Search Results (1,314)

Manipulating the mitochondrial genome remains a significant challenge in genetic engineering, primarily due to the mitochondrial double-membrane structure. While recent advances have expanded the genetic toolkit for nuclear and cytoplasmic targets, precise editing of mitochondrial DNA (mtDNA) has remained elusive. Here we report the first successful mitochondrial import of a catalytically active RNA-guided prokaryotic Argonaute protein from the mesophilic bacterium Alteromonas macleodii (AmAgo). By guiding AmAgo to the single-stranded D- or R-loop region of mtDNA using synthetic RNA guides, we observed a nearly threefold reduction in mtDNA copy number in human cell lines. This proof of concept study demonstrates that a bacterial Argonaute can remain active within the mitochondrial environment and influence mtDNA levels. These findings establish a foundational framework for further development of programmable systems for mitochondrial genome manipulation. Full article

Background/Objectives: Southeastern Europe and Croatia have served as a genetic crossroads between the Near East and Europe since prehistoric times, shaped by numerous and repeated migrations. By integrating 19 newly generated ancient genomes with 285 previously published ancient genomes from Croatia, we investigated patterns of maternal and paternal landscapes from the Neolithic, Bronze, and Iron Ages through to the Antiquity and medieval periods, as well as the modern Croatian population. Methods: Ancient DNA extraction from human remains and library preparation were conducted in dedicated clean-room facilities, followed by high-throughput sequencing on the Illumina platform. Sequencing data were analyzed with established pipelines to determine mitochondrial and Y-chromosomal haplogroups and the genetic sex of individuals. Results: New ancient data reveal a predominantly European maternal profile, dominated by haplogroups H, U, and HV0, whereas Y-chromosomal lineages are characterized by J subclades and R1a, with limited representation of R1b and the absence of I2a. When combined with published ancient Croatian genomes, the results reveal similar haplogroup diversity and patterns, as well as the expansion of mtDNA haplogroup H over time and a substantial increase in Y-chromosome R1a and I2a haplogroup frequency from the prehistoric to the modern period. Conclusions: Although the analyzed samples are heterogeneous and originate from different historical periods, their genetic signatures conform to the broader patterns expected for the region. In a wider context, the ancient Croatian mitochondrial data reveal stronger genetic persistence from prehistory to modern times, unlike paternal lineages, which show significantly higher divergence. Full article

The Xinjiang Uygur Autonomous Region, also known as Xinjiang, China, is notable for its high diversity and abundance of lichens. The purpose of this study was to examine species diversity and the distribution patterns of saxicolous lichens, family Megasporaceae, which includes the genera Aspicilia, Circinaria and Lobothallia, in Xinjiang Province. Morphology, anatomy, chemical analysis and rDNA-ITS sequences for the species were employed for their identification. As a result, 34 crustose and strictly saxicolous species belonging to three genera were found, which included 22 species of the genus Aspicilia, two of which were new to Xinjiang (A. disjecta (Zahlbr.) J.C. Wei and A. pycnocarpa Q. Ren & Lin Liu), eight common species of Circinaria, as well as four species of Lobothallia, two of which (L. determinata (H. Magn.) T.B. Wheeler and L. pruinosa Kou & Q. Ren) are new provincial records. There was a unimodal pattern with respect to lichen species richness; all specimens of the Megasporaceae family were found between 1600 and 5100 m altitude. The 30 species were collected at altitudes between 2601 and 3100 m; only four species were recorded below 2150 m, and seven were found above 4600 m. As far as the type of rocks are concerned, 24 species were found on siliceous rocks and 10 species were found on calcareous rocks. The 24 lichen species contained seven different secondary metabolites; stictic acid, substictic acid and norstictic acid were more common, whereas aspicilin, constictic acid, lecanoric acid and connorstictic acid were found in only a few lichen species. Full article

