Vavilov Journal of Genetics and Breeding
Peer-rewieved journal "Vavilov Journal of Genetics and Breeding"
Institute of Cytology and Genetics of Siberian Branch of the RAS
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Study of the progeny of sorghum mutants obtained using the CRISPR/Cas9 genetic construct directed at inducing mutations in the α-kafirin k1C5 gene
Site-directed mutagenesis using genetic constructs carrying the CRISPR/Cas system is an effective technology that is actively used to solve a variety of problems in plant genetics and breeding. One of these problems is to improve the nutritional value of grain sorghum, a high-yielding heat- and drought-tolerant cereal crop that is becoming increasingly important in the conditions of climate aridization. The main reason for the relatively low nutritional value of sorghum grain is the resistance of its storage proteins, kafirins, to proteolytic digestion. We have previously obtained mutants with improved kafirin in vitro digestibility using the CRISPR/Cas technology in grain sorghum variety Avance. The nucleotide sequence of one of the genes (k1C5) of the gene family encoding the signal polypeptide of 22 kDa α-kafirin was used as a target. The aim of this study was to investigate the manifestation of the main agronomically-important traits in the progeny of these mutants and inheritance of high in vitro protein digestibility, and also sequencing nucleotide sequences encoding the 22 kDa α-kafirin signal polypeptide in a number of plants from the T0 generation and their T1 progeny. It was revealed that four of the six studied T0 plants, as well as their progeny, had the same mutation: a T→C substitution in the 23rd position of the nucleotide sequence of the k1C5 gene encoding the signal polypeptide, which led to a substitution of the coding triplet CTC→CCC (Leu→Pro). This mutation is located off-target, 3’ from the PAM sequence. It is suggested that this mutation may have arisen as a result of Cas9 nuclease errors caused by the presence of multiple PAM sequences located close to each other. It was found that the progeny of two of the three studied mutants (T2 and T3 families), grown in the experimental field conditions, differed from the original variety by a reduced plant height (by 12.4–15.5 %). The peduncle length, 1,000-grain mass, and grain mass per panicle did not differ from the original variety, with the exception of the progeny of the 2C-1.2.5b mutant, which had a reduced grain yield per panicle. Unlike the original variety, plants from the T2 and T3 generations had kernels with a modified type of endosperm (completely floury, or floury with inclusions of vitreous endosperm, or with a thin vitreous layer). The level of grain protein digestibility in the progeny of mutants 2C-2.1.1 #13 and 2C-1.2.5a #14 varied from 77 to 84 %, significantly exceeding the original variety (63.4 ± 2.3 %, p < 0.05). The level of protein digestibility from kernels with modified endosperm was higher than that of kernels with normal vitreous endosperm (84–93 %, p < 0.05). The reasons for the variation in endosperm texture in the progeny of the mutants and its relationship with the high digestibility of kafirins are discussed.
Silencing of the Nicotiana benthamiana phytoendesaturase gene by root treatment of exogenous dsRNA
RNA interference (RNAi) is a powerful tool for gene silencing. It has recently been used to design promising plant protection strategies against pests such as viruses, insects, etc. This generally requires modifying the plant genome to achieve in planta synthesis of the double-stranded RNA (dsRNA), which guides the cellular RNA interference machinery to silence the genes of interest. However, given Russian legislation, the approach in which dsRNA is synthesized by the plant itself remains unavailable for crop protection. The use of exogenously produced dsRNA appears to be a promising alternative, allowing researchers to avoid genetic modification of plants, making it possible to implement potential results in agriculture. Furthermore, exogenous dsRNAs are superior to chemical pesticides (fungicides, insecticides, etc.), which are widely used to control various plant diseases. The dsRNA acts through sequence-specific nucleic acid interactions, making it extremely selective and unlikely to harm off-target organisms. Thus, it seems promising to utilize RNAi technology for agricultural plant protection. In this case, questions arise regarding how to produce the required amounts of pathogen-specific exogenous dsRNA, and which delivery method will be optimal for providing sufficient protection. This work aims to utilize exogenous dsRNA to silence the Nicotiana benthamiana phytoene desaturase gene. Phytoene desaturase is a convenient model gene in gene silencing experiments, as its knockdown results in a distinct phenotypic manifestation, namely, leaf bleaching. The dsRNA synthesis for this work was performed in vivo in Escherichia coli cells, and the chosen delivery method was root treatment through watering, both techniques being as simple and accessible as possible. It is surmised that the proposed approach could be adapted for broader use of RNAi technologies in agricultural crop protection.
