Search Results (71)

The oil-bearing rose (Rosa damascena Mill.), traditionally cultivated in Bulgaria for centuries, and the rose oil produced from it are of major cultural and economic importance. Its distinctive fragrance and rich aromatic profile are highly valued worldwide. In this study, a set of 15 start codon-targeted (SCoT) molecular markers was used to evaluate the genetic diversity and relationships of 38 rose accessions. The analyzed materials included Bulgarian-bred R. damascena cultivars, a locally improved population (‘Population 5’), three oil-bearing species (Rosa alba L., Rosa gallica L., and Rosa centifolia L.), Romanian heritage roses, and an unidentified rose genotype from an old Bulgarian plantation (Rosa sp.). The SCoT primers yielded a cumulative count of 238 bands, with an average of 12.9 bands per primer. The range of diversity markers, such as PIC (0.20–0.78), number of different alleles (1.5–2.00), Shannon’s information index (0.24–0.69), and gene diversity (0.15–0.50), provided evidence of genetic differences among the examined accessions. Analysis of Molecular Variance (AMOVA) revealed higher genetic variation within groups (61%) than among the groups (39%). Multivariate analyses (UPGMA, PCoA, and STRUCTURE) resolved the accessions into major genetic clusters corresponding to their taxonomic identity or breeding history. The unidentified Rosa sp. formed a distinct genetic group, showing closer affinity to R. gallica. The locally improved R. damascena ‘Population 5’ exhibited higher genetic diversity than the Bulgarian cultivars. Overall, our results demonstrate the effectiveness of SCoT markers and the value of local and traditional rose germplasm as reservoirs of genetic diversity. The study provides a molecular framework to support breeding, conservation, and sustainable management of oil-bearing rose genetic resources. Full article

Approaches to delivering gene editing tools in the form of ribonucleoproteins may provide a safety advantage over the delivery of nucleic acids encoding ribonucleoproteins. Virus-based vectors are widely used as a delivery platform. However, the persistence of viral exogenous nucleic acids can cause increased genotoxicity. Virus-like particles (VLPs) do not contain an expression cassette and can act as a platform for the delivery of ready-made ribonucleoprotein complexes. The absence of nucleic acids in VLPs eliminates the risk of insertional mutagenesis compared to widely used lentiviruses or adeno-associated viruses. Therefore, we used VLPs to deliver the ribonucleoprotein complex MmCas12m–TadDE to disrupt the HIV-1 gag gene start codon. We detected VLP morphogenesis using electron microscopy. We confirmed the incorporation of MmCas12m–TadDE into VLPs. We achieved an editing efficiency of about 9% in some cases with minimal off-target effects, which confirms the prospect of using VLPs as a platform for delivering genomic editing tools. Full article

Sugar beet (Beta vulgaris L.) is an economically important crop that accounts for approximately 20% of global sugar production. The success of future breeding programs depends on the effective utilization of existing genetic resources. The aim of this study was to assess the genetic diversity and population structure of 192 sugar beet (Beta vulgaris L.) genotypes, including commercial cultivars and accessions obtained from the USDA gene bank, using SCoT and ISSR molecular markers, and to identify potential genetic resources for sugar beet breeding programs. In this study, a total of 192 sugar beet genotypes, including 187 accessions from the USDA (U.S. Department of Agriculture) gene bank and 5 commercial cultivars, were evaluated for genetic diversity using Start Codon Targeted (SCoT) and Inter Simple Sequence Repeat (ISSR) markers. A total of 68 scorable bands were obtained from five SCoT and three ISSR primers, and all bands were found to be polymorphic (100% polymorphism). Parameters such as polymorphic information content (PIC), Nei’s genetic diversity, and Shannon’s index indicated a high level of variation within the gene pool, with SCoT markers being more informative than ISSR markers. Dendrogram analyses based on Nei’s genetic distance revealed that the populations were separated into two main groups, while the sub-clusterings contained broad genetic variation. STRUCTURE analysis identified four (K = 4) populations for the SCoT data and three (K = 3) populations for the ISSR data; the inclusion of a high number of individuals in the admixture population indicated extensive gene flow. Principal component analysis (PCA) revealed both homogeneous groups and differentiated genotypes contributing to within-population diversity. The results demonstrate that the combined use of SCoT and ISSR markers provides powerful and complementary tools for assessing genetic diversity in sugar beet. The findings provide a solid scientific basis for the development of new, high-yielding and high-quality sugar beet cultivars as well as for the conservation of existing genetic resources. Molecular data constitute an important reference for guiding sugar beet breeding programs and for the effective utilization of genetic resources. Full article