Carpione rhabdovirus (CAPRV) is an emerging virus within the family Rhabdoviridae, posing potential threats to aquaculture species such as golden pompano (Trachinotus anak). However, since the 21st century, and for CAPRV strains isolated from marine fish, only a single CAPRV2023 sequence has previously been available in public databases, with no additional sequences reported. Because the virus undergoes genetic variation, relying on this single sequence likely introduced mismatches or off-target risks in earlier detection assay designs. Notably, the previously developed two-step N-targeting detection assay was designed based solely on that single CAPRV2023 sequence. Consequently, this study involved determining and analyzing the N gene sequences from CAPRV isolates gathered from 2023 to 2025, with the aim of pinpointing conserved regions for assay development, and sequence comparisons subsequently verified the existence of mismatches in the primer–probe binding sites of the previous assay. Since quantitative assays in aquatic virology often define copy numbers utilizing either plasmid DNA templates or RNA templates produced via in vitro transcription, which may lead to variations in amplification kinetics and sensitivity, this study compared both standards to ensure reliable quantification across different nucleic acid types. Based on these findings, a one-step TaqMan quantitative PCR (qPCR) assay was developed and validated using dual nucleic acid standards, namely plasmid DNA and in vitro–transcribed RNA. Compared with conventional two-step qPCR, the one-step format combines cDNA synthesis and subsequent DNA amplification in a single sealed tube, thereby effectively preventing cross-contamination, simplifying the workflow, and improving detection efficiency. The assay exhibited strong linearity (R² > 0.99) and consistent amplification efficiencies between 90% and 110%, demonstrating excellent quantitative performance. The detection limits were 2 copies per reaction for plasmid DNA and 20 copies for in vitro–transcribed RNA templates. No cross-reactivity was observed with other aquatic pathogens, and the assay showed strong repeatability and reproducibility (coefficients of variation below 2.0%), providing a sensitive and reliable tool for epidemiological surveillance and the analysis of CAPRV distribution in marine aquaculture systems of southern China. Full article

Hepatitis B virus (HBV) persists in infected hepatocytes through covalently closed circular DNA (cccDNA), a stable episomal form that serves as the transcriptional template for viral replication. Accurate and sensitive quantification of intrahepatic cccDNA is crucial for evaluating antiviral therapies, particularly those targeting a functional cure. Here, we report the development of a novel, cccDNA-specific detection system combining recombinase polymerase amplification (RPA) with CRISPR/Cas12a-based fluorescence detection. We designed and validated CRISPR RNAs (crRNAs) targeting HBV cccDNA-specific regions conserved across genotypes A–D. Reaction conditions for both RPA and Cas12a detection were optimized to enhance sensitivity, specificity, and accuracy. The system reliably detected as few as 10 copies of cccDNA-containing plasmid per reaction and showed no cross-reactivity with non-cccDNA forms in serum or plasma, indicating assay specificity. When applied to liver tissue samples from 10 HBV-infected and 6 non-HBV patients, the RPA-CRISPR/Cas12a assay exhibited a high sensitivity (90%) and a strong correlation with qPCR results (R² = 0.9155), confirming its accuracy. In the conclusion, the RPA-CRISPR/Cas12a system provides a robust, cost-effective, and scalable platform for sensitive and specific quantification of intrahepatic HBV cccDNA. This method holds promises for research and high-throughput therapeutic screening applications targeting cccDNA clearance. Full article

The aim of this study was to determine fungal diversity and composition in an area of high host diversity and identify the organisms involved in the appearance of symptoms in Pinus needles. Asymptomatic and symptomatic live needle samples were obtained from different Pinus spp. in an arboretum with confirmed presence of brown spot needle blight. The samples were analysed using high-throughput sequencing of fungal ITS2rDNA. Ascomycota dominated all samples, with Lophodermium as the most abundant genus, although it showed lower representation in symptomatic needles. Other genera with recognised pathogenic potential, including Lecanosticta, Pestalotiopsis, Cyclaneusma, Rhizosphaera, Neophysalospora, and Cenangium, were also detected, whereas the Dothistroma genus was absent despite its presence in the region. Alpha diversity was higher in asymptomatic needles, with a significant difference only for the Shannon index, while Bray–Curtis dissimilarity revealed significant shifts in community composition between needle types. Functional guilds were dominated by pathotroph–saprotroph trophic mode, and the functional guild ‘plant pathogen’ was the most abundant across samples. These findings identify fungal genera associated with symptomatic and asymptomatic needles and provide guidance for future targeted isolation and detailed morphological and molecular identification using more resolutive techniques, enabling a deeper understanding of pathogenic community presence and their potential synergistic interactions. Full article