Effect of RNAi-mediated silencing of the TaAOS2 gene on phytohormone accumulation, growth and productivity in bread wheat
Reverse genetics methods are actively used in plant biology to study the functions of specific genes responsible for the adaptation of plants to various environmental stresses. The present study describes the production and primary characterization of transgenic bread wheat with silenced expression of allen oxide synthase (AOS). AOS is a key enzyme involved in the initial step of biosynthesis of stress-related phytohormones known as jasmonates. To induce silencing of AOS in wheat, we designed the RNA interference (RNAi) vector containing an inverted repeat region of the TaAOS2 gene cloned from genome DNA of cv. Chinese Spring. With the help of biolistic-mediated transformation, a number of transgenic Chinese Spring plants have been produced. Real-Time PCR analysis confirmed the suppression of target gene expression, since transgenic dsRNAi lines accumulated only 21–44 % mRNA of TaAOS2 after leaf wounding compared to the wound-induced level in non-transgenic control. Gas chromatography–mass spectrometry revealed that the silencing of TaAOS2 substantially reduced the accumulation of jasmonic acid (JA) and jasmonoyl-isoleucine conjugate (JA-Ile), while the production of other phytohormones, such as abscisic acid and salicylic acid, was not affected. TaAOS2-silenced lines were characterized by shorter leaves at the juvenile stage, demonstrated a tendency towards reduced plant height and decreased grain weight, while the average flowering time and plant fertility (number of seeds per spike) were not affected. The obtained transgenic lines in combination with AOS-overexpressing lines can be used for further detailed analysis of the adaptive responses controlled by the jasmonate hormonal system.
Study of the influence of introgression from chromosome 2 of the At subgenome of cotton Gossypium barbadense L. during backcrossing with the original lines of G. hirsutum L
The creation of chromosome substitution lines containing one pair of chromosomes from a related species is one method for introgression of alien genetic material. The frequency of substitutions in different chromosomes of the genome varies due to the selective transmission of alien chromosomes through the gametes of hybrids. The use of monosomic lines with identified univalent chromosomes and molecular genetic SSR markers at the seedling stage allowed rapid screening of the identity of the alien chromosome in backcross hybrids, significantly accelerating and facilitating the backcrossing process for the creation of new chromosome substitution cotton lines. As a result of studying the process of transmission of chromosome 2 of the At subgenome of the cotton plant G. barbadense L. during backcrossing of four original monosomic lines of G. hirsutum L. with monosomic backcross hybrids with substitution of chromosome 2 of the At subgenome, the following specific consequences of the introgression of this chromosome were revealed: decreased crossability, setting and germination of hybrid seeds; differences in the frequency and nature of transmission of chromosome 2 of the At subgenome of the cotton plant G. barbadensе; regularity of chromosome behavior in meiosis; a high meiotic index; a significant decrease in pollen fertility in backcross monosomic hybrids BC1F1; specific morphobiological characteristics of monosomic backcrossed plants, such as delayed development of vegetative and generative organs; dwarfism; reduced foliage; and poor budding and flowering during the first year of vegetation. All of these factors negatively impact the study and backcrossing of monosomic hybrids and significantly complicate and delay the creation of chromosome-substituted forms concerning chromosome 2 of the At subgenome of cotton, G. barbadense. These specific changes likely occurred as a result of hybrid genome reorganization and introgression of alien chromatin. Furthermore, the effectiveness of using molecular genetic microsatellite (SSR) markers to monitor backcrossing processes and eliminate genetic material from the Pima 3-79 donor line of G. barbadense for the selection of genotypes with alien chromosome substitutions has been demonstrated.
Intrapopulation changes in Puccinia hordei induced by two-component fungicides from different chemical classes
Fungicide resistance is a global problem that reduces the effectiveness and duration of action of these compounds due to changes in the racial composition and virulence of phytopathogen populations. Currently, resistance to 100 active substances has been registered in more than 230 fungal plant pathogens. Leaf rust of barley (Puccinia hordei Otth.) is one of the most widespread and harmful pathogens in the barley pathocomplex; it is recorded in southern Russia every year. There are very few studies on the effect of fungicides on the characteristics of rust fungi populations, and none have been carried out on P. hordei in Russia. This research aimed to analyze the effect of fungicides belonging to the chemical classes of triazoles and strobilurins on intrapopulation changes in P. hordei in terms of pathogenicity (virulence and aggressiveness) under the conditions of the North Caucasus region of Russia. Two-component fungicides approved for use in the Russian Federation were selected for the study: Delaro, SC; Amistar Extra, SC; Amistar Gold, SC. Plants were treated using several application rates: 50, 100, 150 and 200 % (the recommended application rate was determined to be 100 %). Treatment of winter barley plants with fungicides with different application rates revealed intrapopulation changes in the virulence structure of P. hordei. In all treatment variants, the frequency of isolates virulent to the Rph4, Rph5, Rph6+2, Rph12 genes decreased with increasing fungicide application rate and the frequency of isolates virulent to Rph14 increased. No isolates virulent to Rph7 were found in either the original population or the experimental variants. The average virulence of the fungal populations treated with the fungicides in all experimental variants was lower compared to the original population (no treatment (48.5 %)) and depending on the application rate varied from 33.8 % (Amistar Gold, 50 %) to 28.5 % (Amistar Gold, 200 %). Under the influence of the increased application rates of the fungicides, an increase in the duration of the latent period was observed: from 168 h (original population) to 216 h (Delaro, Amistar Gold, 200 %). A decrease in sporulation ability (spore mass per pustule ranged from 0.013 mg (original population) to 0.002 mg (Delaro, Amistar Gold, 200 %)) and in the viability of P. hordei (from 100 % for the original population to 22.5 % in Amistar Gold, 200 % treatment) was found under the action of the fungicides. Thus, a fungicide-treated P. hordei population is characterized by intrapopulation changes in aggressiveness and virulence, which can significantly increase barley yield losses due to a decrease in the effectiveness of chemical protection, as well as an increase in the harmfulness of the pathogen.
