G3: Genes|Genomes|Genetics最新文献

The pileus serves as a primary determinant of market grade and commercial value in Lentinula edodes. To elucidate the molecular mechanisms governing pileus development, we conducted comparative transcriptome analysis via RNA sequencing across 3 distinct developmental stages: Early button stage, Young fruiting body stage, and mature fruiting body stage. Gene expression profiling revealed a substantial number of differentially expressed genes (DEGs) between stages, with 283 conserved DEGs spanning the entire developmental continuum. Systematic mining of these conserved DEGs identified 3 candidate regulatory genes encoding: Alpha-amylase, Heat shock protein 70 (HSP70), Phosphatidylserine decarboxylase. Quantitative PCR validation confirmed the accuracy of both RNA-Seq data and DEG identification. Enzymatic activity assays demonstrated significant stage-specific variations in: Antioxidant enzyme activities, Membrane lipid peroxidation levels. This study provides valuable insights into the molecular framework underlying pileus morphogenesis in Lentinula edodes.

Yellow wood sorrel (Oxalis stricta L.), also known as sourgrass, juicy fruit, or sheep weed, is a member of the Oxalidaceae family. Yellow wood sorrel is commonly considered a weed, and while native to North America, it is distributed across Europe, Asia, and Africa. To date, only 2 other genomes from the Oxalidaceae family have been published, star fruit (Averrhoa carambola L.) and Oxalis articulata Savigny. Here, we present a chromosome-scale assembly for O. stricta, revealing its allotetraploid nature and synteny within its 2 subgenomes as well as synteny with A. carambola and O. articulata. Using Oxford Nanopore Technologies long-read sequences coupled with chromatin capture sequencing, we generated a 436 Mb chromosome-scale assembly of O. stricta with a scaffold N50 length of 36.2 Mb that is anchored to 12 chromosomes across the 2 subgenomes. Assessment of the final genome assembly using the Long Terminal Repeat Assembly Index yielded a score of 13.12, and assessment of Benchmarking Universal Single Copy Orthologs revealed 99.6% complete orthologs; both metrics are suggestive of a high-quality reference genome. Total repetitive sequence content in the O. stricta genome was 39.7% with retroelements being the largest class of transposable elements. Annotation of protein-coding genes yielded 61,550 high-confidence genes encoding 115,089 gene models. Synteny between the 2 O. stricta subgenomes was present in 93 syntenic blocks containing 40,750 genes, of which, 76.47% were present in 1:1 syntenic relationships between the 2 subgenomes. The availability of an annotated chromosome-scale high-quality genome assembly for O. stricta will provide a launching point to understand the high fecundity of this weed and provide further foundation for comparative genomics within the Oxalidaceae.

Identifying and genetically characterizing new sources of resistance in barley (Hordeum vulgare) effective against the virulent wheat stem rust (Puccinia graminis f. sp. tritici [Pgt]) population in the Pacific Northwest (PNW) is critical. Isolates from this population, including Pgt isolate Lsp21, were virulent on barley stem rust resistance (R) genes Rpg1, Rpg2, Rpg3, rpg4, Rpg5, and rpg8. Notably, 10% of the Pgt isolates from the population were virulent on barley line Q21861, which contains Rpg1 and rpg4/5 stacked together. Virulence on these 2 broad and effective stem rust R-genes/loci, when combined, is unprecedented Pgt virulence on barley. To discover novel resistance, 277 wild barley (H. vulgare subsp. spontaneum) accessions from the Wild Barley Diversity Collection (WBDC) were screened with Pgt isolate Lsp21. Twelve percent showed moderate resistance, with WBDC-94 and WBDC-238 from Jordan exhibiting exceptional resistance, likely conferred by Rpg7, previously reported in both. To genetically characterize resistance in the WBDC, a genome-wide association study was conducted using disease reactions to Lsp21 and 37,338 genotyping-by-sequencing SNPs. Twelve resistance-associated loci were identified on chromosomes 1H, 2H, 3H, 5H, 6H, and 7H. Rpg7 was not detected due to its low allele frequency in the panel. Importantly, 7 novel resistance loci, WQRpg-2H01, WQRpg-2H02, WQRpg-3H01, WQRpg-5H01, WQRpg-5H03, WQRpg-7H02, and WQRpg-7H03, were discovered. These new sources of resistance can be integrated into cultivated barley, and the associated SNPs will aid in tracking resistance loci in prebreeding lines, enhancing breeding efforts against the virulent PNW Pgt population.

