Vavilovskii Zhurnal Genetiki i Selektsii最新文献

Throughout history, humans have been attempting to develop the ornamental features of domestic animals in addition to their productive qualities. Many chicken breeds have developed tufts of elongated feathers that jut out from the sides and bottom of the beak, leading to the phenotype known as muffs and beard. It is an incomplete autosomal dominant phenotype determined by the Mb locus localised on chromosome GGA27. This project aimed to analyse the genetic diversity of chicken breeds using full genomic genotyping with the Chicken 60K BeadChip. A total of 53,313 Single Nucleotide Polymorphisms were analysed. DNA was obtained from breeds with the muffs and beard as a marker phenotype: Faverolles (n = 20), Ukrainian Muffed (n = 18), Orloff (n = 20), Novopavlov White (n = 20), and Novopavlov Coloured (n = 15). The Russian White (n = 20) was selected as an alternative breed without the muffs and beard phenotype. The chickens are owned by the Centre of Collective Use "Genetic Collection of Rare and Endangered Breeds of Chickens" (St. Petersburg region, Pushkin), and are also included in the Core Shared Research Facility (CSRF) and/or Large-Scale Research Facility (LSRF). Multidimensional scaling revealed that the Novopavlov White and the Novopavlov Coloured populations formed a separate group. The Ukrainian Muffed and the Orloff have also been combined into a separate group. Based on cluster analysis, with the cross-validation error and the most probable number of clusters K = 4 taken into account, the Orloff was singled out as a separate group. The Ukrainian Muffed exhibited a notable similarity with the Orloff under the same conditions. At K = 5, the populations of the Novopavlov White and the Novopavlov Coloured diverged. Only at K = 6, a distinct and separate cluster was formed by the Ukrainian Muffed. The Russian White had the greatest number of short (1-2 Mb) homozygous regions. If the HOXB8 gene is located between 3.402 and 3.404 Mb on chromosome GGA27, homozygous regions are rarely found in the chickens with the muffs and beard phenotype. Scanning the chicken genome with the Chicken 60K BeadChip provided enough information about the genetic diversity of the chicken breeds for the peculiarities of the development of the ornamental muffs and beard phenotypes in them to be understood. For example, Phoenix bantams, whose tail feathers grow throughout their lives, require greater consideration of husbandry conditions.

Polyamines and indole are small regulatory molecules that are involved in the adaptation to stress in bacteria, including the regulation of gene expression. Genes, the translation of which is under the regulatory effects of polyamines, form the polyamine modulon. Previously, we showed that polyamines upregulated the transcription of genes encoding the ribosome hibernation factors RMF, RaiA, SRA, EttA and RsfS in Escherichia coli. At the same time, indole affected the expression at the transcriptional level of only the raiA and rmf genes. Ribosome hibernation factors reversibly inhibit translation under stress conditions, including exposure to antibiotics, to avoid resource waste and to conserve ribosomes for a quick restoration of their functions when favorable conditions occur. In this work, we have studied the influence of indole on the expression of the raiA and rmf genes at the translational level and regulatory effects of the polyamines putrescine, cadaverine and spermidine on the translation of the rmf, raiA, sra, ettA and rsfS genes. We have analyzed the mRNA primary structures of the studied genes and the predicted mRNA secondary structures obtained by using the RNAfold program for the availability of polyamine modulon features. We have found that all of the studied genes contain specific features typical of the polyamine modulon. Furthermore, to investigate the influence of polyamines and indole on the translation of the studied genes, we have constructed the translational reporter lacZ-fusions by using the pRS552/λRS45 system. According to the results obtained, polyamines upregulated the expression of the rmf, raiA and sra genes, the highest expression of which was observed at the stationary phase, but did not affect the translation of the ettA and rsfS genes, the highest expression of which took place during the exponential phase. The stimulatory effects were polyamine-specific and observed at the stationary phase, when bacteria are under multiple stresses. In addition, the data obtained demonstrated that indole significantly inhibited translation of the raiA and rmf genes, despite the stimulatory effect on their transcrip- tion. This can suggest the activity of a posttranscriptional regulatory mechanism of indole on gene expression.