Effect of the biopesticide Novoсhizol on the expression of defense genes during wheat infection with stem rust Puccinia graminis f. sp. tritici
Stem rust, caused by the fungus Puccinia graminis f. sp. tritici (Pgt), is a harmful disease affecting grain crops. The traditional way to combat this and other infectious plant diseases is to use chemical pesticides. Biopesticides, as well as plant disease resistance inducers – in particular those based on chitosan, a derivative of chitin – are increasingly being considered as an effective and safe alternative. Recently, a globular form of chitosan, Novochizol, has been developed, which has a number of advantages and has shown its effectiveness in preliminary field and laboratory experiments. However, there are no works devoted to the effect of this preparation on the expression of defense genes. Therefore, the aim of this work was to search for genes involved in the response of common wheat (Triticum aestivum L.) to stem rust infection and to evaluate the effect of Novochizol treatment on their transcription during the infection process. The wheat line ISr6-Ra with the stem rust resistance gene Sr6 and two Pgt isolates – an avirulent one, Avr6, and a virulent one, vr6 – were used as a model, allowing us to compare the effects of Novochizol depending on the genetic compatibility in the plant−pathogen pathosystem. To analyze the transcription level of defense genes, leaf material was collected at different time points from 3 to 144 h after inoculation of plants with the pathogen. Quantitative PCR analysis showed an increase in the transcription levels of the CERK1, PR3, PR4, PR5, PR6 and PR9 genes in plants treated with Novochizol and infected with various Pgt isolates compared to untreated infected plants. Pgt isolate Avr6 induced the highest expression of some defense genes (primarily CERK1), which is consistent with the phytopathology data showing the maximum degree of resistance (IT1) to stem rust in Novochizol-treated plants with a combination of Sr6–Avr6 genes. The data obtained confirm that one of the optimal strategies for increasing the resistance of grain crops to fungal pathogens is a combination of selection for specific resistance genes with the use of biological control agents.
Differential expression profile of DREB2 subfamily transcription factor genes in the dynamics of salt stress and post-stress recovery in tomato plants
In response to stress, epigenetic modifications occur in the plant genome, which together form a stress memory that can be inherited and increases the efficiency of the plant's defense response to repeated stress events. Genes whose expression becomes the target of epigenetic modifications serve as biomarkers of stress memory. Their characteristic features are considered to be an expression profile that differs between responses to primary and repeated stress events, as well as long-term retention of changes after the stress is canceled. Tomato (Solanum lycopersicum L.) is an important vegetable crop whose yield decreases with soil salinity. Genes induced by salt stress include genes encoding transcription factors of the DREB2 (DEHYDRATION-RESPONSIVE ELEMENT-BINDING PROTEIN 2) subfamily. In this work, we evaluated the SlDREB2 genes of tomato as possible marker genes of salt stress memory. The expression of the genes SlDREB16, 20, 22, 24, 43, 44 and 46 was determined in the leaves of two plant varieties (Gnom, Otradnyi) with different degrees of salt tolerance in response to 24 h of NaCl exposure and in the dynamics of a long-term (14 days) post-stress recovery period. Significant genotype-specific fluctuations in the levels of gene transcripts were revealed both in the control and in the stressed plants. It was shown that during the long-term memory phase, gene expression returns to the control values either temporarily (SlDREB24, 44 and 46 in the moderately resistant Gnom variety after 7 days; after 14 days, the expression changed again) or slowly (SlDREB16 and 43 in the highly resistant Otradnyi variety after 14 days of recovery). Only two genes (SlDREB22 and 46) showed a similar between varieties pattern of expression fluctuations in the dynamics of stress and recovery, and the SlDREB20 gene was not expressed in either the control or the experiment. The data obtained suggest that the SlDREB2 subfamily genes (except SlDREB20) are involved in the response of S. lycopersicum to salt stress in a genotype-specific manner and can serve as markers of stress memory linked to the epigenetic regulation of tomato adaptation to salt stress. The SlDREB16, 28, 43 and 44 genes may contribute to the determination of differences in the mechanism of regulation of plant response to salt stress between salt-tolerant genotypes of S. lycopersicum. The obtained results can form the basis for further studies of the role of SlDREB2 genes in the epigenetic regulation of tomato plant adaptation to salt stress, which can be used in breeding salt-tolerant varieties.
