the genetic base of the cultivated gene pool has repeatedly been shown as being much narrower than that of the wild gene pool. Therefore, efficient utilization of a species’ genetic resources is important for the crop’s further improvement. Analyzing the information on the germplasm collections preserved in national gene banks can help perceive the worldwide diversity of L. angustifolius genetic resources and understand how they are studied and used. In this context, the data on the narrow-leaved lupine collection held by VIR are presented: its size and composition, the breeding status of accessions, methods of studying and disclosing intraspecific differentiation, the classifications used, and the comparison of this information with available data on other collections. It appeared that VIR’s collection of narrow-leaved lupine, ranking as the world’s second largest, differed significantly from others by the prevalence of advanced cultivars and breeding material in it, while wild accessions prevailed in most collections. The importance of the wild gene pool for the narrow-leaved lupine breeding in Australia, the world leader in lupine production, is highlighted. The need to get an insight into the species’ ecogeographic diversity in order to develop cultivars adaptable to certain cultivation conditions is shown. The data on the testing of VIR’s collection for main crop characters valuable for breeders are presented. Special attention is paid to the study of accessions with limited branching as a promising gene pool for cultivation in relatively northern regions of Russia. They demonstrate lower but more stable productivity, and suitability for cultivation in planting patterns, which has a number of agronomic advantages. Analyzing the work with narrow-leaved lupine genetic resources in different national gene banks over the world helps shape the prospects of further activities with VIR’s collection as the only source of promising material for domestic breeding.

The existing spring forms of wheat-rye amphiploids are characterized by late maturity due to the long duration of the interphase period “germination–heading”. The manifestation of this trait is influenced by Vrn-1 genes. Their dominant alleles also determine the spring type of development. The results of studying the interphase period “germination–heading” of spring octaploid and hexaploid forms of triticale created for use in research and breeding programs under the conditions of forest-steppe of Western Siberia are given in this article. The interphase period of the primary forms 8xVrnA1, 8xVrnB1 and 8xVrnD1 obtained by artificial doubling of the chromosome number of the wheat-rye hybrids made by pollination of three lines of the soft wheat ‘Triple Dirk’ – donors of different dominant Vrn-1 genes – by a winter rye variety ‘Korotkostebel’naya 69’ was determined under the field conditions in the nursery of octaploid (8x) triticale. In the nursery of hexaploid triticale, this trait was studied in the populations of hybrids obtained by hybridization of these three primary forms of octaploid triticale with the hexaploid winter triticale variety ‘Sears 57’. In the offspring of crossing 8хVrnD1× ‘Sears 57’, spring genotypes of 6x triticale bearing Vrn-D1 were selected. This fact was determined by PСR. It means that the genetic material from the chromosome of the fifth homeologous group of the D genome of the bread wheat is included in the plant genotypes. This genome is absent in the winter 6x triticale ‘Sears 57’. The grain content of spikes of the created hexaploid forms of triticale is superiour to that of the maternal octaploid triticale forms. It was shown that plants of the hybrid populations 8xVrnA1× ‘Sears 57’ and 8xVrnD1× ‘Sears 57’ carrying the dominant alleles Vrn-A1a and Vrn-D1a, respectively, have a shorter duration of the “germination–heading” interphase period than the initial parental forms of primary 8x triticale. The short interphase period of “germination–heading” of the 6x triticale is a valuable breading trait for the creation of early maturing and productive genotypes of triticale.

