Whole Genome Sequencing Research Articles

Phenotypic and genomic characterization of ST11-K1 CR-hvKP with highly homologous bla KPC-2 -bearing plasmids in China

ABSTRACT Carbapenem-resistant hypervirulent Klebsiella pneumoniae (CR-hvKP) strains present a significant global public health threat due to their high mortality rates. This study investigated the genomic characteristics of seven ST11-K1 CR-hvKP isolates harboring highly homologous KPC-2-encoding multidrug-resistance plasmids. The strains were isolated from a Chinese tertiary hospital between 2017 and 2020. Whole-genome sequencing and bioinformatic analysis revealed various antibiotic resistance genes (ARGs) and virulence determinants. The bla KPC-2 -bearing plasmids that contain multiple antibiotic-resistance genes were also identified in these strains. ISfinder and Orifinder were applied to identify insertion sequences (IS) and conjugation-related factors among these bla KPC-2 -bearing plasmids. The bla KPC-2 was highly consistent in seven bla KPC-2 -bearing plasmids (IS Kpn6-bla KPC-2 -IS Kpn27 -IS Yps3 -IS 26 ). In addition, we found a region composed of IS IR , Tn 5393 , and IS 26 . It was located upstream of the bla CTX-M-15 gene and presented in six bla KPC-2 -bearing plasmids, with pCR-hvKP221-KPC-P3 as an exception. Conjugation experiments demonstrated the horizontal transfer of resistance plasmids pCR-hvKP128-KPC-P1 and pCR-hvKP132-KPC-P1 across species. Notably, pLVPK-like virulence plasmids carrying virulence gene clusters pCR-hvKP173-Vir-P1, and pCR-hvKP221-Vir-P1 were also detected. A fusional plasmid pCR-hvKP221-Vir-P2, which carries virulence gene clusters and ARGs, was also identified. Five CR-hvKP strains displayed enhanced biofilm formation and high virulence in vivo infection models. Phylogenetic and single nucleotide polymorphism (SNP) analyses indicated a close genetic relationship among the isolates, suggesting a subclade. These findings highlight the complex genetic profiles and potential transmission mechanisms of CR-hvKP strains. IMPORTANCE We reported seven CR-hvKP strains all carried a highly homologous bla KPC-2 integrated IncFⅡ-resistant plasmid, and two strains harbored virulence plasmids. Conjugation experiments confirmed the transferability of these plasmids, indicating a potential for resistance spread. Phylogenetic analysis clarified the relationship among the CR-hvKP isolates. This study provides insights into the phenotypic and genomic characteristics of seven ST11-K1 CR-hvKP strains. The high prevalence and potential for local outbreaks emphasize the need for effective control measures.

De Novo Missense Mutation in FREM1 Identified in a Chinese Patient with Comorbid Congenital Microtia and Pulmonary Hypoplasia

Cases of microtia combined with pulmonary hypoplasia are occasionally in clinics, and its genetic etiology has so far proved inconclusive. Here, aiming to contribute to a better understanding of microtia-related comorbid respiratory anomalies, the authors provide a clinical and genetic description of a rare trio family of which the son suffers combined deformities of right microtia, left pulmonary hypoplasia, and dextrocardia using whole-genome sequence (WGS). A novel potential pathologic compound heterozygosity in the FREM1 gene was identified and validated by the trio and bioinformatics analysis. This case expands the FREM1 mutation genotype and phenotype spectrum, sheds light on the congenital deformities of microtia and pulmonary hypoplasia, and emphasizes the importance of the FREM1 gene in pulmonary and craniofacial development.