SmartCrop: knowledge base of molecular genetic mechanisms of rice and wheat adaptation to stress factors
The study of molecular genetic mechanisms of plant responses to specific growth conditions and stress factors is a central focus of scientific research aimed at developing new valuable crop varieties, particularly rice and wheat. These factors include abiotic stresses (high or low temperatures, drought, salinity, soil metal contamination), biotic stresses (pathogens, pests), as well as plant responses to regulatory factors (fertilizers, hormones, elicitors, and other compounds). Modern research in plant genetics is based on the understanding that the formation of any phenotypic characteristics (molecular genetic, biochemical, physiological, morphological, etc.) is controlled by gene networks – groups of coordinately functioning genes interacting through their products (RNA, proteins, and metabolites). Previously, we developed the ANDSystem intelligent technology designed to extract knowledge from scientific publication texts for the reconstruction of gene networks in biology and biomedicine. In this work, using an adapted version of ANDSystem for plants, we created the SmartCrop knowledge base designed to address challenges related to studying molecular genetic mechanisms of genotype-phenotype-environment interactions for agriculturally valuable rice and wheat crops. SmartCrop is designed to assist researchers in solving tasks such as interpreting omics technology results (establishing connections between gene sets and biological processes, phenotypic traits, etc.); reconstructing gene networks describing relationships between molecular genetic objects and concepts in breeding, phenomics, seed production, phytopathology, diagnostics, protective agents, etc.; identifying regulatory and signaling pathways of plant responses to specific growth conditions and biotic and abiotic stresses; predicting candidate genes for genotyping; searching for markers for marker-assisted selection; and identifying potential targets for substances (including external factors) affecting plants to ensure timely and uniform germination, better vegetative growth, efficient nutrient uptake, and improved stress resistance.
Validation of markers for resistance to Pyrenophora teres f. teres loci on barley chromosomes 3H, 4H, and 6H in the polygenic inheritance of the trait
The causal agent of net blotch Pyrenophora teres Drechs. f. teres (Ptt) is a dangerous pathogen of barley. The development of genetic protection against this disease is a necessary link in resource-saving and environmentally friendly barley cultivation technologies. Effective QTL markers controlling both qualitative and quantitative resistance are required for breeding for resistance to Ptt. As a result of GWAS, we identified barley accessions of different origins, the SNP haplotypes of which were associated with resistance loci simultaneously on different barley chromosomes (VIR catalogue numbers: k-5900, k-8829, k-8877, k-14936, k-30341 and k-18552). The aim of the study was to validate SNP markers (MM) of Ptt resistance loci on chromosomes 3H, 4H and 6H in F2 from crossing six resistant accessions with the susceptible variety Tatum. The observed segregation for resistance in all crossing combinations confirmed the presence of several genetic determinants of resistance in the studied accessions. To study the polymorphism of the parents from the crosses and the correspondence between the phenotypes to the presence/absence of the markers in the segregating populations, primers with a specific 3’-end, CAPS markers, and KASP markers were developed. A significant association (p < 0.05) between the presence of the CAPS marker JHI-Hv50k-2016-391380 HindIII on chromosome 6H and the phenotype of resistance to Ptt in F2 plants was revealed in crosses between the susceptible cultivar Tatum and accessions k-5900, k-8829, k-8877 and k-18552. On chromosome 4H, a significant association with the resistance phenotype in the F2 population from the cross with accession k-8877 was revealed for marker JHI-Hv50k-2016-237924, and in that from the cross with accession k-5900, for marker SCRI_RS_181886. The presence of QTL on chromosome 6H, which controls qualitative resistance in four barley accessions, masks the expression of other genes, which explains the discrepancy between the resistance phenotype and the presence of molecular markers in the segregating populations. Resistance donors and molecular markers with proven efficacy can be used in marker-assisted selection (MAS) to develop barley cultivars resistant to net blotch.
Identification of CtE1 gene nucleotide polymorphisms and development of SNP-based KASP markers in guar (Cyamopsis tetragonoloba (L.) Taub.)
