Identification Of Resistance Genes Research Articles

Antimicrobial Resistance Gene Profiles in Communities Selected with Novel and Previously Encountered Antibiotics

Antibiotics are extensively employed worldwide for therapeutic purposes, often leading to overuse, which constitutes a contributing factor to the emergence of antibiotic resistance (ABR). This phenomenon arises from diminished effectiveness of antibiotics in treating bacterial infections due to resistance development. However, limitations in experimental conditions and equipment making it challenging to pinpoint the precise sources of antibiotic resistance, consequently constraining the breadth of research on ABR. Genomic analysis of antibiotic-resistant bacteria assists in unveiling the origins and pathways of resistance dissemination. This study employs Nanopore-based metagenomic sequencing, enabling direct sequencing of microbial DNA within macrogenomic samples, thereby yielding comprehensive sequence data. Coupled with qPCR and 16S rRNA gene datasets, this approach facilitates the exploration of antibiotic resistance gene distribution and prevalence within microbial communities across different environments Focusing on microbial communities within Scottish farmlands, isolated colonies were exposed to antibiotics (sulfamonomethoxine and erythromycin) for several weeks. By comparing them to untreated control group microorganisms, the identification of antibiotic resistance genes (ABRGs), along with their variations in prevalence and abundance, aims to analyse the impact of these two antibiotics on these species.

Pathogenic potential of ornithogenic <i>Escherichia coli</i> strains detected in the Earth's polar regions

Introduction. Pathogenic strains of Escherichia coli are an important object of surveillance within the One Health concept in the wild, agriculture and human society. Migratory bird colonies and high latitude avian colonies may be points of active intraspecies and interspecies contact between different animal species, accompanied by the spread of pathogens. At the same time, the phylogeography of E. coli in relation to the presence of natural foci of colibacillosis in polar regions remains virtually unstudied. The aim of this study was to assess the pathogenic potential of E. coli strains from the polar regions of the Earth, based on the analysis of the genomes of these bacteria from typical ornithogenic ecosystems of the Arctic and Antarctic. Materials and methods. The study used collections of E. coli isolated from ornithogenic biological material during expeditions to high latitude areas of the Arctic (archipelagos of Novaya Zemlya, Franz Josef Land, Svalbard) and Antarctic (Haswell Archipelago). 16 cultures associated with avian E. coli (12 polar and 4 temperate strains) were selected for genome-wide sequencing using BGI technology. The annotation of the genomes focused on the identification of genes for pathogenicity factors and antimicrobial resistance, as well as the identification of strains belonging to individual genetic lineages using the cgMLST method. Results. The annotation of the genomes allowed their assignment to different sequence types in the multilocus sequencing typing and genome-wide sequencing typing schemes. The analysis of the geographical distribution of the sequence types of polar E. coli strains determined by the cgMLST method showed their global representation in geographically distant regions of the planet. For example, cgST 133718 was observed in Antarctica (strain 17_1myr) and in the UK, and sequence 11903, to which strain 32-1 from the northernmost point of Novaya Zemlya belonged, was previously identified in the USA. All strains studied were characterized by the presence of extensive virulence. Among the pathogenicity factors identified were haemolysins A, E, F, siderophores, including the yersiniabactin gene cluster, a number of adhesion, colonization and invasion factors, as well as the thermostable enterotoxin EAST-1 and genes that characterize enteroaggregative strains of E. coli (the virulence regulator gene eilA and enteroaggregative protein (air)). One of the Arctic strains (33-1) had determinants of antibiotic resistance, in particular the extended-spectrum beta-lactamase gene TEM-1b and the Tn1721 transposon, including tetracycline resistance genes (tetA-TetR), were detected in its genome. Conclusion. The results of the study indicate the circulation of E. coli strains with strong pathogenic potential in high-latitude Arctic and Antarctic ornithogenic ecosystems. The analysis of genomic data indicates the presence of geographically widespread genetic lineages in these regions, which justifies the importance of monitoring epidemic clones of E. coli, along with monitoring for other pathogens, in bird colonies in high-latitude areas.