C-C motif chemokine receptor-2 blockade ameliorates pulmonary hypertension in rats and synergizes with a pulmonary vasodilator

Abstract Aims We investigated whether the disruption of C-C motif chemokine receptor (CCR) 2 may attenuate the development of pulmonary arterial hypertension (PAH) in any rat models with the reversal of the associated pro-inflammatory state and vascular dysfunction, and synergize with a conventional pulmonary vasodilator. Methods and Results Using Ccr2(-/-) rats generated by CRISPR/Cas9, we investigated pulmonary hypertension (PH) in Ccr2(+/+) or Ccr2(-/-) rats treated with monocrotaline (MCT), SU5416/hypoxia (SuHx) and chronic hypoxia (CH). Ccr2(-/-) decreased the right ventricular systolic pressure, an index of right ventricular hypertrophy and mortality rate, and reversed increased expression of inflammatory cytokines/chemokines (interleukin-6, tumor necrosis factor-α, C-C motif chemokine receptor (CCL)-2, interleukin-1β, transforming growth factor-β) in rats 3weeks after MCT injection, but not in SuHx or CH models. Consistently, Ccr2(-/-) decreased indices of pulmonary vascular diseases (PVD) and perivascular macrophage infiltration, as well as reversed impaired bone morphogenetic protein receptor type 2 signaling, increased endothelial apoptosis and impaired nitric oxide signaling and decreased phosphodiesterase-5 (PDE5) expression in lungs in MCT-treated rats. Gene expression of receptors for prostaglandin I2 and endothelin was not changed by Ccr2(-/-) in MCT-treated rats. In cultured pulmonary arterial smooth muscle cells (PASMCs), Ccr2(-/-) suppressed CCL2-induced hyperproliferation and dedifferentiation as well as reversed CCL2-induced decrease in PDE5 expression. The whole-genome RNA sequencing analysis identified differentially expressed genes in CCL2-stimulated Ccr2(-/-) PASMCs, which are related to regulation of cellular differentiation and contraction. Based on studies in rats and cultured PASMCs, we investigated whether a PDE5 inhibitor, tadalafil, synergizes with Ccr2(-/-). Tadalafil administration ameliorated PH and PVDs in MCT-treated Ccr2(-/-) rats but not in Ccr2(+/+) rats. Tadalafil further improved survival in MCT-treated Ccr2(-/-) rats. Conclusion The present findings demonstrated that CCR2 disruption ameliorated PAH in MCT-treated rats, which was associated with the reversal of dysregulated inflammatory pathways and vascular dysfunction and synergized with tadalafil. These findings suggest that CCR2 may be a therapeutic target in intractable PAH patients with a certain CCR2-related inflammatory phenotype and refractory to conventional pulmonary vasodilators.

Prognostic value of circulating tumor DNA in different cancer types detected by ultra-low-pass whole-genome sequencing. A systematic review and patient-level survival data meta-analysis.

Ultra-low-pass whole-genome sequencing (ULP-WGS) (≤0.5× coverage) of plasma cell-free DNA (cfDNA) has emerged as a low-cost promising tool to assess circulating tumor DNA (ctDNA) fraction. This meta-analysis aims to summarize the current findings and comprehensively investigate the prognostic value of baseline ctDNA detected by ULP-WGS in solid tumors. A systematic review was carried out by searching PubMed/MEDLINE and Scopus databases to identify eligible studies conducted between January 2014 and January 2024. Inclusion criteria comprised studies with reported overall survival (OS) and progression-free survival (PFS) outcomes across therapy-naïve patients with different solid tumors. All patients underwent baseline ULP-WGS of plasma cfDNA and were categorized as ctDNA positive (tumor fraction ≥10%) or negative (tumor fraction <10%). A one-stage meta-analysis was performed using patient-level survival data reconstructed from published articles. A Cox proportional hazards model with shared frailty was used to assess the difference in survival between arms. A total of six studies, comprising 620 patients (367 negative ctDNA and 253 positive ctDNA), were included in the OS analysis, while five studies, involving 349 patients (212 negative ctDNA and 137 positive ctDNA), were included in the PFS analysis. The meta-analysis showed that patients with baseline positive ctDNA had a significantly higher risk of death (HR = 2.60, 95% CI: 2.01-3.36) and disease progression (HR = 2.28, 95% CI: 1.71-3.05) compared to those with negative ctDNA. The presence of a positive ctDNA at baseline is associated with increased risk of death and progression in patients with same stage cancer.