The analysis of gene function frequently requires the generation of mutants. Deep-mutational scanning (DMS) has emerged as a powerful tool to decipher important functional residues within genes and proteins. However, methods for performing DMS tend to be complex or laborious. Here, we introduce Tiled-Region Exchange (T-REx) mutagenesis, which is a multiplexed modification of the Extremely Methodical and Parallel Investigation of Randomized Individual Codons mutagenesis approach. Self-encoded removal fragments are cloned in parallel in nonoverlapping gene locations and pooled. In a 1-pot reaction, oligonucleotides are then swapped with their corresponding self-encoded removal fragments in bulk using a single Golden Gate reaction. To aid in downstream phenotyping, the library is then fused with unique DNA barcodes using the Bxb1 recombinase. We demonstrate this approach and its optimizations to show that it is both easy to perform and efficient. This method offers simple and expedient means to create comprehensive mutagenesis libraries.

Hedgehog signaling is a conserved developmental pathway that patterns diverse tissues during vertebrate embryogenesis. In zebrafish, disruptions to the hedgehog pathway cause well-characterized defects in specific cell types including neurons and glia derived from the ventral neural tube. We inhibited hedgehog signaling by overexpressing the Gli3 repressor ubiquitously and performed bulk RNA sequencing of 30 h postfertilization zebrafish embryos. Consistent with known roles of hedgehog signaling, we observed reduced expression of genes marking lateral floor plate, motor neurons, Kolmer-Agduhr cells, dopaminergic neurons, slow muscle cells, and anterior pituitary. Gene set enrichment analysis using marker genes derived from the Daniocell atlas also revealed downregulation of genes marking H+-ATPase-rich ionocytes, which are located in the embryonic skin and are responsible for osmotic homeostasis. Reduced expression of ionocyte-specific transporter genes and the transcription factor foxi3a suggests that Gli activity may play a previously unrecognized role in the specification of this cell type.

Plants adapt to diverse environments through complex gene regulatory networks, with the AT-HOOK MOTIF CONTAINING NUCLEAR LOCALIZED (AHL) gene family playing a crucial role. This research identified and examined the AHL gene family within Brachypodium distachyon, a model plant for Pooideae grasses including essential cereal crops. AHL proteins are conserved across land plants, suggesting an ancient origin and fundamental importance in plant development and adaptation. B. distachyon is an efficient research model for monocot studies due to its compact genome, short life cycle, and genetic manipulation compatibility. While Arabidopsis thaliana has consistently served as a valuable model for studying the AHL gene family, understanding their function in monocots, particularly grasses, is essential for crop improvement. The conserved evolutionary history of AHL proteins makes them an excellent target for comparative genomic research across eudicots and monocots systems. Although AHL functions have been studied in A. thaliana, their roles in Pooideae grasses remain largely unknown. This research identified 22 BdAHL genes classified into 2 monophyletic clades and 3 protein types based on conserved domains. By characterizing BdAHL gene structure, phylogeny, expression, and potential protein interactions, this study lays the groundwork for future functional analyses of Pooideae grasses. BdAHL expression profiles across different tissues and global coexpression patterns were also examined. These findings provide a foundation for future research into specific AHL gene functions in B. distachyon growth, development, and stress responses, potentially enhancing our understanding of AHL function in other Pooideae grasses and aiding crop improvement strategies.

Malaria remains a major global health burden and is caused by protozoan parasites in the genus Plasmodium. Parasites are transmitted to humans during blood feeding by anopheline mosquitoes, and members of the Anopheles gambiae species complex are important vectors in sub-Saharan Africa. Gene-drive technologies offer promising options for disease control by enabling the spread of genetic traits through mosquito populations that block parasite transmission. We report here the development and characterization of four population modification gene-drive strains in Anopheles gambiae s.s. and An. coluzzii carrying compound effector genes. We sought to enhance the effectiveness of existing gene-drive strains to block Plasmodium transmission, thereby reducing vector competence and minimizing the opportunities for selection of resistant parasites. Two compound effector gene modules, TP24 and TP43, were introduced using Cas9 endonuclease and dual guide RNAs into TP13-based gene-drive strains to produce the An. gambiae AgTP24 and AgTP43 strains. The gene-drive cassettes were then introgressed into An. coluzzii to produce AcTP24 and AcTP43. Gene-drive dynamics, gene conversion, and inheritance were high in all strains, with 95% to 100% inheritance of the gene-drive constructs. Life table analyses showed mixed impacts on fitness dependent on the species and copy number (hemi- or homozygosity) of the gene-drive systems. The compound effector molecule gene complexes significantly reduced both parasite prevalence and infection intensities in An. gambiae and An. coluzzii following challenge assays with the human malaria parasite, P. falciparum. These findings highlight the potential of compound effector strategies in gene-drive systems to achieve durable malaria transmission control.