Identification and functional analyses of drought stress resistance genes by transcriptomics of the Mongolian grassland plant Chloris virgata

BackgroundMongolian grasslands, including the Gobi Desert, have been exposed to drought conditions with few rains. In such harsh environments, plants with highly resistant abilities against drought stress survive over long periods. We hypothesized that these plants could harbor novel and valuable genes for enhancing drought stress resistance.ResultsIn this study, we identified Chloris virgata, a Mongolian grassland plant with strong drought resistance. RNA-seq-based transcriptome analysis was performed to uncover genes associated with drought stress resistance in C. virgata. De novo transcriptome assembly revealed 25,469 protein-coding transcripts and 1,219 upregulated genes after 3- and 6-hr drought stress treatments. Analysis by homology search and Gene Ontology (GO) enrichment indicated that abscisic acid (ABA)- and drought stress-related GO terms were enriched. Among the highly induced genes, ten candidate cDNAs were selected and overexpressed in Arabidopsis. When subjected to drought stress, three of these genes conferred strong drought resistance in the transgenic plants. We named these genes Mongolian Grassland plant Drought-stress resistance genes 1, 2, and 3 (MGD1, MGD2, and MGD3). Gene expression analyses in the transformants suggested that MGD1, MGD2, and MGD3 may activate drought stress-related signalling pathways.ConclusionThis study highlighted the drought resistance of C. virgata and identified three novel genes that enhance drought stress resistance.

Identification of novel candidate genes for Ascochyta blight resistance in chickpea

Ascochyta blight, caused by the necrotrophic fungus Ascochyta rabiei, is a major threat to chickpea production worldwide. Resistance genes with broad-spectrum protection against virulent A. rabiei strains are required to secure chickpea yield in the US Northern Great Plains. Here, we performed a genome-wide association (GWA) study to discover novel sources of genetic variation for Ascochyta blight resistance using a worldwide germplasm collection of 219 chickpea lines. Ascochyta blight resistance was evaluated at 3, 9, 11, 13, and 14 days post-inoculation. Multiple GWA models revealed eight quantitative trait nucleotides (QTNs) across timepoints mapped to chromosomes 1, 3, 4, 6, and 7. Of these eight QTNs, only CM001767.1_28299946 on Chr 4 had previously been reported. QTN CM001766.1_36967269 on Chr 3 explained up to 33% of the variation in disease severity and was mapped to an exonic region of the pentatricopeptide repeat-containing protein At4g02750-like gene (LOC101506608). This QTN was confirmed across all models and timepoints. A total of 153 candidate genes, including genes with roles in pathogen recognition and signaling, cell wall biosynthesis, oxidative burst, and regulation of DNA transcription, were observed surrounding QTN-targeted regions. Further gene expression analysis on the QTNs identified in this study will provide insights into defense-related genes that can be further incorporated into breeding of new chickpea cultivars to minimize fungicide applications required for successful chickpea production in the US Northern Great Plains.

Identification and Functional Analysis of the Ph-2 Gene Conferring Resistance to Late Blight (Phytophthora infestans) in Tomato.

Late blight is a destructive disease affecting tomato production. The identification and characterization of resistance (R) genes are critical for the breeding of late blight-resistant cultivars. The incompletely dominant gene Ph-2 confers resistance against the race T1 of Phytophthora infestans in tomatoes. Herein, we identified Solyc10g085460 (RGA1) as a candidate gene for Ph-2 through the analysis of sequences and post-inoculation expression levels of genes located within the fine mapping interval. The RGA1 was subsequently validated to be a Ph-2 gene through targeted knockout and complementation analyses. It encodes a CC-NBS-LRR disease resistance protein, and transient expression assays conducted in the leaves of Nicotiana benthamiana indicate that Ph-2 is predominantly localized within the nucleus. In comparison to its susceptible allele (ph-2), the transient expression of Ph-2 can elicit hypersensitive responses (HR) in N. benthamiana, and subsequent investigations indicate that the structural integrity of the Ph-2 protein is likely a requirement for inducing HR in this species. Furthermore, ethylene and salicylic acid hormonal signaling pathways may mediate the transmission of the Ph-2 resistance signal, with PR1- and HR-related genes potentially involved in the Ph-2-mediated resistance. Our results could provide a theoretical foundation for the molecular breeding of tomato varieties resistant to late blight and offer valuable insights into elucidating the interaction mechanism between tomatoes and P. infestans.