Common winter wheat is the main grain crop cultivated in the North Caucasus. Rust disease damage is one of the factors limiting wheat productivity. There are three species of rust in the region: leaf (Puccinia triticina), stem (P. graminis) and stripe rust (P. striiformis), and their significance varies from year to year. The most common is leaf rust, but in the last decade the frequency of its epiphytotic development has significantly decreased. At the same time, an increase in the harmfulness of stripe rust (P. striiformis) is noted. Stem rust in the region is mainly absent or observed at the end of the wheat growing season to a weak degree. Only in some years with favorable weather conditions its mass development is noted on susceptible cultivars. It is believed that the sources of infection with rust species in the North Caucasus are infested soft wheat crops, wild-growing cereals and exodemic infection carried by air currents from adjacent territories. In the North Caucasus, forage and wild grasses are affected by Puccinia species almost every year. Depending on weather conditions, the symptom expression is noted from late September to December and then from late February to May–June. Potentially, an autumn infection on grasses can serve as a source for infection of winter soft wheat cultivars sown in October. The purpose of these studies is to characterize the virulence of P. triticina, P. graminis, P. striiformis on wild cereals and to assess the specialization of causative agents to winter wheat in the North Caucasus. Infectious material represented by leaves with urediniopustules of leaf, stem and stripe rusts was collected from wild cereals (Poa spp., Bromus spp.) in the Krasnodar Territory in October–November 2019. Uredinium material from P. triticina, P. striiformis, and P. graminis was propagated and cloned. Monopustular Puccinia spp. isolates were used for virulence genetics analysis. In experiments to study the specialization of rust species from wild-growing cereals on common wheat, 12 winter cultivars were used (Grom, Tanya, Yuka, Tabor, Bezostaya 100, Yubileynaya 100, Vekha, Vassa, Alekseich, Stan, Gurt, Bagrat). These cultivars are widely cultivated in the North Caucasus region and are characterized by varying degrees of resistance to rust. Additionally, wheat material was inoculated with Krasnodar populations of P. triticina, P. striiformis, P. graminis from common wheat. In the virulence analysis of P. triticina on cereal grasses, four phenotypes (races) were identified: MCTKH (30 %), TCTTR (30 %), TNTTR (25 %), MHTKH (15 %), and five were identified in P. graminis (RKMTF (60 %), TKTTF, RKLTF, QKLTF, LHLPF (10 % each). Among P. striiformis isolates, three phenotypes were identified using the International and European sets of differentiating cultivars – 111E231 (88 %), 111E247 (6 %) and 78E199 (6 %). Using isogenic Avocet lines, 3 races were also identified, which differed among themselves in virulence to the Yr1, Yr11, Yr18 genes (with the prevalence of virulent ones (94 %)). Composite urediniums’ samples (a mixture of all identified races) of grass rust of each species were used to inoculate winter wheat cultivars. The most common winter wheat cultivars (75 %) were characterized by a resistant response when infected with P. graminis populations from common wheat and cereal grasses. All these cultivars were developed using donors of the rye translocation 1BL.1RS, in which the Lr26, Sr31, and Yr9 genes are localized. The number of winter wheat cultivars resistant to leaf rust in the seedling phase was lower (58 %). At the same time, all the studied cultivars in the seedling phase were susceptible to P. striiformis to varying degrees. The virulence analysis of the leaf, stem and stripe rust populations did not reveal significant differences in the virulence of the pathogens between wild-growing cereals and soft wheat. Urediniomaterial of all studied rust species successfully infested soft wheat cultivars. The results obtained indicate that grasses are rust infection reservoirs for common wheat crops in the North Caucasus.

The size of the nuclear genome in eukaryotes is mostly determined by mobile elements and noncoding sequences and may vary within wide limits. It can differ significantly both among higher-order taxa and closely related species within a genus; genome size is known to be uncorrelated with organism complexity (the so-called C-paradox). Less is known about intraspecific variation of this parameter. Typically, genome size is stable within a species, and the known exceptions turn out be cryptic taxa. The Eisenia nordenskioldi complex encompasses several closely related earthworm species. They are widely distributed in the Urals, Siberia, and the Russian Far East, as well as adjacent regions. This complex is characterized by significant morphological, chromosomal, ecological, and genetic variation. The aim of our study was to estimate the nuclear genome size in several genetic lineages of the E.  nordenskioldi complex using flow cytometry. The genome size in different genetic lineages differed strongly, which supports the hypothesis that they are separate species. We found two groups of lineages, with small (250–500 Mbp) and large (2300–3500 Mbp) genomes. Moreover, different populations within one lineage also demonstrated variation in genome size (15–25 %). We compared the obtained data to phylogenetic trees based on transcriptome data. Genome size in ancestral population was more likely to be big. It increased or decreased independently in different lineages, and these processes could be associated with changes in genome size and/or transition to endogeic lifestyle.

The article describes a new phenomenon in the breeding group of mini-pigs at the Institute of Cytology and Genetics (ICG, Novosibirsk): polydactyly (extra digits), which is unusual because the additional digits are situated at the lateral surface of legs or at the lateral and medial ones. This anomaly was first found here in 2017 in adult animals intended for culling due to incorrect positioning of the legs caused by flexor tendon laxity and resulting in weight-bearing on the palmar surface of the proximal phalanges (“bear’s paw”). Therefore, the polydactyly of mini-pigs has a pronounced negative selection effect. A visual survey of the livestock was conducted, and a description of the detected anomaly was compiled. The polydactyly in mini-pigs is a stand-alone trait and is not part of any syndromes. Individuals with polydactyly may have extra digits either on pectoral or on pectoral and pelvic limbs. On thoracic limbs, there may be either one lateral digit or a lateral digit and a medially located rudimentary hooflet. On pelvic limbs, only lateral extra digits can occur. Anatomical and morphological analyses showed that the lateral extra digit is an anatomically complete (“mature”) structure, whereas the medial rudimentary digit consists of only a hooflet without other structures characteristic of normal digits. Cytological examination revealed no specific karyotypic features, except for Robertsonian translocation Rb 16;17 previously reported for the mini-pigs of the same livestock. Cytological findings indicated that the polydactyly and Robertsonian translocation are not linked genetically. Genealogical analysis and results of crosses are consistent with a working hypothesis of recessive inheritance of the trait. Overall, the study shows that this type of polydactyly is anatomically and morphologically unique and not typical of Sus scrofa. In this species, only polydactyly types with medial accessory toes have been described and are usually inherited as a dominant trait with incomplete penetrance. In our case, the results of test crosses indicate recessive inheritance of the trait with varying expression and incomplete penetrance, because of which poorly expressed phenotypes are not visually detectable.