Whole-genome sequencing confirms multiple species of Galapagos giant tortoises.

Galapagos giant tortoises are endemic to the Galapagos Archipelago, where they are found in isolated populations. While these populations are widely considered distinguishable in morphology, behavior, and genetics, the recent divergence of these taxa has made their status as species controversial. Here, we apply multispecies coalescent methods for species delimitation to whole genome resequencing data from 38 tortoises across all 13 extant taxa to assess support for delimiting these taxa as species. In contrast to previous studies based solely on divergence time, we find strong evidence to reject the hypothesis that all Galapagos giant tortoises belong to a single species. Instead, a conservative interpretation of model-based and divergence-based results indicates that these taxa form a species complex consisting of a minimum of 9 species, with most analyses supporting 13 species. There is mixed support for the species status of taxa living on the same island, with some methods delimiting them as separate species and others suggesting multiple populations of a single species per island. These results make clear that Galapagos giant tortoise taxa represent different stages in the process of speciation, with some taxa further along in that evolutionary process than others. Our study provides insight into the complex process of speciation on islands, which is urgently needed given the threatened status of island species around the world. Las tortugas gigantes de las Galápagos son endémicas del Archipiélago de Galápagos, donde se encuentran como poblaciones aisladas. Aunque estas poblaciones se consideran distinguibles en cuanto a morfología, comportamiento y genética, la divergencia reciente de estos taxones hace que su estatus como especies sea controvertido. Aquí aplicamos métodos de coalescencia de especies múltiples con datos de resecuenciación de genomas completos de 38 tortugas de los 13 taxones existentes para evaluar el sustento de la delimitación de estos taxones como especies. En contraste con estudios previos basados únicamente en el tiempo de divergencia, encontramos evidencia sólida para rechazar la hipótesis de que todas las tortugas gigantes de las Galápagos pertenecen a una sola especie. En cambio, una interpretación conservadora de los resultados basados en modelos y divergencia indica que estos taxones forman un complejo de especies que consiste de un mínimo de 9 especies, con la mayoría de los análisis respaldando la existencia de 13 especies. Hay sustento mixto para designar como especies a los taxones que habitan la misma isla, con algunos métodos delimitándolos como especies y otros sugiriendo la existencia de poblaciones múltiples de una sola especie por isla. Estos resultados dejan en claro que los taxones de tortugas gigantes de las Galápagos representan diferentes etapas del proceso de especiación, con algunos taxones más avanzados en ese proceso evolutivo que otros. Nuestro estudio ofrece una perspectiva sobre el complejo proceso de especiación en islas, la cual es urgentemente necesaria dado lo amenazadas que se encuentran las especies insulares en todo el mundo.

Genomic Prediction Using Imputed Whole-Genome Sequence Data in Australian Angus Cattle.

Whole-genome sequence (WGS) data was used to estimate genomic breeding values for growth and carcass traits in Australian Angus cattle. The study aimed to compare the accuracy and bias of genomic predictions with three marker densities, including 50K, high-density (HD) and WGS. The dataset used in this study consisted of animals born between 2013 and 2022. Body weight traits included birthweight, weight at 400 days and weight at 600 days of age. The carcass traits were carcass weight, carcass intramuscular fat and carcass marbling score. The accuracy and bias of prediction were assessed using the cross-validation. Further, for the growth traits, animals in the validation group were subdivided into two subgroups, which were moderately or highly related to the reference. Genomic best linear unbiased prediction (GBLUP) was used to compare genomic predictions with the three marker densities. The prediction accuracies were generally similar across the marker densities, ranging between 0.61 and 0.68 for the body weight traits and between 0.40 and 0.52 for the carcass traits. However, the accuracies marginally decreased as the marker density increased for all the traits studied. A similar lack of difference was found when considering the accuracy by the relatedness subgroups. The results indicated that no meaningful difference in prediction accuracy was estimated when comparing the three marker densities due to the population structure. In conclusion, there was no substantial improvement in genomic prediction when using the WGS in this study.