Comparative proteomic and metabolomic analysis of resistant and susceptible Kentucky Bluegrass cultivars in response to infection by powdery mildew

BackgroundPoa pratensis is a predominant cool-season turfgrass utilized in urban landscaping and ecological management. It is extensively employed in turf construction and in the regulation of ecological environments. However, it is susceptible to powdery mildew, a prevalent disease in humid regions. Currently, the primary control measure for powdery mildew involves the application of pesticides, a practice that is both costly and environmentally detrimental. Developing superior disease-resistant cultivars represents a more cost-effective and sustainable strategy for managing turfgrass diseases. Furthermore, an in-depth investigation into the response mechanisms of P. pratensis to powdery mildew infection could significantly advance research on the identification of disease resistance genes and the molecular breeding of resistant varieties.ResultsIn this study, we first assessed the disease incidence across various disease-resistant P. pratensis cultivars and subsequently examined alterations in their in vivo redox states. We employed isobaric tags for relative and absolute quantification (iTRAQ) proteomics alongside non-targeted metabolomics to elucidate the response mechanisms of P. pratensis to powdery mildew invasion. A comprehensive analysis of the shared KEGG pathways among differentially abundant proteins (DAPs) and differentially enriched metabolites (DEMs) led to the identification of four common KEGG pathways. Notably, the phenylpropanoid biosynthesis pathway, enriched in both examined P. pratensis cultivars, was selected for further investigation. This analysis indicated that lignin biosynthesis plays a crucial role in the response of P. pratensis to powdery mildew infection.ConclusionsThe findings of this study enhance our understanding of the mechanisms underlying powdery mildew resistance in P. pratensis and serve as a valuable reference for the selection of powdery mildew-resistant cultivars, as well as for the identification and application of associated disease resistance genes.Clinical trial numberNot applicable.

Serotyping and Identification of Antimicrobial Resistance Genes in Avian Pathogenic Escherichia Coli Isolated from Poultry Flocks in Jiangxi Province, China.

Avian pathogenic Escherichia coli (APEC) is an important bacterial pathogen that causes severe respiratory and systemic infections in poultry. Our previous research investigated the prevalence and antimicrobial resistance phenotypes of APEC isolated from poultry flocks in Jiangxi Province, China. The present study aims to further identify the serotypes and the carbapenem-resistant gene blaNDM in APEC strains. Serotype investigations revealed that the most dominant serotype was O24 (53.2%), followed by O78 (11.9%), O2 (3.2%), O18 (2.4%), O45 (0.8%), and O88 (0.8%). Serotypes O1, O30, and O65 were not detected, and 35 strains (27.8%) were un-typed. The identified genes blaNDM-5 and blaNDM-1 shared a close phylogenetic distance with Klebsiella sp. and Acinetobacter sp. isolated from river and human feces, respectively. Two APEC strains carrying blaNDM-5 and blaNDM-1 were subjected to whole-genome sequencing and analysis. The results showed that blaNDM-5 was associated with the mobile genetic element IS5 and blaNDM-1 was associated with the mobile genetic element ISAba125. Current study findings can be helpful for effective vaccine development and provide a deep understanding of APEC infections and antimicrobial resistance in poultry flocks.

Genomic surveillance of multidrug-resistant organisms based on long-read sequencing