Lipin-1 is a member of the evolutionarily conserved family of proteins and is expressed predominantly in adipose tissue and skeletal muscle. On the one hand, lipin-1 is an enzyme that catalyzes the dephosphorylation of phosphatidic acid to diacylglycerol (DAG) and thus participates in the metabolic pathways of biosynthesis of storage lipids in the cell, membrane phospholipids, and intracellular signaling molecules. On the other hand, lipin-1 is able to be transported from the cytoplasm to the nucleus and is a coactivator of lipid metabolism gene transcription. It was shown, using the analysis of single nucleotide polymorphism (SNP) associations, that the lipin-1 coding gene (LPIN1) is a promising candidate gene for milk production traits in Holstein and Brown Swiss cows. However, it is unclear how much of its effect depends on the breed. The Yaroslavl dairy cattle breed was created in the 18-19 centuries in Russia by breeding northern Great Russian cattle, which were short and poor productive, but well adapted to local climatic conditions and bad food base. It was shown by whole genome genotyping and sequencing that the Yaroslavl breed has unique genetics compared to Russian and other cattle breeds. The aim of the study was to assess the frequency of alleles and genotypes of three SNPs in the LPIN1 gene and to study the association of these SNPs with milk production traits in Yaroslavl cows. Blood samples from 142 cows of the Yaroslavl breed were obtained from two farms in the Yaroslavl region. Genotyping of SNPs was carried out by polymerase chain reaction-restriction fragment length polymorphism method. Associations of SNPs with 305-day milk yield, fat yield, fat percentages, protein yield, and protein percentages were studied from the first to the fourth lactation. Statistical tests were carried out using a mixed linear model, taking into account the relationship between individuals. We identified three SNPs - rs110871255, rs207681322 and rs109039955 with a frequency of a rare allele of 0.042-0.261 in Yaroslavl cows. SNP rs110871255 was associated with fat yield during the third and fourth lactations. SNP rs207681322 was associated with milk yield for the second, third and fourth lactations, as well as protein yield for the third lactation. Thus, we identified significant associations of SNPs rs207681322 and rs110871255 in the LPIN1 gene with a number of milk production traits during several lactations in Yaroslavl cows.

Commercial panels of microsatellite (STR) loci are intended for DNA analysis of the domestic dog (Canis lupus familiaris) and, therefore, when genotyping the Grey wolf (Canis lupus lupus), most markers reveal significant deviations from the Hardy-Weinberg equilibrium and have a low informative value, which complicates their use in a forensic examination. The aim of this study was to select STR markers that equally effectively reflect population polymorphism in the wolf and the dog, and to create a universal panel for the identification of individuals in forensic science. Based on the study of polymorphisms of 34 STR loci, a CPlex panel of 15 autosomal loci and two sex loci was developed, which is equally suitable for identifying wolfs and dogs. Analysis of molecular variance (AMOVA) between samples revealed significant differentiation values (FST = 0.0828, p < 0.05), which allows the panel to be used for differentiating between wolf and dog samples. For the first time in the forensic examination of objects of animal origin in the Republic of Belarus, population subdivision coefficients (θ-values) were calculated for each of the 15 STR loci of the test system being reported. It was shown that the values of the genotype frequency, when averaged over all studied animals without and with considering the θ-value, differ by three orders of magnitude (3.39 · 10-17 and 4.71 · 10-14, respectively). The use of population subdivision coefficients will provide the researcher with the most relevant results of an expert identification study. The test system was validated in accordance with the protocol of the Scientific Working Group on DNA Analysis Methods. A computational tool was developed to automate the analysis of genetic data on the wolf and dog in the forensic examination; two guides were approved for practicing forensic experts. This methodology is being successfully used in expert practice in investigating cases of illegal hunting, animal abuse and other offenses in the Republic of Belarus.