Whole-genome sequencing of copy number variation analysis in Ethiopian cattle reveals adaptations to diverse environments.

Genomic structural variations (GSVs), notably copy number variations (CNVs), significantly shape genetic diversity and facilitate adaptation in cattle populations. Despite their importance, the genome-wide characterization of CNVs in indigenous Ethiopian cattle breeds-Abigar, Fellata, and Gojjam-Highland remains largely unexplored. In this study, we applied a read-depth approach to whole genome sequencing (WGS) data to conduct the first comprehensive analysis of CNVs in these populations. We identified 3,893 CNV regions (CNVRs) covering 19.15Mb (0.71% of the cattle genome). These CNVRs ranged from 1.60kb to 488.0kb, with an average size of 4.92kb. These CNVRs included deletions (1713), duplications (1929), and mixed events (251) showing notable differences in distribution among the breeds. Four out of five randomly selected CNVRs were successfully validated using real time polymerase chain reaction (qPCR). Further analyses identified candidate genes associated with high-altitude adaptation (GBE1 and SOD1), heat stress adaptation (HSPA13, DNAJC18, and DNAJC8) and resistance to tick infestations (BoLA and KRT33A). In addition, variance stabilizing transformation (VST) statistics highlighted population-specific CNVRs, emphasizing the unique genetic signatures of high-altitude adaptation in the Gojjam-Highland cattle breed. Among the detected CNVRs, 4.93% (192 out of 3,893) overlapped with 520 quantitative traits loci (QTLs) associated with six economically important trait categories suggesting that these CNVRs may significantly contribute to the genetic variation underlying these traits. Our comprehensive analysis reveals significant CNVRs associated with key adaptive traits in Ethiopian cattle breeds highlighting their genetic diversity and resilience. These findings offer valuable insights into the genetic basis of adaptability and can inform sustainable breeding practices and conservation efforts. Future research should prioritize the functional validation of these CNVRs and their integration into breeding programs to enhance traits such as disease resistance and environmental adaptability.

A new blood parasite of the accentor birds: description, molecular characterization, phylogenetic relationships and distribution.

Haemoproteus bobricklefsi sp. nov. (Haemosporida, Haemoproteidae) was found in the dunnock Prunella modularis and represents the first blood parasite described in accentor birds of the Prunellidae. The description is based on the morphology of blood stages and includes information about a barcoding segment of the mitochondrial cytochrome b gene (lineage hDUNNO01) and the full mitochondrial genome, which can be used for identification and diagnosis of this infection. The new parasite can be readily distinguished from described species of haemoproteids parasitizing passeriform birds due to markedly variable position of nuclei in advanced and fully grown macrogametocytes. Illustrations of blood stages of the new species are given, and phylogenetic analyses based on partial mitochondrial cytochrome b gene sequences and the full mitochondrial genome identified the closely related lineages. DNA haplotype networks showed that transmission occurs in Europe and North America. This parasite was found in the dunnock in Europe and several species of the Passerellidae in North America. It is probably of Holarctic distribution, with the highest reported prevalence in the UK. The parasite distribution seems to be geographically patchy, with preference for areas of relatively cool climates. Phylogenetic analysis suggests that H. bobricklefsi sp. nov. belongs to the Parahaemoproteus subgenus and is probably transmitted by biting midges belonging to Culicoides (Ceratopogonidae). The available data on molecular occurrence indicate that this pathogen is prone to abortive development, so worth attention in regard of consequences for bird health.

The Genomic SSR Millets Database (GSMDB): enhancing genetic resources for sustainable agriculture.