BackgroundMultidrug-resistant organisms (MDRO) pose a significant threat to public health worldwide. The ability to identify antimicrobial resistance determinants, to assess changes in molecular types, and to detect transmission are essential for surveillance and infection prevention of MDRO. Molecular characterization based on long-read sequencing has emerged as a promising alternative to short-read sequencing. The aim of this study was to characterize MDRO for surveillance and transmission studies based on long-read sequencing only.MethodsGenomic DNA of 356 MDRO was automatically extracted using the Maxwell-RSC48. The MDRO included 106 Klebsiella pneumoniae isolates, 85 Escherichia coli, 15 Enterobacter cloacae complex, 10 Citrobacter freundii, 34 Pseudomonas aeruginosa, 16 Acinetobacter baumannii, and 69 methicillin-resistant Staphylococcus aureus (MRSA), of which 24 were from an outbreak. MDRO were sequenced using both short-read (Illumina NextSeq 550) and long-read (Nanopore Rapid Barcoding Kit-24-V14, R10.4.1) whole-genome sequencing (WGS). Basecalling was performed for two distinct models using Dorado-0.3.2 duplex mode. Long-read data was assembled using Flye, Canu, Miniasm, Unicycler, Necat, Raven, and Redbean assemblers. Long-read WGS data with > 40 × coverage was used for multi-locus sequence typing (MLST), whole-genome MLST (wgMLST), whole-genome single-nucleotide polymorphisms (wgSNP), in silico multiple locus variable-number of tandem repeat analysis (iMLVA) for MRSA, and identification of resistance genes (ABRicate).ResultsComparison of wgMLST profiles based on long-read and short-read WGS data revealed > 95% of wgMLST profiles within the species-specific cluster cut-off, except for P. aeruginosa. The wgMLST profiles obtained by long-read and short-read WGS differed only one to nine wgMLST alleles or SNPs for K. pneumoniae, E. coli, E. cloacae complex, C. freundii, A. baumannii complex, and MRSA. For P. aeruginosa, differences were up to 27 wgMLST alleles between long-read and short-read wgMLST and 0–10 SNPs. MLST sequence types and iMLVA types were concordant between long-read and short-read WGS data and conventional MLVA typing. Antimicrobial resistance genes were detected in long-read sequencing data with high sensitivity/specificity (92–100%/99–100%). Long-read sequencing enabled analysis of an MRSA outbreak.ConclusionsWe demonstrate that molecular characterization of automatically extracted DNA followed by long-read sequencing is as accurate compared to short-read sequencing and suitable for typing and outbreak analysis as part of genomic surveillance of MDRO. However, the analysis of P. aeruginosa requires further improvement which may be obtained by other basecalling algorithms. The low implementation costs and rapid library preparation for long-read sequencing of MDRO extends its applicability to resource-constrained settings and low-income countries worldwide.

Performance of a hybrid capture-based target enrichment next-generation sequencing for the identification of respiratory pathogens and resistance-associated genes in patients with severe pneumonia.

Severe pneumonia remains the leading infectious cause of death worldwide. The time-consuming nature and suboptimal sensitivity of sputum cultures hamper prompt pathogen detection for tailored treatments. Advanced techniques such as polymerase chain reaction (PCR) and next-generation sequencing (NGS) offer rapid genetic pathogen detection and identification of antimicrobial resistance (AMR) genes. However, the performance of hybrid capture-based target enrichment NGS, e.g., Respiratory Pathogen ID/AMR Enrichment Panel (RPIP), for pathogen detection in patients with severe pneumonia remains uncertain. A prospective study involving adults with severe pneumonia was conducted. Respiratory samples from the lower respiratory tract were collected via bronchoalveolar lavage, bronchial washing, or endotracheal tube suction. The performance of RPIP in pathogen and AMR-associated gene detection was compared to that of conventional culture methods and the multiplex PCR-based FilmArray Pneumonia Panel (FilmArray-PN). A total of 83 subjects were enrolled. The most prevalent pathogens detected by RPIP were Rothia mucilaginosa, Stenotrophomonas maltophilia, Pseudomonas aeruginosa; herpes simplex virus-1, cytomegalovirus, and Epstein-Barr virus, and Pneumocystis jirovecii. Overall, the positive and negative agreement rates for bacterial detection were 63.6% and 97.5% between RPIP and culture methods, respectively, and 55.8% and 99.4% between FilmArray-PN and culture methods, respectively. Compared to FilmArray-PN, RPIP exhibited significantly better detection rates for bacteria (P = 0.029), viruses (P < 0.001), and fungi (P < 0.001) and identified additional blaOXA, blaCMY as extended-spectrum β-lactamase genes and blaOXA, blaSHV as carbapenemase genes. In conclusion, RPIP can sensitively profile respiratory pathogens and is a promising tool for detecting multiple microorganisms and AMR-associated genes in patients with severe pneumonia.IMPORTANCESensitive pathogen detection is pivotal for timely treatment by tailoring adequate antimicrobial agents. Unlike conventional phenotypic approach, novel measures using molecular interrogation appear promising. This study aimed to elucidate the efficacy of a hybrid capture-based target enrichment next-generation sequencing technique (Respiratory Pathogen ID/AMR Enrichment Panel, RPIP) as exemplified in a cohort with severe pneumonia. Pathogen landscape in the population was illustrated by these three methodologies. As compared with multiplex polymerase chain reaction-based FilmArray Pneumonia Panel and conventional culture, RPIP demonstrated significantly improved sensitivity in identifying bacteria, viruses, and fungi. The RPIP also exhibited better performance in identifying different pathogens in patients co-infected with multiple microorganisms. Additionally, the genotypes contributing to antimicrobial resistance were determined by RPIP. The study facilitated the implementation of molecular diagnosis by presenting real-world data, whereas future studies are mandated to generalize such an approach toward different clinical settings.