Polyglutamine expansion in Ataxin-2 (ATXN2) is responsible for rare, dominantly inherited Spinocerebellar Ataxia type 2 (SCA2). Together with its paralog Ataxin-2-like (ATXN2L), both proteins have received much interest, since the deletion of their yeast and fly orthologs alleviates TDP-43-triggered neurotoxicity in Amyotrophic Lateral Sclerosis models. Their typical structure across evolution combines LSm with LSm-Associated Domains and a PAM2 motif. To understand the physiological regulation and functions of Ataxin-2 homologs, the phylogenesis of sequences was analyzed. Human ATXN2 harbors multiple alternative start codons, e.g., from an intrinsically disordered sequence (IDR) present since armadillo, or from the polyQ sequence that arose since amphibians, or from the LSm domain since primitive eukaryotes. Multiple smaller isoforms also exist across the C-terminus. Therapeutic knockdown of polyQ expansions in human ATXN2 should selectively target exon 1B. PolyQ repeats developed repeatedly, usually framed and often interrupted by (poly)Pro, originally near PAM2. The LSmAD sequence appeared in algae as the characteristic Ataxin-2 feature with strong conservation. Frequently, Ataxin-2 has added domains, likely due to transcriptional readthrough of neighbor genes during cell stress. These chimerisms show enrichment of rRNA processing; nutrient store mobilization; membrane strengthening via lipid, protein, and glycosylated components; and cell protrusions. Thus, any mutation of Ataxin-2 has complex effects, also affecting membrane resilience. Full article

Rye (Secale cereale L.) is a small-grain cereal traditionally cultivated under low-input conditions, where locally adapted populations have contributed substantially to the maintenance of genetic diversity. Despite this importance, Greek rye germplasm has received limited attention at the molecular level. In the present study, 33 rye accessions, including gene bank landraces, locally cultivated populations and one commercial variety, were analyzed using inter-simple sequence repeat (ISSR), start codon-targeted (SCoT), and exon-based amplified polymorphism (EBAP) markers. All three marker systems generated high proportions of polymorphic loci and comparable estimates of expected heterozygosity, indicating considerable genetic variability within the studied material. Multivariate analyses revealed moderate population structuring and consistently identified a small number of genetically divergent accessions, most notably T-492, K-163, and K-166. No clear clustering according to geographical origin was detected, as in most cases of landraces or local populations. Taken together, the results provide a detailed molecular overview of Greek rye germplasm—which has never been performed before for Greek rye genetic material—and offer a useful basis for conservation priorities and future pre-breeding efforts. Full article

Lavender has been cultivated in Bulgaria for over a century. The high essential oil content and quality of Bulgarian lavender varieties have established the country as a leading global producer. Studies into the crop’s genetic diversity are essential for selecting varieties best suited to specific environmental conditions, maximizing resilience and yield. Therefore, identifying appropriate genetic markers to monitor lavender diversity is a key prerequisite for developing effective crop selection strategies, particularly in response to the challenges posed by global climate change. In this study, we evaluate the versatility of markers for assessing genetic diversity of lavender genotypes. A total of 96, 97 and 96 bands were recorded using the 13 Start Codon Targeted Polymorphism (SCoT), 13 Inter-Simple Sequence Repeat (ISSR) and 14 Cis-Element Aligned Polymorphism (CEAP) primers, respectively. All amplification programs used were successful in the studied genotypes. Additionally, four informative primers of each marker system were applied for assessment of the within-field genetic variability in two lavender plantations from Bulgaria. This is the first report on the combined use and comparison of CEAP, SCoT and ISSR primers in lavender genotypes in Bulgaria. Full article