Background/Objectives: Multidrug-resistant tuberculosis remains a critical public health challenge in Kazakhstan, yet the genomic determinants contributing to its emergence are still insufficiently understood. Although the quantity of genomic studies from Central Asia and the wider post-Soviet region has increased in recent years, the involvement of DNA repair and genome maintenance pathways in the development of resistance within Kazakhstan has not been comprehensively explored. Methods: In this study, we performed whole-genome analysis of 175 Mycobacterium tuberculosis clinical isolates collected across Kazakhstan between 2010 and 2022 to evaluate the contribution of single-nucleotide polymorphisms in DNA replication, repair, and recombination (3R) genes to the evolution of drug resistance. Results: Alongside well-established resistance mutations in gyrA, we identified recurrent variants in 3R-associated loci (genes involved in DNA replication, repair, and recombination)—including polA, uvrC and ligC—that were enriched among drug-resistant isolates, suggesting a broader role for genome maintenance pathways in facilitating resistance evolution under treatment pressure. Conclusions: These findings provide the first region-specific genomic insights into 3R gene variation in Kazakhstani M. tuberculosis isolates. Full article

Accurate authentication of herbal medicine Descurainiae Semen, the tiny seeds of Descurainia sophia, is challenging due to their morphological similarity to various adulterants. To develop a precise and reliable molecular identification method, we conducted comparative analyses of rDNA-ITS sequences using D. sophia and five adulterant species and subsequently developed species-specific sequence-characterized amplified region (SCAR) markers. The discriminatory power and detection limits of these markers were evaluated using serially diluted genomic DNA from each species and commercially available Descurainiae Semen, respectively. The SCAR markers developed in this study enabled the detection of adulterant contamination at levels as low as 0.01–1%. Among several potential adulterants tested using 17 herbal medicines, Erysimum macilentum was found to be the most common adulterant in commercial products, with a ratio of 88%. The SCAR-PCR assay established in this study provides a rapid and accurate tool for identifying D. sophia and illegal adulterants at the species level and at very low contamination levels, thereby supporting improved quality control and enhancing consumer confidence in the herbal medicine industry. Full article

×Trititrigia cziczinii Tzvelev is a promising crop developed through distant hybridization between Elytrigia intermedia (Host) Nevski (=Thinopyrum intermedium (Host) Barkworth & D.R. Dewey) and Triticum aestivum L., followed by backcrossing with wheat. This study elucidates the genomic composition of this hybrid and its parental taxa using molecular phylogenetic analysis of nuclear (ITS, ETS) and chloroplast (trnK–rps16, ndhF) DNA markers, complemented by Next-Generation Sequencing (NGS) of the 18S–ITS1–5.8S rDNA region. Results from Sanger sequencing revealed that the primary nuclear ribosomal DNA (rDNA) of the hybrid originates from Triticum aestivum; a finding strongly supported by both Bayesian inference and Maximum Likelihood analyses. Chloroplast DNA data unequivocally indicate maternal inheritance from T. aestivum. In contrast, ETS sequence analysis showed phylogenetic affinity to Elytrigia intermedia, suggesting complex genomic reorganization or chimeric sequence formation in the hybrid. NGS data corroborate the dominance of T. aestivum-like ribotypes in the hybrid’s rDNA pool, with only a minor fraction identical to the main ribotype of E. intermedia. Genetic structure analysis further revealed geographic heterogeneity in the genomic composition of E. intermedia populations. The predominance of the wheat genome in ×T. cziczinii is likely a consequence of stabilizing backcrosses and illustrates a case of rDNA elimination from one parental genome during hybridization. This research underscores the complex genomic dynamics in artificial hybrids and the utility of multi-marker phylogenetic approaches for clarifying their origins. Full article