The global population surge demands increased food production and nutrient-rich options to combat rising food insecurity. Climate-resilient crops are vital, with millets emerging as superfoods due to nutritional richness and stress tolerance. Given limited genomic information, a comprehensive genetic resource is crucial to advance millet research. Whole-genome sequencing provides an unprecedented opportunity, and molecular genetic methodologies, particularly simple sequence repeats (SSRs), play a pivotal role in DNA fingerprinting, constructing linkage maps, and conducting population genetic studies. SSRs are composed of repetitive DNA sequences where one to six nucleotides are repeated in tandem and distributed throughout the genome. Different millet species exhibit genomic variations attributed to the presence of SSRs. While SSRs have been identified in a few millet species, the existing information only covers some of the sequenced genomes. Moreover, there is an absence of complete gene annotation and visualization features for SSRs. Addressing this disparity and leveraging the de-novo millet genome assembly available from the NCBI, we have developed the Genomic SSR Millets Database (GSMDB; https://bioinfo.icgeb.res.in/gsmdb/). This open-access repository provides a web-based tool offering search functionalities and comprehensive details on 6.747645 million SSRs mined from the genomic sequences of seven millet species. The database, featuring unrestricted public access and JBrowse visualization, is a pioneering resource for the research community dedicated to advancing millet cultivars and related species. GSMDB holds immense potential to support myriad studies, including genetic diversity assessments, genetic mapping, marker-assisted selection, and comparative population investigations aiming to facilitate the millet breeding programs geared toward ensuring global food security. Database URL: https://bioinfo.icgeb.res.in/gsmdb/.

Whole-Genome Sequence and Characterization of Ralstonia solanacearum MLY102 Isolated from Infected Tobacco Stalks

Background/Objectives: Bacterial wilt disease is a soil-borne disease caused by Ralstonia solanacearum that causes huge losses to crop economies worldwide. Methods: In this work, strain MLY102 was isolated and further identified as R. solanacearum from a diseased tobacco stalk. The genomic properties of MLY102 were explored by performing biochemical characterization, genome sequencing, compositional analysis, functional annotation and comparative genomic analysis. Results: MLY102 had a pinkish-red color in the center of the colony surrounded by a milky-white liquid with fluidity on TTC medium. The biochemical results revealed that MLY102 can utilize carbon sources, including D-glucose (dGLU), cane sugar (SAC) and D-trehalose dihydrate (dTRE). Genome sequencing through the DNBSEQ and PacBio platforms revealed a genome size of 5.72 Mb with a G+C content of 67.59%. The genome consists of a circular chromosome and a circular giant plasmid with 5283 protein-coding genes. A comparison of the genomes revealed that MLY102 is closely related to GMI1000 and CMR15 but has 498 special genes and 13 homologous genes in the species-specific gene family, indicating a high degree of genomic uniqueness. Conclusions: The unique characteristics and genomic data of MLY102 can provide important reference values for the prevention and control of bacterial wilt disease.

Assessing the Impact of Bedaquiline, Clofazimine, and Linezolid on Mycobacterial Genome Integrity

Tuberculosis (TB) presents significant medical challenges, largely due to the genetic diversity of Mycobacterium tuberculosis, which enhances the resilience and resistance of the pathogen to first-line treatments. In response to the global rise of drug-resistant TB, second-line antitubercular drugs like bedaquiline (BDQ), linezolid (LZD), and clofazimine (CFZ) have become critical treatment options. Understanding the molecular changes these drugs induce is essential for optimizing TB therapy. To contribute to this effort, we investigated their impact on genome maintenance and stability using Mycobacterium smegmatis as a model organism. Using mutation accumulation assays and whole-genome sequencing, we found that the second-line antibiotics did not significantly increase mutation rates, unlike the positive control UV treatment. However, upon BDQ treatment, we detected mutations in transporter proteins and transcription factors without any increase in the minimal inhibitory concentration. Additionally, BDQ and CFZ were found to alter DNA repair pathways and reduce cellular dNTP levels, particularly CFZ, which depleted dGTP, impacting DNA synthesis. CFZ also upregulated DNA repair enzymes, enhancing error-free repairs. Despite minimal mutagenic effects, both drugs displayed distinct impacts on cellular mechanisms, suggesting additional modes of action.

Unraveling the DNA methylation landscape in dog blood across breeds.