Study of RNA-protein interactions and identification of RNA targets are among the key aspects of understanding RNA biology. Currently, various methods are available to investigate these interactions with, RNA immunoprecipitation (RIP) being the most common. The search for RNA targets has largely been conducted using antibodies to an endogenous protein or to GFP-tag directly. Having to be dependent on the expression level of the target protein and having to spend time selecting highly specific antibodies make immunoprecipitation complicated. Expression of the GFP-fused protein can lead to cytotoxicity and, consequently, to improper recognition or degradation of the chimeric protein. Over the past few years, multifunctional tags have been developed. SNAP-tag and HaloTag allow the target protein to be studied from different perspectives. Labeling of the fusion protein with custom-made fluorescent dyes makes it possible to study protein expression and to localize it in the cell or the whole organism. A high-affinity substrate has been created to allow covalent binding by chimeric proteins, minimizing protein loss during protein isolation. In this paper, a HaloTag-based method, which we called Halo-RPD (HaloTag RNA PullDown), is presented. The proposed protocol uses plants with stable fusion protein expression and Magne® HaloTag® magnetic beads to capture RNA-protein complexes directly from the cytoplasmic lysate of transgenic Arabidopsis thaliana plants. The key stages described in the paper are as follows: (1) preparation of the magnetic beads; (2) tissue homogenization and collection of control samples; (3) precipitation and wash of RNA-protein complexes; (4) evaluation of protein binding efficiency; (5) RNA isolation; (6) analysis of the RNA obtained. Recommendations for better NGS assay designs are provided.

Improving the nutritional value of grain sorghum, a drought- and heat-tolerant grain crop, is an important task in the context of global warming. One of the reasons for the low nutritional value of sorghum grain is the resistance of its storage proteins (kafirins) to proteolytic digestion, which is due, among other things, to the structural organization of protein bodies, in which γ-kafirin, the most resistant to proteases, is located on the periphery, encapsulating more easily digested α-kafirins. The introduction of genetic constructs capable of inducing RNA silencing of the γ-kafirin (gKAF1) gene opens up prospects for solving this problem. Using Agrobacterium-mediated genetic transformation of immature embryos of the grain sorghum cv. Avans we have obtained a mutant with improved digestibility of endosperm proteins (up to 92 %) carrying a genetic construct for RNA silencing of the gKAF1 gene. The goal of this work was to study the stability of inheritance of the introduced genetic construct in T2-T4 generations, to identify the number of its copies, as well as to trace the manifestation of agronomically valuable traits in the offspring of the mutant. The mutant lines were grown in experimental plots in three randomized blocks. The studied lines were characterized by improved digestibility of kafirins, a modified type of endosperm, completely or partially devoid of the vitreous layer, an increased percentage of lysine (by 75 %), reduced plant height, peduncle length, 1000-grains weight, and grain yield from the panicle. In T2, a line with monogenic control of GA resistance was selected. qPCR analysis showed that in different T3 and T4 plants, the genetic construct was present in 2-4 copies. In T3, a line with a high digestibility of endosperm proteins (81 %) and a minimal decrease in agronomically valuable traits (by 5-7 %) was selected.