The rapid emergence of antimicrobial resistance in bacterial pathogens threatens the efficacy of nearly all available antibiotics. One evolution-informed strategy to limit the emergence of resistance is the exploitation of collateral sensitivity, whereby resistance to one compound results in increased sensitivity to another. Here, we investigate the collateral sensitivity relationship between the cephalosporin antibiotic ceftazidime and the natural product borrelidin A in Escherichia coli. Previously, we found that borrelidin A prevented the evolution of clinical ceftazidime resistance during laboratory selection. In this study, we further characterized the genomic and phenotypic consequences of evolution under collateral sensitivity in these evolved populations. Co-dosing with 128µM borrelidin A significantly reduced the evolution of ceftazidime resistance, while preserving fitness in the absence of drug. Overall co-dosed strains had reduced resistance and cross-resistance to all tested antibiotics. Whole-genome sequencing revealed that co-dosing suppressed the accumulation of single nucleotide polymorphism and insertion/deletion mutations in known resistance-associated genes yet resulted in an increased number of mobile-element insertion mutations. In one case we find a possible novel borrelidin A resistance mutation. Our results suggest that ceftazidime-borrelidin A co-dosing limits both resistance and cross-resistance through selection against costly resistance mutations. While borrelidin A itself is cytotoxic, our findings highlight the promise of targeting bacteria-specific vulnerabilities to curb the emergence of multidrug resistance. These findings contribute to the growing body of evidence supporting collateral sensitivity-informed approaches as practical strategies to mitigate antimicrobial resistance in bacterial populations.

C. elegans insulin/insulin-like growth factor 1 signaling, IIS, affects diverse physiological processes through the DAF-16/FOXO transcription factor. Despite its ubiquitous presence in somatic cells, DAF-16's effects exhibit prevalent tissue specificity as well as tissue crosstalk. This implies that tissue-specific DAF-16 transcriptional programs contribute to functional diversity of IIS. To further investigate this possibility, we sought muscle-cell-specific DAF-16 transcriptional targets. Using fluorescence-activated cell sorting to enrich for body wall muscle cells from young hermaphroditic adults, we compared the muscle cell mRNA transcriptomes under conditions of high and low IIS activity, with and without DAF-16. We further analyzed DAF-16a's binding sites in muscle and intestine cells by chromatin-immunoprecipitation sequencing. Combined output of these analyses is 12 candidate DAF-16 targets enriched for muscle cells. Transcriptional and translational reporters for three out of the four top candidates - a secreted protein C54F6.5, a calcium-binding protein CEX-1/calexcitin, and a fatty acid metabolic enzyme MLCD-1/MCD - showed DAF-16-dependent activation specifically in body wall muscle cells. Notably, reporters for C54F6.5 and cex-1 exhibit DAF-16-independent, constitutive expression in non-muscle cells, explaining their low rank or absence from the DAF-16 target lists generated by whole-animal microarray or mRNA-sequencing analyses. These results highlight the need to examine FOXO targets in a cell-type-specific manner.

The New World screwworm, Cochliomyia hominivorax, is an obligate parasite of warm-blooded animals and a major pest of livestock and wildlife in the Americas. The first genome assembly for C. hominivorax enabled substantial progress in key areas including gene expression related to fly behavior and physiology and gene editing technologies. However, the first genome was sequenced prior to several technological advances that result in fewer errors and better genome annotations. Here, we used the trio-binning approach to produce haplotype-resolved genome assemblies of C. hominivorax. A single male progeny from the cross of a Panama line male with a production strain female was sequenced using PacBio HiFi and scaffolded using Hi-C chromatin conformation, while Illumina NextSeq 2000 was used for short read sequencing of both parents to facilitate trio-binning. We produced a linear haploid reference assembly by transferring a copy of the X chromosome and mitochondrial genome to the paternal haplotype. This assembly is comprised of five autosomes, two sex chromosomes, the mitogenome, and 75 unplaced scaffolds spanning 455.6 Mb, which is closer to the predicted size based on flow cytometry (443.8 Mb) than the previous assembly of 534.4 Mb. NCBI's external Eukaryotic Genome Annotation Pipeline (EGAPx) was used to annotate the protein coding and non-coding genes in the linear haploid reference and the maternal haplotype assemblies. Due to the better resolution of the sex chromosomes and updated genome annotations, these improved assemblies will advance future experiments aimed at understanding sex determination, gene expression, and the evolution of parasitism in the Calliphoridae.