Benefits and costs of meiotic recombination are a matter of discussion. Because recombination breaks allele combinations already tested by natural selection and generates new ones of unpredictable fitness, a high recombination rate is generally beneficial for the populations living in a fluctuating or a rapidly changing environment and costly in a stable environment. Besides genetic benefits and costs, there are cytological effects of recombination, both positive and negative. Recombination is necessary for chromosome synapsis and segregation. However, it involves a massive generation of double-strand DNA breaks, erroneous repair of which may lead to germ cell death or various mutations and chromosome rearrangements. Thus, the benefits of recombination (generation of new allele combinations) would prevail over its costs (occurrence of deleterious mutations) as long as the population remains sufficiently heterogeneous. Using immunolocalization of MLH1, a mismatch repair protein, at the synaptonemal complexes, we examined the number and distribution of recombination nodules in spermatocytes of two chicken breeds with high (Pervomai) and low (Russian Crested) recombination rates and their F1 hybrids and backcrosses. We detected negative heterosis for recombination rate in the F1 hybrids. Backcrosses to the Pervomai breed were rather homogenous and showed an intermediate recombination rate. The differences in overall recombination rate between the breeds, hybrids and backcrosses were mainly determined by the differences in the crossing over number in the seven largest macrochromosomes. The decrease in recombination rate in F1 is probably determined by difficulties in homology matching between the DNA sequences of genetically divergent breeds. The suppression of recombination in the hybrids may impede gene flow between parapatric populations and therefore accelerate their genetic divergence.

The consumption of food rich in sugar and fat provokes obesity. Prenatal conditions have an impact on taste preferences and metabolism in the adult offspring, and this impact may manifest differently in different sexes. An increase in blood leptin level in pregnant females reduces the risk of obesity and insulin resistance in the offspring, although the mechanisms mediating this effect are unknown. Neither is it known whether maternal leptin affects taste preferences. In this study, we investigated the effect of leptin administration to pregnant mice on the development of diet-induced obesity, food choice, and gene expression in the liver and muscles of the offspring with regard to sex. Leptin was administered to female mice on days 11, 12, and 13 of pregnancy. In male and female offspring, growth rate and intake of standard chow after weaning, obesity development, gene expression in the liver and muscles, and food choice when kept on a high-calorie diet (standard chow, lard, sweet cookies) were recorded. Leptin administration to pregnant females reduced body weight in the female offspring fed on the standard diet. When the offspring were given a high-calorie diet, leptin administration inhibited obesity development and reduced the consumption of cookies only in males. It also increased the consumption of standard chow and the mRNA levels of genes for the insulin receptor and glucose transporter type 4 in the muscles of both male and female offspring. The results demonstrate that an increase in blood leptin levels in pregnant females has a sex-specific effect on the metabolism of the offspring increasing resistance to obesity only in male offspring. The mechanism underlying this effect includes a shift in food preference in favor of a balanced diet and maintenance of insulin sensitivity in muscle tissues.

Wart (a disease caused by Synchytrium endobioticum) and golden cyst potato nematode (Globodera rostochiensis), which parasitize the roots of the host plant, cause significant damage to potato crop. Both of these disease factors are quarantined in the Russian Federation, and each registered variety is tested for resistance to their most common races and pathotypes. The main method of opposing such diseases is by the development of resistant varieties. An important step in this process is the selection of resistant genotypes from the population and the estimation of the resistance of hybrids obtained by crosses during the breeding process. Conducting a permanent phenotypic evaluation is associated with difficulties, for example, it is not always possible to work with pathogens, and phenotypic evaluation is very costly and time consuming. However, the use of DNA markers linked to resistance genes can significantly speed up and reduce the cost of the breeding process. The aim of the study was to screen the GenAgro potato collection of ICG SB RAS using known diagnostic PCR markers linked to golden potato cyst nematode and wart resistance. Genotyping was carried out on 73 potato samples using three DNA markers 57R, CP113, Gro1-4 associated with nematode resistance and one marker, NL25, associated with wart resistance. The genotyping data were compared with the data on the resistance of the collection samples. Only the 57R marker had a high level of correlation (Spearman R = 0.722008, p = 0.000000, p < 0.05) between resistance and the presence of a diagnostic fragment. The diagnostic efficiency of the 57R marker was 86.11 %. This marker can be successfully used for screening a collection, searching for resistant genotypes and marker-assisted selection. The other markers showed a low correlation between the presence of the DNA marker and resistance. The diagnostic efficiency of the CP113 marker was only 44.44 %. Spearman’s correlation coefficient (Spearman R = –0.109218, p = 0.361104, p < 0.05) did not show significant correlation between resistance and the DNA marker. The diagnostic efficiency of the NL25 marker was 61.11 %. No significant correlation was found between the NL25 marker and resistance (Spearman R = –0.017946, p = 0.881061, p < 0.05). The use of these markers for the search for resistant samples is not advisable.