The most important part of the plant antioxidant system is the ascorbate-glutathione cycle (AGC), the activity of which is observed upon exposure to a range of stressors, including lack of O2, and oxidative stress occurring immediately after the restoration of oxygen access, hereafter termed reaeration or post-anoxia. The operation of the AGC (enzymes and low-molecular components) in wheat (Triticum aestivum, cv. Leningradka, non-resistant to hypoxia) and rice (Oryza sativa, cv. Liman, resistant) seedlings after 24 h anoxia and 1 h or 24 h reaeration was studied. Significant accumulation of oxidized forms of ascorbate and glutathione was revealed in the non-resistant plant (wheat) after 24 h of anoxia and reaeration, indicating the development of oxidative stress. In the resistant plant (rice), reduced forms of these antioxidants prevailed both in normoxia and under stress, which may indicate their intensive reduction. In wheat, the activities of ascorbate peroxidase and dehydroascorbate reductase in shoots, and monodehydroascorbate reductase and glutathione reductase in roots decreased under anoxia and reaeration. The activity of antioxidant enzymes was maintained in rice under lack of oxygen (ascorbate peroxidase, glutathione reductase) and increased during post-anoxia (AGC reductases). Anoxia stimulated accumulation of mRNA of the organellar ascorbate peroxidase genes OsAPX3, OsAPX5 in shoots, and OsAPX3-5 and OsAPX7 in roots. At post-anoxia, the contribution of the OsAPX1 and OsAPX2 genes encoding the cytosolic forms of the enzyme increased in the whole plant, and so did that of the OsAPX8 gene for the plastid form of the enzyme. The accumulation of mRNA of the genes OsMDAR2 and OsMDAR4 encoding peroxisomal and cytosolic monodehydroascorbate reductase as well as the OsGR2 and OsGR3 for cytosolic and organellar glutathione reductase was activated during reaeration in shoots and roots. In most cases, O2 deficiency activated the genes encoding the peroxisomal, plastid, and mitochondrial forms of the enzymes, and upon reaeration, an enhanced activity of the genes encoding the cytoplasmic forms was observed. Taken together, the inactivation of AGC enzymes was revealed in wheat seedlings during anoxia and subsequent reaeration, which disrupted the effective operation of the cycle and triggered the accumulation of oxidized forms of ascorbate and glutathione. In rice, anoxia led to the maintenance of the activity of AGC enzymes, and reaeration stimulated it, including at the level of gene expression, which ensured the effective operation of AGC.

Gravitropism is an adaptive reaction of plants associated with the ability of various plant organs to be located and to grow in a certain direction relative to the gravity vector, while usually the asymmetric distribution of the phytohormone auxin is a necessary condition for the gravitropical bending of plant organs. Earlier, we described significant morphological changes in phloem fibers with a thickened cell wall located on different sides of the stem in the area of the gravitropic curvature. The present study is the first work devoted to the identification of genes encoding auxin transporters in cells at different stages of development and during gravity response. In this study, the flax genes encoding the AUX1/LAX, PIN-FORMED, PIN-LIKES, and ABCB auxin transporters were identified. A comparative analysis of the expression of these genes in flax phloem fibers at different stages of development revealed increased expression of some of these genes at the stage of intrusive growth (LusLAX2 (A, B), LuxPIN1-D, LusPILS7 (C, D)), at the early stage of tertiary cell wall formation (LusAUX1 (A, D), LusABCB1 (A, B), LusABCB15-A, LusPIN1 (A, B), LusPIN4-A, and LusPIN5-A), and at the late stage of tertiary cell wall development (LusLAX3 (A, B)). It was shown that in the course of gravitropism, the expression of many genes, including those responsible for the influx of auxin in cells (LusAUX1-D), in the studied families increased. Differential expression of auxin transporter genes was revealed during gravity response in fibers located on different sides of the stem (upper (PUL) and lower (OPP)). The difference was observed due to the expression of genes, the products of which are responsible for auxin intracellular transport (LusPILS3, LusPILS7-A) and its efflux (LusABCB15-B, LusABCB19-B). It was noted that the increased expression of PIN genes and ABCB genes was more typical of fibers on the opposite side. The results obtained allow us to make an assumption about the presence of differential auxin content in the fibers of different sides of gravistimulated flax plants, which may be determined by an uneven outflow of auxin. This study gives an idea of auxin carriers in flax and lays the foundation for further studies of their functions in the development of phloem fiber and in gravity response.