Lavender (Lavandula angustifolia Mill.) is a traditional and economically important crop in Bulgaria. The cultivated areas are primarily planted with seven Bulgarian varieties: ‘Hemus’, ‘Druzhba’, ‘Sevtopolis’, ‘Yubileina’, ‘Raya’, ‘Hebar’, and ‘Karlovo’. Except for ‘Karlovo’, these cultivars are widely grown due to their proven agronomic performance and adaptability across different regions of the country. However, their genetic diversity and relationships have not been deeply examined. In this study, 13 Start Codon Targeted (SCoT) markers were used to assess the genetic diversity of those seven Bulgarian cultivars. This research provides the first report on the application of SCoT markers in lavender accessions. The results revealed considerable polymorphism, confirming the effectiveness of the SCoT marker system for L. angustifolia. The obtained data indicate moderate genetic diversity among the cultivars, supported by the effective number of alleles and polymorphic information content. Cluster analysis (UPGMA) and Principal Coordinates Analysis (PCoA) demonstrated clear genetic differentiation, grouping the cultivars according to their genetic proximity. These findings provide valuable baseline information for future selection, conservation, and genetic evaluation of Bulgarian lavender. Full article

Translation of mRNAs is a tightly regulated process in gene expression. In mRNA, the 5′ untranslated region (5′ UTR) controls ribosome recruitment and frequently contains structured elements that modulate translation efficacy. This study investigates stable structural motifs within the 5′ UTR of DDX23 mRNA, encoding a protein relevant for anticancer therapy, as potential regulators and targets for antisense oligonucleotides (ASOs). Despite bioinformatic predictions and transcriptomic validations suggesting RNA G-quadruplex (rG4) formation, comprehensive structural analysis using a light-up assay and CD, UV, and NMR spectroscopy revealed that most putative rG4-forming sequences do not fold into stable rG4 structures, although one of them exists in an equilibrium between rG4 and an alternative, likely hairpin, conformation. Reporter assays using a robust G4 stabilizer also argue against the significant regulatory role of rG4s in DDX23 mRNA translation. Instead, we identified and characterized a stable hairpin structure with potential regulatory function. Based on these findings, we designed fully locked nucleic acid (LNA)-modified ASOs to target this hairpin and regions flanking the upstream open reading frame (uORF) and start codon of the coding sequence. A reporter assay demonstrated that cap-proximal targeting achieved robust translation inhibition up to 80%. In contrast, targeting the efficiently translated uORF was ineffective, presumably due to steric hindrances from the ribosomal complex. The study yields crucial design principles for translation-regulating ASOs: avoid targeting regions shielded by efficient uORF translation and carefully tune ASO-RNA duplex stability to surpass endogenous structures without disrupting regulatory mechanisms. These findings provide insights into the regulation of DDX23 expression and establish a framework for developing ASO-based therapeutics with broad implications for mRNA targeting in anticancer applications. Full article

The success of plant tissue cryopreservation strongly depends on maximizing explant survival during storage in liquid nitrogen and recovery, which requires species-specific protocol optimization and ongoing refinement. This study examined the effect of Plant Vitrification Solution 3 (PVS3) supplemented with nanoparticles (NPs) or melatonin (MEL) on the recovery of Lamprocapnos spectabilis (L.) Fukuhara explants after cryostorage. Treatments with ZnO + Ag NPs, as well as different MEL concentrations, were applied to evaluate their influence on explant survival, photosynthetic efficiency, and genetic stability. The highest recovery (40–44%) was obtained with PVS3 containing 50 mg L⁻¹ ZnO + 0.1% Ag NPs and PVS3 supplemented with 8 mg L⁻¹ MEL, which was 17.5–20% higher than in the control. These treatments, however, did not ensure the highest photosynthetic efficiency of recovered plants. PVS additives likely support recovery by slowing metabolism and reducing oxidative stress, with lower photosynthetic activity suggesting a lag phase in plastid regeneration. Using the Start Codon Targeted (SCoT) marker system, no significant genetic alterations were detected in recovered plants of any tested variant. These findings demonstrate the feasibility of optimizing cryopreservation protocols for L. spectabilis and encourage further research on combined NPs and MEL treatments or alternative nanocarriers. Full article

Backgrounds: Accurate annotation of open reading frames (ORFs) is fundamental for understanding gene function and post-transcriptional regulation. A critical but often overlooked aspect of transcriptome annotation is the selection of authentic translation start sites. Many genome annotation pipelines identify the longest possible ORF in alternatively spliced transcripts, using internal methionine codons as putative start sites. However, this computational approach ignores the biological reality that ribosomes select start codons based on sequence context, not ORF length. Methods: Here, we demonstrate that this practice leads to systematic misannotation of nonsense-mediated decay (NMD) targets in the Arabidopsis thaliana Araport11 reference transcriptome. Using TranSuite software to identify authentic start codons, we reanalyzed transcriptomic data from an NMD-deficient mutant. Results: We found that correct ORF annotation more than doubles the number of identifiable NMD targets with premature termination codons followed by downstream exon junctions, from 203 to 426 transcripts. Furthermore, we show that incorrect ORF annotations can lead to erroneous protein structure predictions, potentially introducing computational artefacts into protein databases. Conclusions: Our findings underscore the importance of biologically informed ORF annotation for accurate assessment of post-transcriptional regulation and proteome prediction, with implications for all eukaryotic genome annotation projects. Full article

