Ecological genetics

Transgenic plants occupy an important place in modern agriculture in many countries. The global area under genetically modified crops continues to increase each year and currently exceeds 200 million hectares. Increasing attention in the scientific data is devoted to assessing the potential environmental consequences of cultivating transgenic plants. Particular concern is associated with the possible effects of insecticidal proteins on non-target fauna and on microbial communities of the rhizosphere and phyllosphere. Another important issue is the escape of transgenes into the environment, which may occur through the uncontrolled introduction of seeds of transgenic crops into biocenoses, as well as through cross-pollination between transgenic plants and non-transgenic varieties of the same species or related plant species. The emergence and spread of spontaneous hybrids of transgenic varieties have been monitored by several researchers over multiple years of experiments; however, conducting long-term studies of these processes remains problematic. In this context, naturally transgenic plants, which originated in nature as a result of genetic transformation hundreds of thousands to millions of years ago, may serve as an interesting model for studying the evolutionary fate of transgenes, both under stabilizing selection in their favor and in its absence. Recent studies have expanded the list of naturally transgenic plants. It currently includes more than two hundred. Of particular interest in this context are genera in which the diversity of natural transgene sequences has been studied in detail across a large number of species, ecotypes, and populations. These include Nicotiana, Camellia, Arachis, Vaccinium, Cuscuta, Ipomoea, and several others. Within representatives of these genera, different evolutionary trajectories of natural transgenes can be observed. Some transgenes remain intact and expressed, leading to the emergence of new biosynthetic pathways in plants. Others gradually lose their integrity, accumulate point mutations, and over time undergo more substantial rearrangements, up to complete elimination. Such trends have been reported for certain genes in species belonging to the genera Nicotiana, Camellia, Arachis, and Vaccinium. Taken together, these findings suggest that in the absence of selective pressure favoring traits controlled by transgenes, plants not only accumulate point mutations but may also gradually eliminate transgenes.

BACKGROUND: Genetic modification of pea remains challenging, which can likely be attributed to its low regeneration efficiency. To obtain transgenic pea plants, shoot regeneration followed by rooting is usually applied. Another regeneration pathway, somatic embryogenesis, is not used for pea genome modification due to very low frequency of this process. If a gene stimulating somatic embryogenesis was identified, it could be used as a morphogenic regulator to enable regeneration of pea plants from genetically modified callus cells. The search for such genes relies on the development of a cultivation system, allowing production of a significant amount of callus tissue in which a potential morphogenic regulator is ectopically expressed.

AIM: The aim of study was to evaluate the possibility of obtaining pea callus tissue expressing foreign DNA with or without usage of selective agents, specifically, kanamycin and hygromycin B.

METHODS: In this study, we combined agrobacterial transformation protocol with a method of callus induction from pea shoot apex explants. To evaluate the effectiveness of this transformation system with different selective agents, we used two different reporters: RUBY and DsRed.

RESULTS: Our results demonstrate that transformation of pea shoot apices using the developed system yields a significant percentage of calli containing tissue regions which express foreign DNA. Addition of the antibiotics as selective agents doesn’t increase frequency of calli expressing introduced DNA.

CONCLUSION: Results obtained in this study suggest that searching for regeneration stimulators is feasible in Pisum sativum without usage of selective agents.

BACKGROUND: Russian aquaculture develops rapidly in terms of commercial rainbow trout production that requires massive juvenile stocks. In the last decade, the industry for the production of juvenile trout from fertilized eggs has been formed and is now expanding. Most fish farmers are striving to carry out this stage under a fully controlled water regime in recirculating aquaculture system. Today, the problem of ongoing monitoring of the bacterial pathogens and control of their numbers in such recirculating aquaculture system does not have a solution for practical fish farms. Given that the use of antibiotics is strictly regulated in food production, a project was launched to develop a bacteriophage specifically targeting highly pathogenic bacterial species.

AIM: Study various samples taken from the recirculating aquaculture system to determine the optimal site and method for bacteria collection and nucleic acid extraction.

METHODS: Samples were collected during 2025 at recirculating aquaculture system for rainbow trout Oncorhynchus mykiss in the Leningrad Region and the Republic of Karelia. The bacteria studied in this article are Aeromonas hydrophila. The bacterial content was assessed using traditional bacterial cultivation methods. DNA was extracted from various sample types and subjected to isothermal amplification targeting A. hydrophila. For samples confirmed to contain A. hydrophila, additional processing methods were employed following initial lysis in a buffer composed SDS and NaOH. Both chemical techniques, such as precipitation with alcohols and nanoparticles, and physical methods, including heating and syringe pipetting, were utilized in this procedure.

RESULTS: The study found that certain chemical methods (nanoparticle precipitation) for isolating bacterial DNA from the pre-lysed samples were just as effective as physical methods (syringe pipetting and heating). The most informative sample types for pathogen detection sites from the recirculating aquaculture system were swabs taken from pipes of the fish tanks drain.