The breeding of remontant rose cultivars that are resistant to diseases and adverse conditions, with high decorative value and continuous flowering is the most important task during work with the gene pool of garden roses. Currently, intercultivar hybridization within a single garden group has largely outlived its usefulness. It is necessary to breed for highly decorative forms or cultivars that have outstanding resistance, morphological characters and patterns of seasonal rhythms, and use these plants as parental forms in further breeding. This study represents a comparative analysis of rose cultivars from two garden groups, Grandiflora (Gurzuf, Lezginka, Korallovy Syurpriz, Queen Elizabeth, Komsomolsky Ogonyok, Love) and Rosa Kordesii (Letniye Zvyozdy, Dortmund, Gutsulochka). These cultivars proved themselves during many years of testing in harsh climatic conditions. The objectives of the study were to determine the genetic relationship within the groups and to assign phenotypically different cultivars to one or another garden group. The analysis was carried out by morphological, phenological and ISSR markers. According to the phenological observations on the Grandiflora cultivars, Komsomolsky Ogonyok had later budding and flowering stages. Polymorphic data generated from the ISSR markers showed that this cultivar was the most distant from the others and formed a separate cluster on the dendrogram. A comparison of the morphological characters (flower diameter, number of petals, peduncle length, bush height) showed a significant difference ( p < 0.05) between Komsomolsky Ogonyok and the other Grandiflora cultivars. A dendrogram based on a molecular analysis showed a lack of close relationships between Komsomolsky Ogonyok and the Kordesii group, which formed a separate cluster. A pairwise comparison of the morphological characters in Komsomolsky Ogonyok with the Kordesii group revealed a significant ( p <0.05) difference in three of the four characters studied. The exceptions were flower diameter when comparing with Dortmund and Letniye Zvyozdy and peduncle length when comparing with Gutsulochka. Although Komsomolsky Ogonyok has a pattern of seasonal development similar to Dortmund in the Kordesii group, the molecular analysis did not assign the former to this group of roses. The cultivars that have valuable characters that no average rose does and that are phenotypically different from such roses represent the most valuable breeding material.

Bacterial stress adaptive response is formed due to changes in the cell gene expression profile in response to alterations in environmental conditions through the functioning of regulatory networks. The mutual influence of network signaling molecules represented by cells' natural metabolites, including indole and second messengers (p) ppGpp and cAMP, is hitherto not well understood, being the aim of this study. E. coli parent strain BW25141 ((p) ppGpp+) and deletion knockout BW25141ΔrelAΔspoT which is unable to synthesize (p)ppGpp ((p)ppGpp0) were cultivated in M9 medium supplemented with different glucose concentrations (5.6 and 22.2 mM) in the presence of tryptophan as a substrate for indole synthesis and in its absence. The glucose content was determined with the glucose oxidase method; the indole content, by means of HPLC; and the cAMP concentration, by ELISA. The onset of an increase in initially low intracellular cAMP content coincided with the depletion of glucose in the medium. Maximum cAMP accumulation in the cells was proportional to the concentration of initially added glucose. At the same time, the (p) ppGpp0 mutant showed a decrease in maximum cAMP levels compared to the (p)ppGpp+ parent, which was the most pronounced in the medium with 22.2 mM glucose. So, (p)ppGpp was able to positively regulate cAMP formation. The promoter of the tryptophanase operon responsible for indole biosynthesis is known to be under the positive control of catabolic repression. Therefore, in the cells of the (p)ppGpp+ strain grown in the tryptophan-free medium that were characterized by a low rate of spontaneous indole formation, its synthesis significantly increased in response to the rising cAMP level just after glucose depletion. However, this was not observed in the (p)ppGpp0 mutant cells with reduced cAMP accumulation. When tryptophan was added to the medium, both of these strains demonstrated high indole production, which was accompanied by a decrease in cAMP accumulation compared to the tryptophan-free control. Thus, under glucose depletion, (p)ppGpp can positively regulate the accumulation of both cAMP and indole, while the latter, in its turn, has a negative effect on cAMP formation.