Polemonium L. (Polemoniaceae) is a widespread genus native to subarctic and arctic regions of the Northern Hemisphere. The taxonomy and genome relationships within Polemonium are still unclear. We analyzed genomes of three species from each Polemonium caeruleum and Polemonium pulcherrimum complex using bioinformatic analysis by RepeatExplorer2/TAREAN pipelines of next-generation sequencing data. The repeatomes of all studied species were similar in type and number of repeats. Satellite DNAs (satDNAs) demonstrated high sequence identity within the studied species. FISH chromosome mapping of 45S rDNA, 5S rDNA, and two satDNAs Pol_C 33 and Pol_C 46 allowed us to construct the species karyograms and assess the genome diversity within the P. caeruleum complex and P. pulcherrimum complex, and also confirm the taxonomic status of P. kiushianum as an independent species. Our findings demonstrate a close genomic relationship among the species from P. caeruleum and P. pulcherrimum complexes, indicating the presence of a common ancestral genome; additionally, our results provide cytogenetic evidence for the monophyletic origin of these sections and also complex evolutionary history of the genus Polemonium. The developed approach may be a valuable framework for further investigation of the chromosomal organization of karyotypes in other species of the genus Polemonium. Full article

The chloroplast (cp) genome of Panicum bisulcatum (Thumb.), a significant agricultural weed, was sequenced and characterized to elucidate its genomic architecture, evolutionary dynamics, and phylogenetic relationships. The complete cp genome was assembled as a circular DNA molecule of 138,489 bp, exhibiting a typical quadripartite structure comprising a large single-copy (LSC, 82,260 bp), a small single-copy (SSC, 12,569 bp), and a pair of inverted repeats (IR, 21,830 bp each) regions. It encodes 135 genes, including 89 protein-coding genes, 49 tRNAs, and 8 rRNAs. Functional annotation revealed that most genes are involved in photosynthesis and genetic system. A total of 51 simple sequence repeats (SSRs) and 62 long repeats (LRs) were identified, providing potential molecular markers. Comparative analysis of IR boundaries highlighted both conserved features and species-specific expansion/contraction events among Panicum species. Phylogenomic analysis robustly placed P. bisulcatum within the genus Panicum, showing a closest relationship with P. incomtum and confirming the monophyly of the genus. Furthermore, single nucleotide polymorphism (SNP) analysis with its closest relative, P. incomtum, revealed 4659 SNPs, with a dominance of synonymous substitutions, indicating the action of purifying selection. This study provides the first comprehensive cp genomic resource for P. bisulcatum, which will facilitate future studies in species identification, phylogenetic reconstruction, population genetics, and the development of sustainable management strategies for this weed. Full article

Background/Objectives: Conventional next-generation sequencing (NGS) workflows often require more than two weeks to complete, delaying treatment decisions and limiting access to precision oncology in community settings. This report aimed to demonstrate the feasibility of performing rapid, comprehensive cell-free DNA (cfDNA)-based genomic profiling by introducing a fully automated NGS workflow in a community hospital environment. Case Presentation: A postoperative patient with pancreatic ductal adenocarcinoma and liver metastasis underwent cfDNA-based liquid biopsy using plasma collected in PAXgene^® Blood ccfDNA Tubes. Gene analysis was performed using the Oncomine Precision Assay GX5 on the Ion Torrent Genexus™ System (Thermo Fisher Scientific). Three pathogenic hotspot mutations—KRAS G12R, TP53 M246I/M246K, and GNA11—and one copy number gain in PIK3CA were identified, whereas no variants were detected in a healthy volunteer control. The total turnaround time from plasma separation to report generation was approximately 27 h, requiring only 40 min of total hands-on time. Discussion: This rapid, automated workflow enabled comprehensive cfDNA analysis within a clinically practical timeframe, overcoming key limitations of conventional multi-step NGS workflows that typically require external sample shipment and specialized personnel. The results confirm the technical feasibility of conducting high-quality molecular testing in a regional hospital setting. Conclusions: This report demonstrates that fully automated cfDNA-based NGS can achieve clinically meaningful genomic profiling within 27 h in a community hospital. This advancement addresses the time and cost barriers of traditional NGS analysis and represents a significant step toward promoting precision medicine in community healthcare. Full article