Introduction: Stevia rebaudiana Bertoni has recently gained significant attention due to the presence of intensely sweet yet low-calorie steviol glycosides (SGs) in its leaves, making it a promising natural sugar alternative with applications in the food, pharmaceutical, and cosmetics industries. The primary goal of this study was to determine whether generating somaclonal variation from plant material obtained by indirect regeneration results in further genetic changes identifiable using the SCoT marker (Start Codon Targeted). Methods: In the first stage, callus tissue was initiated from first-generation somaclones on MS medium supplemented with 4.0 mg/L 6-benzylaminopurine (BAP), 2.0 mg/L 1-naphthaleneacetic acid (NAA), and 2.0 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D). Their morphogenetic potential was analyzed on four media with different BAP and Kinetin concentrations. Donor plants, first and second generation somaclones, were also analyzed for genetic diversity using SCoT markers. Results: All first-generation somaclones demonstrated a very high callus initiation capacity, ranging from 95 to 100%. It was found that for most of the studied somaclones, the greatest number of shoots were developed by explants grown in a medium supplemented with 0.5 mg/L BAP and 0.25 mg/L Kin. The studied group of somaclones exhibits a high degree of polymorphism (55.2%). The analysis of genetic similarity of somaclones presented in the form of individual dendrograms indicates that in most cases, greater genetic diversity was revealed as a result of indirect regeneration in the first generation of somaclones compared to the second. Indirect organogenesis allows for the production of subsequent generations of genetically unstable somaclones, creating the potential for obtaining new phenotypic variants useful in plant breeding. Full article

Eryngium foetidum L. belongs to the Apiaceae family and is a perennial herb. The entire plant is rich in essential oils, which have a distinctive aroma similar to cilantro. This plant exhibits significant biological activity and possesses characteristics such as disease resistance and antimicrobial properties, showing great potential in medical and food applications. Additionally, its essential oil has substantial commercial value. Mitochondria play a crucial role as organelles within plant cells; however, the mitochondrial genome of E. foetidum remains underexplored. To fill this research gap, we conducted sequencing and assembly of the mitochondrial genome of E. foetidum, aiming to uncover its genetic mechanisms and evolutionary trajectories. Our investigation reveals that the mitochondrial genome of E. foetidum is a circular structure, similar to that of other species, with a length of 241,660 bp and a GC content of 45.35%, which is within the range observed in other organisms. This genome encodes 59 genes, comprising 37 protein-coding sequences, 18 tRNA genes, and 4 rRNA genes. Comparative analysis highlighted 16 homologous regions between the mitochondrial and chloroplast genomes, with the longest segment spanning 992 bp. By analyzing 37 protein-coding genes (PCGs), we identified 479 potential RNA editing sites, which induce the formation of stop codons in the nad3 and atp6 genes, as well as start codons in the ccmFC, atp8, nad4L, cox2, cox1, and nad7 genes. Meanwhile, the genome shows a preference for A/T bases and A/T-ending codons, with 32 codons having a relative synonymous codon usage (RSCU) value greater than 1. The codon usage bias is relatively weak and mainly influenced by natural selection. Most PCGs are under purifying selection (Ka/Ks < 1), while only a few genes, such as rps7 and matR, may be under positive selection. Phylogenetic analysis of mitochondrial PCGs from 21 species showed E. foetidum at the basal node of Apiaceae, consistent with the latest APG angiosperm classification and chloroplast genome-based phylogenetic relationships. In summary, our comprehensive characterization of the E. foetidum mitochondrial genome not only provides novel insights into its evolutionary history and genetic regulation but also establishes a critical genomic resource for future molecular breeding efforts targeting mitochondrial-associated traits in this economically important species. Full article