DNA methylation is a covalent bond modification that is observed mainly at cytosine bases in the context of CG pairs. DNA methylation patterns reflect the status of individual tissues, such as cell composition, age, and the local environment, in mammals. Genetic factors also impact DNA methylation, and the genetic diversity among various dog breeds provides a valuable platform for exploring this topic. Compared to those in the human genome, studies on the profiling of methylation in the dog genome have been less comprehensive. Our study provides extensive profiling of DNA methylation in the whole blood of three dog breeds using whole-genome bisulfite sequencing. The difference in DNA methylation between breeds was moderate after removing CpGs overlapping with potential genetic variation. However, variance in methylation between individuals was common and often occurred in promoters and CpG islands (CGIs). Moreover, we adopted contextual awareness methodology to characterize DNA primary sequences using natural language processing (NLP). This method could be used to effectively separate unmethylated CGIs from highly methylated CGIs in the sequences that are identified by the conventional criteria. This study presents a comprehensive DNA methylation landscape in the dog blood. Our observations reveal the similar methylation patterns across dog breeds, while CGI regions showed high variations in DNA methylation level between individuals. Our study also highlights the potential of NLP approach for analyzing low-complexity DNA sequences, such as CGIs.

The discovery of an overseen pygmy backswimmer in Europe (Heteroptera, Nepomorpha, Pleidae).

The Pleidae, or pygmy backswimmers, is a family of aquatic bugs (Hemiptera, Heteroptera, Nepomorpha) containing four genera. Here, we describe Plea cryptica sp. nov. and redescribe its sister species, Plea minutissima Leach, 1817. Whereas the morphological distinction of these closely related species is only possible for males, molecular data clearly separate them. As part of our taxonomic study, we provide comprehensive molecular data including more than 200 DNA barcodes from all over Europe, complete nuclear ribosomal DNA, full mitochondrial genome data, and 3D scans for both species. Furthermore, the same molecular markers are also presented for Neoplea striola (Fieber, 1844). We used Maximum Likelihood (ML) analyses to reconstruct the phylogeny of the Pleidae and Notonectoidea based on available mitogenomic data. Our study represents a successful implementation of the proposed concept of taxonomics, using data from high-throughput sequencing technologies for integrative taxonomic studies, and allowing high confidence for both biodiversity and ecological research.

Complete genome sequence of tsaoko stripe mosaic virus, a novel macluravirus found in Amomum tsaoko.

A novel macluravirus, tentatively named "tsaoko stripe mosaic virus" (TkSMV), was identified in Amomum tsaoko through high-throughput sequencing. The complete genome sequence of TkSMV was determined using RT-PCR and RACE. The genome sequence consists of 8218 nucleotides, excluding the poly(A) tail, and contains a large open reading frame encoding a polyprotein of 2625 amino acids with a molecular weight of approximately 297.13 kDa. TkSMV is most closely related to Alpinia oxyphylla mosaic virus, sharing 71.5% nucleotide and 75.9% amino acid sequence identity. These values are below the species demarcation threshold for the family Potyviridae. These results suggest that TkSMV should be considered a distinct member of the genus Macluravirus.

A novel picorna-like virus in the flatworm Stenostomum leucops (Catenulida).

We present the genome sequence and organization and evidence of persistence of a new picorna-like virus infecting the flatworm Stenostomum leucops. The complete genome sequence belongs to a virus with a positive single-stranded RNA genome with two open reading frames (ORFs) flanked by untranslated regions and a polyadenylated C-terminus. The ORFs encode proteins with conserved motifs typical of members of the order Picornavirales. Phylogenetic analysis confirmed membership in this viral order, and it was found to be closely related to viruses found in Biomphalaria (Mollusca) in France and a virus detected in a metagenomic analysis of water sources from the USA, suggesting widespread distribution. RT-PCR analysis revealed that this virus can be detected in a laboratory-grown worm isolate for at least five years, suggesting persistent infection. However, no apparent deleterious effects were observed in the worms in culture, suggesting a possible commensal relationship between the virus and the worms.