Guar (Cyamopsis tetragonoloba (L.) Taub), is an important short-day legume crop, whose cultivation is limited at high latitudes due its photoperiod sensitivity, that negatively impacts flowering and maturation of this industrial oriented crop. In its close relative, soybean, the E1 gene has been highly associated with the regulation of flowering time under long-day conditions. In this study we investigated the natural diversity of the E1 homologue gene (CtE1) in a panel of 144 guar accessions. For this purpose, the CtE1 gene was amplified and sequenced using Illumina. As a result, five novel SNPs were identified in the 5’-untranslated region, coding region, and 3’-untranslated region of the CtE1 gene. One non-synonymous SNP was located in the coding region causing a conservative Arg→Lys substitution. Based on the identified SNP, five KASP markers linked to polymorphism in the target gene were developed and tested in the guar collection. No significant associations were detected between discovered SNPs and available data on variability in flowering time or vegetation period length in the cohort of 144 accessions. These findings suggest that natural variation of the CtE1 gene in the studied germplasm collection has minimal effect on flowering or maturation. The limited functional allelic diversity observed in the CtE1 gene of guar compared to the E1 gene in soybean likely reflects differences in their evolutionary histories, domestication bottlenecks, and selection pressures.
Using a wheat line with wild emmer genetic material to improve modern Triticum aestivum L. varieties by a complex of economically useful traits
Wild emmer Triticum dicoccoides samples have a high content of protein and microelements in their grain, but when crossed with common wheat varieties, undesirable properties of a wild relative (low yield, spike fragility and difficult threshing) can be transmitted to the hybrid along with valuable traits. The possibility of improving economically useful traits of modern common wheat varieties using a wheat line with wild emmer genetic material (l29), combining high cytological stability with improved nutritional value and productivity, was studied. The F4–F5 hybrids obtained as a result of crossing in the forward and reverse directions of four common spring wheat varieties with l29 were studied. A C-banding technique and genotyping with SSR markers were used to determine the introgression fragments of T. dicoccoides genetic material. Cytological stability was assessed based on the study of chromosome behavior in microsporogenesis. The grain content of macro- (K, P, Ca and Mg) and microelements (Zn, Fe, Cu and Mn) was established by atomic emission spectrometry with inductively coupled plasma; the grain quality indices were measured on an Infra LUM FT-12 analyzer. The C-banding and microsatellite analysis data indicate a high frequency of alien genetic material introgression in the genome of hybrid forms. All variants of the l29 introgression of wild emmer material (1BL, 2BS, 3B, 5B and 6AL) were identified among the progeny of eight crossing combinations. The recombinant chromosome 3B was found in all hybrid combinations. The hybrids were characterized by a high level of cytological stability (the meiotic index was 90.0–98.0 %). The effectiveness of using a wheat line with T. dicoccoides genetic material to enhance modern varieties in terms of the content of protein, gluten and mineral composition of grain without reducing productivity was shown. Secondary introgression hybrids, exceeding the initial varieties by a set of grain quality characteristics and not inferior to them in terms of basic productivity indicators, were obtained.
Flax transposons: unraveling their impact on domestication and agronomic trait variation
Flax is an important agricultural crop with multifunctional uses. Diversified breeding for oil content in seeds and fiber in stems has led to the emergence of two morphotypes – fiber flax and oilseed flax. Previously, using single nucleotide polymorphisms (SNPs), we characterized the genetic diversity of 306 flax samples from the collection of the Russian Federal Research Center for Bast Crops. However, larger structural variations, such as mobile genetic elements, also play a significant role in shaping agronomically important plant traits and can be used for further flax improvement. Here, we used the same flax collection to predict sites of new transposon insertions and to assess the role of such insertions in the formation of agronomically important traits, as well as in the process of flax domestication. We discovered 588,480 new transposon insertion sites not present in the reference flax genome (NCBI assembly ASM22429v2), the majority of which were attributed to retrotransposons of the Copia and Gypsy superfamilies, while among DNA transposons, insertion sites of the MULE-MuDR, hAT, and CMC-EnSpm superfamilies were most common. Unlike SNPs, which were significantly more numerous in oilseed flax than in fiber flax, we did not find such a substantial difference in the number of insertions of different transposon families per plant among samples of different morphotypes. Analysis of genomic regions affected by recent breeding efforts revealed a total of 61 candidate regions, of which 18 regions overlapped with QTLs associated with important agronomic traits. Interestingly, 5 regions of reduced genetic diversity in kryazhs and cultivars compared to landraces were also identified as regions of reduced diversity when using single nucleotide polymorphisms as markers. A genomewide association study (GWAS) identified 50 TE insertions associated with different phenotypic traits, with many associations confirmed by multiple models or detected in data from multiple years. Thus, transposon insertion sites are an important source of genetic diversity in flax, alongside single nucleotide polymorphisms, making them suitable for further crop improvement in breeding.