Molecular Identification of Resistance and Pathogenicity Genes of E. coli Isolated From Broiler Chicken Farms

Molecular Identification of Resistance and Pathogenicity Genes of E. coli Isolated From Broiler Chicken Farms

Introgression and Mapping of a Novel Bacterial Blight Resistance Gene xa49(t) from Oryza rufipogon acc. CR100098A into O. sativa.

Bacterial blight (BB) caused by Xanthomonas oryzae pv. oryzae is one of the epidemic diseases in rice. Rapid changes in the pathogenicity of the X.oryzae pv. oryzae pathogen demand the identification and characterization of novel BB resistance genes. Here, we report the transfer and mapping of a new BB resistance gene from Oryza rufipogon acc. CR100098A. Inheritance studies on the BC2F2 population, BC2F3 progenies, and backcross-derived recombinant inbred lines derived from a cross between Pusa44/O. rufipogon acc. CR100098A//2*PR114 showed that a single recessive gene confers resistance in O. rufipogon acc. CR100098A. Bulked segregant analysis using 203 simple sequence repeat (SSR) markers localized the BB resistance gene on chromosome 11 bracketed between two SSR markers, RM27235 and RM2136. Using PR114 and O. rufipogon acc. CR100098A genotyping by sequencing data, 86 KASP markers within the bracketed region were designed and tested for bulked segregant analysis. Only five KASP markers showed polymorphism between parents, and three were associated with the target gene. Seventy-seven new SSR markers were designed from the same interval. A total of 33 polymorphic markers were analyzed on the whole population and mapped the BB gene in an interval of 2.8 cM flanked by SSR markers PAU11_65 and PAU11_44 within a physical distance of 376.3 kb. The BB resistance gene mapped in this study is putatively new and designated as xa49(t). Fourteen putative candidate genes were identified within the xa49(t) region having a role in biotic stress resistance. The linked markers to the xa49(t) gene were validated in other rice cultivars for its successful deployment in BB resistance breeding.

Global emergence of Escherichia coli with PBP3 insertions.

Escherichia coli producing metallo-β-lactamases with penicillin-binding protein 3 (PBP3) insertions have reduced susceptibility to aztreonam-avibactam and cefiderocol. Here, we analysed high-quality E. coli genomes for PBP3 insertions. E. coli genomes (n = 167 518) were retrieved from EnteroBase with CheckM2 for quality control, fastANI for species confirmation, multi-locus sequencing typing for sequence type (ST) determination and AMRFinderPlus for resistance gene identification. For PBP3 insertion analysis, we used Prokka for predicting coding sequences, BLAST+ for comparing resulted protein sequences and SnpEff for annotating variants. Among the included 159 341 genomes, PBP3 insertions with 11 variants were found in 2.01% (n = 3198). The predominant variant is a duplication of 334-337 amino acids (aa) (94.75%, n = 3030), comprising YRIN (65.92%, n = 2108) and its single-aa-variant YRIK (28.83%, n = 922), followed by a similar PYRI duplication of 333-336 (4.16%, n = 133). The less common ones are a TIPY duplication of 331-334 (n = 24) and its single-aa-variant TVPY's duplication: TVVPY (n = 1), TVPYTVPY (n = 1) and TVPYPVPY (n = 1). Rare duplications include VGDR of 106-109 (n = 3), ANALNIPL of 114-121 (n = 3), AL of 567-568 (n = 1) and TG of 584-585 (n = 1). Insertion variants were detected across 62 countries on six continents, primarily in human samples, and associated with 85 STs, concentrated in high-risk clones ST410 (29.18%, n = 1923), ST167 (23.40%, n = 1740) and ST405 (10.56%, n = 1334), with 83.32% (n = 2218) encoding metallo-β-lactamase NDM. Global spread of E. coli harbouring PBP3 insertion, often with NDM β-lactamase, high-risk ST410, ST167 and ST405 clones and various hosts, underscores the escalating antimicrobial resistance crisis and the urgency for a 'One Health' strategy.