Neural network-based predictions of antimicrobial resistance phenotypes in multidrug-resistant Acinetobacter baumannii from whole genome sequencing and gene expression.

Whole genome sequencing (WGS) potentially represents a rapid approach for antimicrobial resistance genotype-to-phenotype prediction. However, the challenge still exists to predict fully minimum inhibitory concentrations (MICs) and antimicrobial susceptibility phenotypes based on WGS data. This study aimed to establish an artificial intelligence-based computational approach in predicting antimicrobial susceptibilities of multidrug-resistant Acinetobacter baumannii from WGS and gene expression data. Antimicrobial susceptibility testing (AST) was performed using the broth microdilution method for 10 antimicrobial agents. In silico multilocus sequence typing (MLST), antimicrobial resistance genes, and phylogeny based on cgSNP and cgMLST strategies were analyzed. High-throughput qPCR was performed to measure the expression level of antimicrobial resistance (AMR) genes. Most isolates exhibited a high level of resistance to most of the tested antimicrobial agents, with the majority belonging to the IC2/CC92 lineage. Phylogenetic analysis revealed undetected transmission events or local outbreaks. The percentage agreements between AMR phenotype and genotype ranged from 70.08% to 89.96%, with the coefficient of agreement (κ) extending from 0.025 and 0.881. The prediction of AST employed by deep neural network models achieved an accuracy of up to 98.64% on the testing data set. Additionally, several linear regression models demonstrated high prediction accuracy, reaching up to 86.15% within an error range of one gradient, indicating a linear relationship between certain gene expressions and the corresponding antimicrobial MICs. In conclusion, neural network-based predictions could be used as a tool for the surveillance of antimicrobial resistance in multidrug-resistant A. baumannii.

Whole-genome sequencing reveals genetic structure and adaptive genes in Nepalese buffalo breeds.

Indigenous buffaloes, as the important livestock species contributing to economy of the country, are the lifeline of livelihood in Nepal. They are distributed across diverse geographical regions of the country and have adapted to various feeding, breeding, and management conditions. The larger group of these native buffalo breeds are present in narrow and stiff hilly terrains. Their dispersal indicates a possible environmental adaptation mechanism, which is crucial for the conservation of these breeds. We utilized whole-genome sequencing (WGS) to investigate the genetic diversity, population structure, and selection signatures of Nepalese indigenous buffaloes. We compared 66 whole-genome sequences with 118 publicly available sequences from six river and five swamp buffalo breeds. Genomic diversity parameters indicated genetic variability level in the Nepalese buffaloes comparable to those of Indian breeds, and population genetic structure revealed distinct geography-mediated genetic differentiation among these breeds. We used locus-specific branch length analysis (LSBL) for genome-wide scan, which revealed a list of potentially selected genes in Lime and Parkote breeds that inhabit the hilly region. A gene ontology (GO) analysis discovered that many GO terms were associated with cardiac function regulation. Furthermore, complementary analyses of local selection signatures, tissue expression profiles, and haplotype differences identified candidate genes, including KCNE1, CSF1R, and PDGFRB, related to the regulation of cardiac and pulmonary functions. This study is a comprehensive WGS-based genetic analysis of the native Nepalese buffalo breeds. Our study suggested that the Nepalese "hilly" buffaloes, especially the Lime and Parkote breeds, have undergone some characteristic genetic changes and evolved increased cardiac and pulmonary function for their adaptation to the steep hilly terrains of the country.

Complete genome sequences of mycobacteriophages Ashwin from cluster O and Chanagan from cluster A1.

Ashwin and Chanagan are double-stranded siphoviral mycobacteriophages isolated from a single soil sample from a lavender flowerpot in Johannesburg, South Africa. Both are capable of infecting and lyzing Mycobacterium smegmatis and are predicted to contain 122 and 91 open reading frames, respectively, most without known function.

First report of paprika as a natural host plant for tomato chlorosis virus in Korea.