The influence of allelic variants of the Vrn-A1 gene on the duration of the vegetation period in Triticum dicoccoides
The duration of the vegetation period (DVP) is an important agronomic trait in cereal. Тhe main influence on it in wheat is exerted by Vrn genes, which determine the growth habit (spring vs. winter) and DVP. In the present study, 137 wild emmer Triticum dicoccoides (Körn. ex Aschers. et Graebn.) Schweinf. accessions were evaluated according to the growth habit trait, among which 39 spring ones were identified. The nucleotide sequences of the promoter region of the Vrn-A1 gene were established in the spring accessions by sequencing. Five allelic variants of Vrn-A1 genes previ ously found in T. dicoccoides were identified, namely Vrn-A1b.1, Vrn-A1b.2, Vrn-A1b.4, Vrn-A1d, Vrn-A1u. Three spring accessions PI355457, PI190919, PI560817 simultaneously contained two alleles of the Vrn-A1 gene: Vrn-A1d and previously undescribed functional allelic variant designated by the authors as Vrn-A1b.8. The promoter region of this allele had several deletions relative to the intact variant. One of such deletions covered 8 bp of the VRN box. In a single ex periment, under controlled greenhouse conditions, the relationship between the allelic variants of the Vrn-A1 gene and the duration of the vegetation period of the T. dicoccoides’ spring accessions was studied using the 2B-PLS (Two-Block Partial Least Squares) analysis. The correlation coefficient (r) between these traits was 0.534. The correlation coefficient between the duration of the vegetation period of wild emmer plants and the regions of origin of the studied accessions was also calculated (r = 0.478). It was shown that accessions with identical alleles of the Vrn-A1 gene and originating from the same region can differ significantly from each other in the duration of the vegetation period. The presence of phenotypic differences with the same allelic composition of the Vrn-A1 gene indicates the contribution of other hereditary factors localized in the genomes of these accessions, which determines their value as new donors of genetic resources that contribute to the expansion of the biodiversity of common and durum wheat commercial cultivars.
Frame-based mathematical models – a tool for the study of molecular genetic systems
This paper reviews existing approaches for reconstructing frame-based mathematical models of molecular genetic systems from the level of genetic synthesis to models of metabolic networks. A frame-based mathematical model is a model in which the following terms are specified: formal structure, type of mathematical model for a particular biochemical process, reactants and their roles. Typically, such models are generated automatically on the basis of description of biological processes in terms of domain-specific languages. For molecular genetic systems, these languages use constructions familiar to a wide range of biologists in the form of a list of biochemical reactions. They rely on the concepts of elementary subsystems, where complex models are assembled from small block units (“frames”). In this paper, we have shown an example with the generation of a classical repressilator model consisting of three genes that mutually inhibit each other’s synthesis. We have given it in three different versions of the graphic standard, its characteristic mathematical interpretation and variants of its numerical calculation. We have shown that even at the level of frame models it is possible to identify qualitatively new behaviour of the model through the introduction of just one gene into the model structure. This change provides a way to control the modes of behaviour of the model through changing the concentrations of reactants. The frame-based approach opens the way to generate models of cells, tissues, organs, organisms and communities through frame-based model generation tools that specify structure, roles of modelled reactants using domain-specific languages and graphical methods of model specification.
Computational prediction of the interaction network between long non-coding RNAs and microRNAs in maize based on the transcriptome of the fuzzy tassel mutant line
Long non-coding RNAs (lncRNAs) play an important role in the regulation of gene expression, including interactions with microRNAs (miRNAs), acting as molecular “sponges”. Bioinformatics methods are generally used to predict such interactions. To refine computational predictions, additional evidence based on the co-expression of miRNAs and lncRNAs can be incorporated. In the present study, we investigated potential interactions between lncRNAs and miRNAs in the maize mutant line fuzzy tassel (fzt), which is characterized by reduced expression of certain miRNAs due to a mutation in the Dicer-like1 (DCL1) gene in shoot and tassel tissues. Transcriptome assembly was performed based on RNA-seq data from maize shoot and tassel tissues of control and mutant lines, with data obtained from the NCBI SRA archive. In the shoot, 10 lncRNAs with significantly altered expression levels between control and mutant groups were identified, 9 of which were upregulated in the mutant plants. In the tassel, 34 differentially expressed lncRNAs were identified, with 20 showing increased expression in the mutant line. For lncRNAs with increased expression and miRNAs with decreased expression in the mutant line, potential interactions were predicted using the machine learning algorithm PmliPred. The IntaRNA program was used to confirm possible complementary binding for the identified miRNA–lncRNA pairs, which enabled the construction of competing endogenous RNA (ceRNA) networks. Structural analysis of these networks revealed that certain lncRNAs are capable of binding multiple miRNAs simultaneously, supporting their regulatory role as “sponges” for miRNAs. The results obtained deepen our understanding of post-transcriptional regulation in maize and open new perspectives for breeding strategies aimed at improving stress tolerance and crop productivity.