Bronchiectasis Exacerbations: Are We Doing Everything We Can?

At the European Respiratory Society (ERS) Congress 2024, two experts in bronchiectasis, Pieter Goeminne, Department of Respiratory Diseases, Vitaz Saint-Nicholas Hospitals, Belgium, and Michal Shteinberg, Pulmonology Institute and CF Center – Carmel Medical Center; Israel Institute of Technology; and The B. Rappaport Faculty of Medicine, Haifa, Israel, discussed bronchiectasis’ pathogenesis and exacerbations, along with unmet needs regarding diagnosis and treatment. Bronchiectasis is a chronic and progressive inflammatory disease with a rising prevalence. Commonly associated conditions/related comorbidities of bronchiectasis include post-infective diseases and other airway conditions (such as chronic obstructive pulmonary disease [COPD] and asthma), although the cause of bronchiectasis may remain unknown in over a third of patients. Development of bronchiectasis involves the intersection of four pathogenic components: chronic infections, airway ciliary dysfunction, chronic inflammation (mostly neutrophilic), and structural lung damage, commonly known as the ‘vicious vortex.’ In particular, bronchiectasis development, progression, and exacerbation also involve upregulated and dysregulated neutrophil function. Exacerbations in bronchiectasis are marked by symptoms of increased cough, sputum changes, decreased lung function, and fatigue, among others. Careful clinical examination and awareness of bronchiectasis symptoms are needed to properly diagnose and treat the initial condition and prevent exacerbations. Triggers for exacerbations can be endogenous, such as neutrophil or eosinophil increases, as well as exogenous, including the presence of infectious agents and pollution. Research regarding treatment for bronchiectasis is limited, but European guidelines recommend airway clearance techniques and antibiotics during exacerbations. To enable more targeted treatment for bronchiectasis from first occurrence, to limit exacerbations, and during an exacerbation, there are unmet needs for better identification of resistant genes, treatments for pathogens and inflammation, and biomarkers of exacerbation triggers.

Influence of cultivar selection on blast and brown spot diseases in rice: Molecular screening of blast resistance genes &amp;nbsp;

The prevalence of blast and brown spot diseases in rice is a substantial threat to national food security. This study investigated the distribution patterns of blast and brown spot, comparing their occurrence and distribution with respect to cultivar selection and conducting molecular screening for the identification of blast resistance genes. The research was conducted over five cultivating seasons from Yala 2017 to Yala 2019 in the Northern Province of Sri Lanka. Incidence percentages of the two diseases were calculated in 114 randomly selected fields across the five districts; Jaffna, Mullaitheevu, Kilinochchi, Mannar, and Vavuniya. Molecular markers were used to screen for nine major blast-resistant genes in 25 commonly cultivated rice cultivars. The results showed a significant shift in the disease over the period of study. While blast disease incidence declined after Maha 2017, brown spot incidence increased steadily from Yala 2017, peaking in Yala 2019. Interestingly, farmers’ cultivar preferences, often diverging from the Department of Agriculture recommendations, exhibited a strong correlation with disease occurrence. The cultivar Attakkari was identified as a highly susceptible cultivar, which had only three R genes and a major contributor to the progression of blast before Maha 2017. Despite higher brown spot incidence percentages observed in cultivars At362, Bw367, and Bg450, compared to Bg360, intensive post-Maha 2017 cultivation of Bg360 increased the average brown spot incidence to 43%. Cultivars grown after Maha 2017, with over five R genes, showed lower blast disease incidence, suggesting a genetic link to susceptibility. This lower incidence of blast was also observed in the disease evaluation test, where we used the same cultivars. Hence, this study highlights rice cultivar selection as a decisive factor influencing disease occurrence. Given Sri Lanka’s robust germplasm for blast-resistant genes, strategic cultivar selection has the potential for effective disease management.