CONCLUSION: The ability to obtain microorganism identification results using nucleic acid amplification methods outside the laboratory makes them highly promising for use as rapid diagnostics for bacterial pathogens in practical aquaculture. Following the completion of sample collection and the creation of a collection of pathogenic organisms, the development plan includes the development of a genetically modified bacteriophage for bacterial population control.

BACKGROUND: Preeclampsia is a serious pregnancy disorder that arises after 20 weeks’ gestation and is characterized by hypertension, proteinuria and edema; it can progress to eclampsia with severe complications and death. Several studies have reported amyloid like aggregates in placenta and urine from preeclampsia patients that stain with Congo red. Mass spectrometry of these aggregates identified Albumin, Ceruloplasmin, Interferon alpha inducible protein 6, Serotransferrin, Alpha 1 antitrypsin, immunoglobulin light chains (κ) and Aβ peptides. Except for Aβ, and immunoglobulin light chains, the ability of these proteins to form amyloids in vivo has not been demonstrated.

AIM: To evaluate the in vivo amyloidogenic potential of proteins identified in preeclampsia associated urinary aggregates using the yeast based assay.

METHODS: Candidate human proteins (including Aβ42 as positive control) were cloned in frame with the N-terminal prion domain of S. cerevisiae Sup35 (Sup35N) into a yeast expression plasmid. Constructs were under control the copper inducible CUP1 promoter. The constructs were transformed into a S. cerevisiae ade1-14 strain (nonsense mutation in ADE1) and lacking endogenous prions. Expression was induced by growth colonies on −Ura medium containing 150 µM CuSO₄ for 48 h. Colonies were then replica plated to adenine deficient (−Ade) medium, and growth was monitored up to 21 days as a readout of Sup35 conversion to the [PSI⁺] prion and of the amyloidogenicity of the Sup35N fusion.

RESULTS: Analysis indicates that none of the human proteins tested—except the Aβ42 peptide—have demonstrated amyloidogenic potential in vivo.

CONCLUSION: None of the full-length human proteins detected in urine from preeclampsia patients–albumin, ceruloplasmin, IFN-α-inducible protein 6, serotransferrin, and alpha-1-antitrypsin showed amyloidogenic activity in a yeast-based phenotypic assay; only Aβ42 functioned as an effective seed. Future work will assess amyloidogenicity of individual domains of these proteins.

Cystinuria is a hereditary nephropathy caused by defects in the SLC3A1 and SLC7A9 genes, which encode the subunits of the heterodimeric transporter rBAT–b^0,+AT. This complex mediates cystine reabsorption in the proximal renal tubules. Consequently, dysfunction of this complex leads to cystine accumulation in urine and the formation of cystine stones. A major challenge in patient management still remains delayed diagnosis, typically after the manifestation of urolithiasis, which particularly underscores the need to develop preventive and molecularly oriented approaches. The absence of effective therapies capable of modifying the course of the disease thus highlights the relevance of establishing adequate experimental models. Animal models—primarily murine—are clearly indispensable tools for both dissecting the molecular basis of cystinuria and for preclinical evaluation of potential novel therapeutic strategies. This comprehensive review summarizes all current data on animal models of cystinuria, with emphasis on their ability to reproduce important key aspects of the disease associated with dysfunction of the rBAT–b^0,+AT transport complex.

BACKGROUND: The CRISPR/Cas9 technology enables the generation of genetically modified founder animals (F0 generation) already at the stage of zygote editing. However, the resulting offspring are often mosaic, meaning they carry different mutations in different cells, which complicates accurate genotyping using standard tissue samples. To establish a stable knockout line, it is necessary to identify F0 individuals carrying the target mutations, which requires subsequent crossing with wild-type mice and a large-scale analysis of the F1 offspring, involving significant costs. Therefore, the efficient prediction of inheritable mutations at the F0 generation stage is a critical task.

AIM: A comparative evaluation of the efficiency of the TIDE and ICE bioinformatic algorithms for analyzing Sanger sequencing data to accurately predict inheritable mutations in the vldlr gene in F0 mosaic mice.

METHODS: The study was conducted on five F0 mosaic mice with mutations in the vldlr gene, obtained by microinjection of CRISPR/Cas9 components into zygotes. Genomic DNA was isolated from ear tissue, the target region of the vldlr gene was amplified by PCR and sequenced by the Sanger method. The resulting chromatograms were analyzed using the TIDE and ICE algorithms. The predictions were validated by crossing the F0 mice with wild-type mice and analyzing the inheritance of mutations in the F1 generation.

RESULTS: The comparison of the algorithms showed that both programs correctly predicted all mutations that were subsequently detected in the F1 generation. In total, 31 F1 offspring were analyzed.

CONCLUSION: Both tools are suitable for primary screening. The analysis of the offspring confirmed that all actually inherited mutations were predicted by both methods.