Study of insecticidal and fungicidal potential of endophytic bacteria of wheat, soybean and rapeseed by bioinformatic analysis methods
Endophytic bacteria play a key role in agricultural ecosystems, as they can affect the availability of various compounds, crop yield and growth, and provide resistance to diseases and pests. Therefore, the study of endophytes of agriculturally important crop plants is a promising task in the field of biological plant protection. Understanding the mechanisms of interaction between endophytic bacteria and plants will allow the use of these microorganisms as bioagents in the future and thus reduce dependence on chemical pesticides. In this paper, samples obtained from the leaves and/or roots of wheat, rapeseed and soybean are considered. Whole-genome sequencing of the isolates was performed. Using an analytical pipeline, the genomes of 15 strains of endophyte bacteria of cultivated plants were assembled and characterized. Their insecticidal and fungicidal potential was analyzed. Gene repertoire analysis performed with GenAPI showed a high degree of correspondence between the gene repertoires of strain BZR 585 against Alcaligenes phenolicus, BZR 762 and BZR 278 against Alcaligenes sp., BZR 588 and BZR 201P against Paenochrobactrum pullorum. All strains, with the exception of BZR 162, BZR 588 and BZR 201P, were found to contain genes encoding proteins with fungicidal activity, such as iturins, fengycins and surfactins. All strains also contained genes encoding proteins with insecticidal activity, namely GroEL, Spp1Aa1, Spp1Aa2, Vpb1Ab1, Vpb4Ca1, HldE, mycosubtilin, fengycin and bacillomycin. The obtained genomic data are confirmed by the results of previous experimental studies: high insecticidal activity of a number of strains (BZR 1159, BZR 936, BZR 920, etc.) against Galleria mellonella, Tenebrio molitor and Cydia pomonella, as well as fungicidal properties against Fusarium, Alternaria, Trichothecium, was demonstrated. This shows the practical significance of the identified genetic determinants for the creation of new biocontrol agents.
A housekeeping gene search to analyze expression changes of individual genes in Macaca mulatta
Rhesus macaques (Macaca mulatta) are the most common non-human primates living in captivity. The use of rhesus macaques as model objects is determined, first of all, by their phylogenetic and physiological closeness to humans, and, as a consequence, the possibility of extrapolating the obtained results to humans. Currently, it is known that a number of biochemical changes occur under various physiological conditions, including at the transcriptomic level. The real-time polymerase chain reaction is a widely used universal method for gene expression analysis. Carrying out such studies always requires a preliminary selection of “housekeeping genes” (HKGs) – genes necessary for the implementation of basic functions in the cell and stably expressed in different cell types and under different conditions. At present, there are only two systematic studies on the search for HKGs in the rhesus macaque brain, and therefore in this work a search and systematization of HKGs for this species were carried out. As a result, two panels of promising HKGs for M. mulatta were formed: an extended panel, consisting of 56 genes, and a small panel, consisting of 8 genes: ARHGDIA, CYB5R1, NDUFA7, RRAGA, TTC1, UBA6, VPS72, and YWHAH. Both panels of potential HKGs do not have pseudogenes in macaques or humans, are characterized by stable and sufficient expression in the brain of rhesus macaques and can be used to analyze expression not only in the brain but also in peripheral blood. However, it should be noted that the data have not been experimentally verified and require verification in laboratory conditions.
Genes representing the stress-dependent component in arterial hypertension development
Hypertension is among the major risk factors of many cardiovascular diseases. Chronic psychoemotional stress is one of its key causes. Studies of molecular mechanisms of human hypertension development are conducted in animals, including artificial rat strains that model various forms of the disease. The RatDEGdb database, used in our work, includes 144 hypothalamic genes that represent the common response to single short-term restraint stress in hypertensive ISIAH and normotensive WAG rats. These rat genes were annotated with changes in the expression of the human orthologs using data on 17,458 differentially expressed genes (DEGs) from patients with hypertension compared to normotensive subjects. We applied principal component analysis to orthologous pairs of DEGs identified in hypertensive patients and rat hypothalamic DEGs upon single short-term restraint stress. Two principal components, corresponding to a linear combination of log2 expression changes associated with the similarity (PC1) and difference (PC2) in the response to psychoemotional stress in two rat strains, on the one hand, and different forms of human hypertension, on the other, explained 64 % and 33 % of the variance in differential gene expression, respectively. The significant correlation revealed between PC1 and PC2 values for the group of DEGs with stress-induced downregulation indicates that psychoemotional stress and hypertension share a common molecular mechanism. Functional annotation suggests that stress-induced downregulation of genes involved in the plasma membrane function and, simultaneously, interactions with the extracellular matrix is the most likely contribution of psychoemotional stress to the development of the hypertensive status inpatients, and the SMARCA4 transcription factor is the most likely mediator in the epigenetic modification affecting gene expression under chronic stress. Peripheral blood markers for the diagnosis of psychoemotional stress are proposed.