Molecular identification of antibiotic resistance genes in E. coli isolated from dairy cattle.

Molecular identification of antibiotic resistance genes in E. coli isolated from dairy cattle.

Identification of quantitative trait loci and candidate genes for pod shatter resistance in Brassica carinata

BackgroundUnderstanding the genetic control of pod shatter resistance and its association with pod length is crucial for breeding improved pod shatter resistance and reducing pre-harvest yield losses due to extensive shattering in cultivars of Brassica species. In this study, we evaluated a doubled haploid (DH) mapping population derived from an F1 cross between two Brassica carinata parental lines Y-BcDH64 and W-BcDH76 (YWDH), originating from Ethiopia and determined genetic bases of variation in pod length and pod shatter resistance, measured as rupture energy. The YWDH population, its parental lines and 11 controls were grown across three years for genetic analysis.ResultsBy using three quantitative trait loci (QTL) analytic approaches, we identified nine genomic regions on B02, B03, B04, B06, B07 and C01 chromosomes for rupture energy that were repeatedly detected across three growing environments. One of the QTL on chromosome B07, flanked with DArTseq markers 100,046,735 and 100,022,658, accounted for up to 27.6% of genetic variance in rupture energy. We observed no relationship between pod length and rupture energy, suggesting that pod length does not contribute to variation in pod shatter resistance. Comparative mapping identified six candidate genes; SHP1 on B6, FUL and MAN on chromosomes B07, IND and NST2 on B08, and MAN7 on C07 that mapped within 0.2 Mb from the QTL for rupture energy.ConclusionThe results suggest that favourable alleles of stable QTL on B06, B07, B08 and C01 for pod shatter resistance can be incorporated into the shatter-prone B. carinata and its related species to improve final seed yield at harvest.

Genetic Mapping and Characterization of the Clubroot Resistance Gene BraPb8.3 in Brassica rapa.

Clubroot, a significant soil-borne disease, severely impacts the productivity of cruciferous crops. The identification and development of clubroot resistance (CR) genes are crucial for mitigating this disease. This study investigated the genetic inheritance of clubroot resistance within an F2 progeny derived from the cross of a resistant parent, designated "377", and a susceptible parent, designated "12A". Notably, "377" exhibited robust resistance to the "KEL-23" strain of Plasmodiophora brassicae, the causative agent of clubroot. Genetic analyses suggested that the observed resistance is controlled by a single dominant gene. Through Bulked Segregant Analysis sequencing (BSA-seq) and preliminary gene mapping, we localized the CR gene locus, designated as BraPb8.3, to a 1.30 Mb genomic segment on chromosome A08, flanked by the markers "333" and "sau332-1". Further fine mapping precisely narrowed down the position of BraPb8.3 to a 173.8 kb region between the markers "srt8-65" and "srt8-25", where we identified 22 genes, including Bra020861 with a TIR-NBS-LRR domain and Bra020876 with an LRR domain. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) analyses confirmed that both Bra020861 and Bra020876 exhibit increased expression levels in the resistant parent "377" following inoculation with P. brassicae, thereby underscoring their potential as key genes implicated in BraPb8.3-mediated clubroot resistance. This study not only identifies molecular markers associated with BraPb8.3 but also enriches the genetic resources available for breeding programs aimed at enhancing resistance to clubroot.