Bats (Chiroptera) represent nearly one-fifth of all mammalian species and play vital ecological roles as pollinators, pest controllers, and reservoirs of zoonotic pathogens. Accurate identification of bat species is essential for biodiversity monitoring, conservation, and disease surveillance. Traditional methods based on morphology or acoustic calls are often limited by overlapping features, while DNA barcoding using the cytochrome oxidase I (COI) gene can be hindered by sequence variability. In this study, we developed a simple, single-step PCR assay targeting a short, variable region of the mitochondrial 12S rRNA gene. Alignment of sequences from 232 bat species allowed the design of a single primer pair producing a 203–224 bp amplicon that successfully distinguished all species analyzed. The assay achieved 100% amplification success across 241 bat samples, with 97.2% concordance between molecular and morphological identification. Two samples showed sequence divergence suggestive of an undescribed species. Overall, ten bat species from six genera were identified, with Eptesicus fuscus being the most frequent. This assay offers a practical and robust approach for bat identification, supporting biodiversity assessment and pathogen surveillance in ecological and public health research. Full article

Accurate species identification in blackberries (Rubus spp.) is difficult because of morphological similarity and frequent hybridization. We studied 56 wild accessions from the Sirius Federal Territory (Russia), representing coastal and foothill ecosystems of the Black Sea region. Multilocus DNA barcoding with the plastid rbcL gene and nuclear ITS1 and ITS2 regions revealed signals of hybridization and hidden diversity. The rbcL marker showed low variation, grouping most accessions into two clusters with several singletons, which limited its use for distinguishing species. In contrast, ITS1 and ITS2 showed higher variation, forming six clusters and eight singletons, and allowed for clear separation of taxa such as Rubus caesius L., R. irritans Focke, and R. amabilis Focke. Accession 3 carried a raspberry (closely to R. corchorifolius L.fil) plastid haplotype, pointing to a hybrid origin. We also found groups of nearby plants with identical mutations, which likely reflect clonal spread with fixed somatic changes or the persistence of recent hybrid lineages. At the same time, accessions collected up to 140 km apart did not form separate clusters, showing weak geographic structuring along the coast. The results demonstrate that multilocus barcoding can reveal not only species boundaries but also evolutionary processes among Rubus such as hybridization, clonal propagation, and early stages of speciation. Full article

Background/Objectives: Sites of ribosomal RNA genes are the most widely documented regions of chromosomes in various groups of eukaryotes, including insects. Data on the number and chromosomal location of 45S rDNAs (25S, 5.8S, and 18S rDNA) are actively used to study the diversity of karyotypes, the organization of individual chromosomes, and the evolution of entire genomes. In true bugs (suborder Heteroptera), the number and chromosomal distribution of 18S rDNA loci are currently known for less than 0.5% of described species. Although some patterns of rDNA distribution can already be identified both in individual taxa of true bugs and in the suborder as a whole, there are still negligible data. In order to expand our understanding of the diversity of rDNA distribution in Heteroptera, we studied for the first time the location of 18S rDNA in 13 species from 13 genera (seven families) of the infraorders Cimicomorpha and Pentatomomorpha (=Terheteroptera, the terminal group of Heteroptera). Methods: Fluorescence in situ hybridization (FISH) with an 18S rDNA probe was used in our study. Results: In total, we have identified three main types of rDNA arrangement: (1) on autosomes, (2) on the X chromosome, and (3) on autosomes and on the X chromosome simultaneously. In most of the studied species, 18S rDNA loci were detected in the terminal position on one pair of autosomes. Conclusions: This study contributed to the understanding of the chromosomal distribution of rDNA loci in the infraorders Cimicomorpha and Pentatomomorpha and confirmed the importance of rDNA in the reorganization of the genomes of Heteroptera as a whole. Full article