Overcoming the problem of heterologous proteins folding to improve the efficiency of yeast bioproducers
In the last few decades, yeasts have been successfully engineered to be an excellent microbial cell factory for producing recombinant proteins with desired properties. This was due to their cost-effective characteristics and the successful application of genomic modification technologies. In addition, yeasts have a conserved post-translational modification pathway among eukaryotic organisms, which ensures the correct folding of recombinant proteins. However, the folding machinery cannot always cope with the load caused by the overexpression of recombinant genes, leading to the accumulation of misfolded proteins, the formation of aggregates and low production. Therefore, the protein-folding capacity of the endoplasmic reticulum (ER) remains one of the main limitations for heterologous protein production in yeast host organisms. However, thanks to many years of effective research of the fundamental mechanisms of protein folding, these limitations have been largely overcome. The study of folding in both model organisms and bioproducers has allowed to identify the molecular factors and cellular mechanisms that determine how a nascent polypeptide chain acquires its three-dimensional functional structure. This knowledge has become the basis for developing new effective techniques for engineering highly productive yeast strains. In this review, we examined the main cellular mechanisms associated with protein folding, such as ER transition, chaperone binding, oxidative folding, glycosylation, protein quality control. We discuss the effectiveness of applying this knowledge to the development of various engineering techniques aimed at overcoming bottlenecks in the protein folding system. In particular, selection of optimal signal peptides, coexpression with chaperones and foldases, modification of protein quality control, inhibition of proteolysis, and other techniques have allowed to enhance the ability of yeast bioproducers to effectively secrete heterologous proteins.
Cellulases: key properties, natural sources, and industrial applications
This review focuses on cellulases, a subclass of hydrolases that catalyse the breakdown of the polysaccharide cellulose. Cellulases are of immense practical significance, given that cellulose-containing materials are utilised across a multitude of industrial sectors. An overview of the fundamental properties and structure of cellulases is provided. However, primary attention is paid to the industrial application of these enzymes, with other aspects discussed within this context. The most practically significant bacterial and fungal cellulases are analysed, with their key benefits and differences being emphasised. Particular attention is paid to extremophilic (specifically thermo-, psychro-, and halophilic) cellulases, as they possess properties essential for modern technological processes. Given that practical application necessitates mass production and an optimal combination of enzymatic characteristics, the creation of effective producers and the modification of cellulase properties are also assessed. Finally, key trends in cellulase production approaches and their future application potential are summarised.
Breeding for the absence of proanthocyanidins in grain of barley (Hordeum vulgare L.): molecular genetic and technological aspects
Phenolic compounds constitute a significant group of secondary metabolites in barley grain and influence its technological qualities when used in brewing, feed production, and food manufacturing. Proanthocyanidins – polymeric flavonoids localized in the seed coat – play a particularly important role among them. These compounds are responsible for several production issues, such as colloidal haze in beer and browning of groats after heat treatment. Although proanthocyanidins possess health-beneficial properties based on their antioxidant activity, they can act as antinutritional factors due to their ability to bind proteins. In this regard, the breeding of barley varieties completely lacking proanthocyanidins in the grain was initiated, primarily for use in the brewing industry. Initially, it was assumed that their absence would not be critical for the plant, since wheat, corn, and rice varieties lacking proanthocyanidins in the grain had been identified. However, accumulated evidence indicates that proanthocyanidins perform important physiological functions: they contribute to the maintenance of seed dormancy, provide protection against fungal and bacterial pathogens and pests, and their absence negatively affects agronomic traits. For instance, proanthocyanidin free barley mutants obtained through induced mutagenesis exhibit reduced productivity and pathogen resistance, an increased risk of pre-harvest sprouting, and deterioration of several technologically important properties. Nevertheless, these mutant lines are actively used in breeding programs to develop varieties for various purposes. This review aims to systematize and analyze global experience in breeding proanthocyanidin-free barley varieties, describing achieved results to identify the most successful approaches and define future research directions. The work examines challenges faced by breeders when using mutant lines, as well as strategies that have helped minimize negative side effects. It is demonstrated that through targeted crossing and optimal selection of mutant alleles, competitive varieties have been developed that combine the required technological qualities with satisfactory agronomic performance, meeting the demands of both the brewing and food industries.