Identification and Screening of Leaf Rust Resistance Genes in Wheat Cultivars Through Microsatellites

Wheat is one of the important staple food being grown worldwide and in Pakistan. The wheat rust-causing fungi are very eco-adaptive and evolve rapidly to overcome genetic resistance of wheat varieties. Among all rust-causing fungi, Puccinia triticina causes leaf rust in wheat, and inflicts heavy yield losses. Deploying resistant varieties against leaf rust is the most effective, environmentally-friendly and economic way to control the disease. Therefore, new sources of genetic resistance are continually sought to develop rust resistance in wheat varieties, and nowadays rust resistant varieties have been developed through the accumulation of slow rusting or minor genes that would perform better in fields. Hence, the purpose of this study is to screen a few of the available leaf rust-resistant germplasm in field and evaluate their field response on genetic basis. For this purpose, minor genes were detected through the amplification of simple sequence repeat (SSR) in the selected wheat genotypes through screened primers. Field experiment was conducted. Molecular studies to identify the minor genes were performed. Meteorological data was recorded at the observatory laboratory of the same area. All the epidemiological factors (Temperature (maximum, minimum), Relative Humidity, and Pan Evaporation) showed significant correlation in tested 5 varieties ZARDANA89, ZARLASHTA99, RASKOH 05, BHAKKAR-2000, GA-2002 which were conducive for disease development except rainfall. PCR amplification of Xgwm118, and Xgwm165, showed the range of alleles in 14 genotypes. It was observed that 22 verities showed resistance, 3 were moderately resistant, 3 varieties were moderately susceptible, 8 varieties were moderately susceptible, and 5 varieties were moderately susceptible against leaf rust pathogen.

Decoding Pseudomonas aeruginosa: Genomic insights into adaptation, antibiotic resistance, and the enigmatic role of T6SS in interbacterial dynamics

Pseudomonas aeruginosa demonstrates a remarkable capacity for adaptation and survival in diverse environments. Furthermore, its clinical importance is underscored by its intrinsic and acquired resistance to a wide range of antimicrobial agents, posing a substantial challenge in healthcare settings. Amidst this complex landscape of resistance, the Type VI Secretion System (T6SS) in P. aeruginosa adds yet another layer of intricacy and allows bacteria to engage in interbacterial competition, potentially influencing their resilience and pathogenicity. Whole genome sequencing (WGS) was conducted on the five isolates under investigation, enabling the identification of antibiotic resistance genes (ARGs) and mutations associated with resistance. All isolates exhibit class C and D β-lactamases, displaying variant differences. The Resistance-nodulation-division (RND) antibiotic efflux pumps, crucial for multidrug resistance, have been encoded chromosomally. When exploring the role of the T6SS in urinary tract infections involving other bacteria, it was noted that P. aeruginosa isolates exhibited reduced counts when co-cultivated with other bacteria. The downregulation of the tssJ gene, associated with the T6SS under bacterial stress, and the exclusion of several cluster genes in this study suggest the hypothesis of a basal state rather than an attack/defence mechanism in the initial contact.

Culture and amplification-free nanopore sequencing for rapid detection of pathogens and antimicrobial resistance genes from urine.

Urinary Tract Infections (UTIs) are among the most prevalent infections globally. Every year, approximately 150million people are diagnosed with UTIs worldwide. The current state-of-the-art diagnostic methods are culture-based and have a turnaround time of 2-4 days for pathogen identification and susceptibility testing. This study first establishes an optical density culture-based method for spiking healthy urine samples with the six most prevalent uropathogens. Urine samples were spiked at clinically significant concentrations of 103-105 CFU/ml. Three DNA extraction kits (BioStic, PowerFood, and Blood and Tissue) were investigated based on the DNA yield, average processing time, elution volume, and the average cost incurred per extraction. After DNA extraction, the samples were sequenced using MinION and Flongle flow cells. The Blood and Tissue kit outperformed the other kits based on the investigated parameters. Using nanopore sequencing, all the pathogens and corresponding genes were only identified at a spike concentration of 105 CFU/ml, achieved after 10min and 3 hours of sequencing, respectively. However, some pathogens and antibiotic-resistance genes (ARG) could be identified from spikes at 103 colony formation units (CFU/mL). The overall turnaround time was five hours, from sample preparation to sequencing-based identification of pathogen ID and antimicrobial resistance genes. This study demonstrates excellent promise in reducing the time required for informed antibiotic administration from 48 to 72h to five hours, thereby reducing the number of empirical doses and increasing the chance of saving lives.