The Chinese holly (Ilex chinensis Sims.), an evergreen tree species native to China, is distributed mainly in regions south of the Qinling Mountains and Huai River. This research aimed to characterize the molecular profiles and genetic relationships of 40 Chinese holly genotypes via inter-simple sequence repeat (ISSR) and start codon targeted (SCoT) polymorphism markers. Genetic diversity analysis revealed that the ISSR markers detected 111 polymorphic bands from 13 primers, with a polymorphism rate of 88.10%. The analysis generated parameters such as the observed allele number (Na = 1.876), effective allele number (Ne = 1.461), Shannon’s information index (I = 0.271), and expected heterozygosity (H = 0.411). In comparison, the SCoT markers produced 65 polymorphic bands from the 6 primers, resulting in a 100% polymorphism rate, with Na = 2.000, Ne = 1.695, I = 0.393, and H = 0.575. Cluster analysis classified the 40 genotypes into two main clusters with genetic similarity coefficients of 0.69 (ISSR) and 0.55 (SCoT). The ISSR markers presented the greatest similarity between the ZSS and ZLS genotypes, whereas the ZZDH and ZWW genotypes presented lower similarity. Conversely, the SCoT markers identified ZZP and ZJDS as the most similar, with ZLJ and ZHX showing less similarity. These results provide a theoretical basis for hybrid breeding, germplasm innovation, and conservation strategies of Chinese holly in China. Full article

Background: Tissue culture might be a method supplementing traditional plant propagation in various fields, like agriculture, medicine, industry, and the active conservation of plant species. For the purpose of plant restoration, it is important that the obtained progenies are identical with the mother plants to ensure the true-to-typeness of the future population. Methods: In the present study, the stability of Aldrovanda vesiculosa regenerants obtained in vitro through phenotypic and genetic analysis was estimated. Clones of aldrovanda plants were cultivated in tissue culture in the 1/10 MS liquid medium under the same conditions for over a year, with five weeks of subculturing. Results: It was observed that two clones formed plants that displayed atypical growth structures, the shoots were shorter with many lateral shoots, and they had a lower fresh weight. They also formed fewer and smaller snap-traps, which, in the case of carnivorous plants, determines the capability of catching prey. The 35 in vitro regenerated plants and 5 specimens obtained from the natural habitat were subjected to genetic analyses with two molecular markers: start codon targeted (SCoT) polymorphism and sequence-related amplified polymorphism (SRAP). Despite the visible morphological variants, the genetic stability of all the regenerants with the individuals from natural stands was confirmed. All of them were monomorphic except three bands that were obtained for reference, where individuals were amplified with SCoT28 and me12-em13 SRAP primers. Conclusions: As shown in the presented research, it might be recommended to use different methods to evaluate the stability of in vitro cultivated plants. Full article

Conventional methods for generating knock-out or knock-in mammalian cell models using CRISPR-Cas9 genome editing often require tedious single-cell clone selection and expansion. In this study, we develop and optimise rapid and robust strategies to engineer homozygous fluorescent reporter knock-in cell pools with precise genome editing, circumventing clonal variability inherent to traditional approaches. To reduce false-positive cells associated with random integration, we optimise the design of donor DNA by removing the start codon of the fluorescent reporter and incorporating a self-cleaving T2A peptide system. Using fluorescence-assisted cell sorting (FACS), we efficiently identify and isolate the desired homozygous fluorescent knock-in clones, establishing stable cell pools that preserve parental cell line heterogeneity and faithfully reflect endogenous transcriptional regulation of the target gene. We evaluate the knock-in efficiency and rate of undesired random integration in the electroporation method with either a dual-plasmid system (sgRNA and donor DNA in two separate vectors) or a single-plasmid system (sgRNA and donor DNA combined in one vector). We further demonstrate that coupling our single-plasmid construct with an integrase-deficient lentivirus vector (IDLV) packaging system efficiently generates fluorescent knock-in reporter cell pools, offering flexibility between electroporation and lentivirus transduction methods. Notably, compared to the electroporation methods, the IDLV system significantly minimises random integration. Moreover, the resulting reporter cell lines are compatible with most of the available genome-wide sgRNA libraries, enabling unbiased CRISPR screens to identify key transcriptional regulators of a gene of interest. Overall, our methodologies provide a powerful genetic tool for rapid and robust generation of fluorescent reporter knock-in cell pools with precise genome editing by CRISPR-Cas9 for various research purposes. Full article