Paprika (Capsicum annuum var. grossum (L.) Sendtn.), also called bell pepper or sweet pepper, is a valuable greenhouse crop that is mostly consumed as fresh fruit in Korea. In 2022, it was cultivated on 726 ha in Korea and 82,042 tons were produced (KOSIS, 2023). In April 2023, interveinal chlorosis and yellowing were observed on the lower and middle leaves of greenhouse paprika in Jinju and Haman, South Korea, and many whiteflies (Bemisia tabaci) were present. The disease incidence was 30-40% on five farms (approximately 4.5 ha) in the two areas. The typical symptoms and presence of whiteflies in greenhouses indicated potential infection by the whitefly-transmitted crinivirus, tomato chlorosis virus (ToCV). Six samples collected from symptomatic plants were examined by leaf dip using transmission electron microscopy (TEM) and revealed filamentous virus particles about 800 nm long. To confirm the TEM results, six symptomatic paprika leaf samples were subject to RT-PCR using the specific primers ToCV-M-4F and ToCV-M-4R to detect ToCV (Choi, 2023). All tested samples were positive for ToCV. To confirm the presence of other viruses and obtain the complete genome sequences, one of the six ToCV-positive samples (ToCV-PAP-JJ6) was subject to high-throughput sequencing (HTS). Total RNA was extracted from symptomatic leaves using the RNeasy Plant Mini Kit (QIAGEN, Germany) and a transcriptome library was generated using the TruSeq Stranded Total RNA LT Sample Prep Kit (Illumina, San Diego, CA, USA) according to standard protocols. HTS was performed on an Illumina NovaSeq 6000 system (Macrogen, Korea). De novo transcriptome assembly of the 85,668,854 reads with Trinity software (r20140717) yielded 817,303 contigs of 201 to 38,987 nucleotides (nt). BLASTn and BLASTx analysis of the contigs against the NCBI viral reference database showed that three large contigs were virus sequences, two of which were homologous to ToCV-RNA1 and -RNA2 and one to bell pepper alphaendornavirus (BPEV). The ToCV-RNA1 (genome 8,594 nt, 11,212 mapped reads, mean read coverage 196.2 times) and ToCV-RNA2 (genome 8,242 nt, 40,122 mapped reads, mean read coverage 734.9 times) contigs showed 100% coverage and 99% base pair matching (8,583/8,594 and 8,233/8,242, respectively) with known genome sequences of ToCV-RNA1 (MG813908) and ToCV-RNA2 (KP114534) isolated from tomato in Korea (Lee et al. 2018). The sequences were deposited in GenBank as isolates ToCV-PAP-JJ6 RNA1 (OR865222) and ToCV-PAP-JJ6 RNA2 (OR865223). The BPEV contig (genome 14,728 nt, 2,928,519 mapped reads, mean read coverage 15,012.4 times) was detected at very high read depth and RT-PCR for BPEV confirmed that all six samples were coinfected with ToCV. BPEV was recently reported in paprika and red pepper in Korea (Jo et al., 2022). The BPEV-infected pepper cultivars have been reported not to produce any symptoms (Escalante & Valverde 2019). ToCV was first reported in tomato greenhouses in Korea in 2013 and is a problematic virus in tomato crops, along with tomato yellow leaf curl virus (Jo et al. 2023; Lee et al. 2018). Due to the continuous presence of ToCV, whiteflies, and natural weed hosts (Kil et al. 2015) in tomato greenhouses, ToCV has spread to other crops, including paprika. To the best of our knowledge, this is the first report of ToCV infecting paprika in Korea. A nationwide study is needed to prevent the spread of ToCV, a new threat to paprika farms.

Draft genome sequences of multi-drug-resistant Escherichia coli strains isolated from ulcerative colitis patients.

The whole-genome sequences of four multi-drug-resistant Escherichia coli strains, namely, DAK02, DAK03, DAK05, and DAK10, are reported here. The E. coli strains were isolated from fecal samples of ulcerative colitis patients from Northern India. The size of the draft genome sequence ranged from 4,809 to 5,000 kb.