Functional Genomic Studies Research Articles

Expanding the BonnMu sequence-indexed repository of transposon induced maize (Zea mays L.) mutations in dent and flint germplasm.

The BonnMu resource is a transposon tagged mutant collection designed for functional genomics studies in maize. To expand this resource, we crossed an active Mutator (Mu) stock with dent (B73, Co125) and flint (DK105, EP1, and F7) germplasm, resulting in the generation of 8064 mutagenized BonnMu F2-families. Sequencing of these Mu-tagged families revealed 425 924 presumptive heritable Mu insertions affecting 36 612 (83%) of the 44 303 high-confidence gene models of maize (B73v5). On average, we observed 12 Mu insertions per gene (425 924 total insertions/36 612 affected genes) and 53 insertions per BonnMu F2-family (425 924 total insertions/8064 families). Mu insertions and photos of seedling phenotypes from segregating BonnMu F2-families can be accessed through the Maize Genetics and Genomics Database (MaizeGDB). Downstream examination via the automated Mutant-seq Workflow Utility (MuWU) identified 94% of the presumptive germinal insertion sites in genic regions and only a small fraction of 6% inserting in non-coding intergenic sequences of the genome. Consistently, Mu insertions aligned with gene-dense chromosomal arms. In total, 42% of all BonnMu insertions were located in the 5' untranslated region of genes, corresponding to accessible chromatin. Furthermore, for 38% of the insertions (163 843 of 425 924 total insertions) Mu1, Mu8 and MuDR were confirmed to be the causal Mu elements. Our publicly accessible European BonnMu resource has archived insertions covering two major germplasm groups, thus facilitating both forward and reverse genetics studies.

Predicting the role of β-GAL genes in bean under abiotic stress and genome-wide characterization of β-GAL gene family members.

Β-Gals are a subgroup of the glycoside hydrolase (GH) family of enzymes, which possess the Glyco_hydro_35 (GH35) domain. Although studies have been conducted on the β-Gal gene family in numerous plant species, no such research has been conducted on beans. The purpose of this study was to determine the gene expression levels of β-Gal genes in the leaf tissue of P. vulgaris under salt and drought stress using quantitative real-time polymerase chain reaction (qRT-PCR) and to perform a comprehensive analysis of β-Gal gene family members using bioinformatics tools. In the bean genome, 25 Pvul-βGAL proteins with amino acid numbers ranging from 291 to 1119, molecular weights from 32.94 to 126.56kDa, and isoelectric points from 5.46 to 9.08 were identified. Both segmental and tandem duplication have occurred in β-Gal genes in the bean genome, and Pvul-BGAL genes have been subject to negative selection in the evolutionary process. For a deeper comprehension of the evolutionary proximity of Pvul-BGAL genes, a phylogenetic tree and synteny map were drawn together with Arabidopsis thaliana and Glycine max β-Gal genes. The expression profiles of β-Gal genes in different tissues of the bean were determined in silico. In addition, the expression profiles of β-Gal genes in the leaves of bean plants subjected to drought and salt stress were analyzed, and the role of β-Gal genes in salt and drought stress was estimated. In this study, the role of β-Gal gene family in abiotic stress response and the characterization of β-Gal genes in beans were determined for the first time and will provide a basis for future functional genomics studies.

Three Novel Spider Genomes Unveil Spidroin Diversification and Hox Cluster Architecture: Ryuthela nishihirai (Liphistiidae), Uloborus plumipes (Uloboridae) and Cheiracanthium punctorium (Cheiracanthiidae).

Spiders are a hyperdiverse taxon and among the most abundant predators in nearly all terrestrial habitats. Their success is often attributed to key developments in their evolution such as silk and venom production and major apomorphies such as a whole-genome duplication. Resolving deep relationships within the spider tree of life has been historically challenging, making it difficult to measure the relative importance of these novelties for spider evolution. Whole-genome data offer an essential resource in these efforts, but also for functional genomic studies. Here, we present de novo assemblies for three spider species: Ryuthela nishihirai (Liphistiidae), a representative of the ancient Mesothelae, the suborder that is sister to all other extant spiders; Uloborus plumipes (Uloboridae), a cribellate orbweaver whose phylogenetic placement is especially challenging; and Cheiracanthium punctorium (Cheiracanthiidae), which represents only the second family to be sequenced in the hyperdiverse Dionycha clade. These genomes fill critical gaps in the spider tree of life. Using these novel genomes along with 25 previously published ones, we examine the evolutionary history of spidroin gene and structural hox cluster diversity. Our assemblies provide critical genomic resources to facilitate deeper investigations into spider evolution. The near chromosome-level genome of the 'living fossil' R. nishihirai represents an especially important step forward, offering new insights into the origins of spider traits.

CRISPR-GRIT: Guide RNAs with Integrated Repair Templates Enable Precise Multiplexed Genome Editing in the Diploid Fungal Pathogen Candida albicans.

Candida albicans, an opportunistic fungal pathogen, causes severe infections in immunocompromised individuals. Limited classes and overuse of current antifungals have led to the rapid emergence of antifungal resistance. Thus, there is an urgent need to understand fungal pathogen genetics to develop new antifungal strategies. Genetic manipulation of C. albicans is encumbered by its diploid chromosomes requiring editing both alleles to elucidate gene function. Although the recent development of CRISPR-Cas systems has facilitated genome editing in C. albicans, large-scale and multiplexed functional genomic studies are still hindered by the necessity of cotransforming repair templates for homozygous knockouts. Here, we present CRISPR-GRIT (Guide RNAs with Integrated Repair Templates), a repair template-integrated guide RNA design for expedited gene knockouts and multiplexed gene editing in C. albicans. We envision that this method can be used for high-throughput library screens and identification of synthetic lethal pairs in both C. albicans and other diploid organisms with strong homologous recombination machinery.

FlyRNAi.org 2025 update-expanded resources for new technologies and species.

The design, analysisand mining of large-scale 'omics studies with the goal of advancing biological and biomedical understanding require use of a range of bioinformatics tools, including approaches tailored to needs specific to a given species and/or technology. The FlyRNAi database at the Drosophila RNAi Screening Center and Transgenic RNAi Project (DRSC/TRiP) Functional Genomics Resources (https://fgr.hms.harvard.edu/tools) supports an increasingly broad group of technologies and species. Recently, for example, we expanded the database to include additional new data-centric resources that facilitate mining and analysis of single-cell transcriptomics. In addition, we have applied our approaches to CRISPR reagent and gene-centric bioinformatics approaches in Drosophila to arthropod vectors of infectious diseases. Building on our previous comprehensive reports on the FlyRNAi database, here we focus on new and updated resources with a primary focus on data-centric tools. Altogether, our suite of online resources supports various stages of functional genomics studies for Drosophila and other arthropods, and facilitate a wide range of reagent design, analysis, data miningand analysis approaches by biologists and biomedical experts studying Drosophila, other common genetic model species, arthropod vectorsand/or human biology.

Fine-tuning of a CRISPRi screen in the seventh pandemic Vibrio cholerae

BackgroundVibrio cholerae O1 El Tor, the etiological agent responsible for the last cholera pandemic, has become a well-established model organism for which some genetic tools are available. While CRISPRi technology has been applied to V. cholerae, improvements were necessary to upscale it and enable pooled screening by high-throughput sequencing in this bacterium.ResultsIn this study, we present a genome-wide CRISPR-dCas9 screen specifically optimized for the N16961 El Tor model strain of V. cholerae. This approach is characterized by a tight control of dCas9 expression and activity, as well as a streamlined experimental setup. Our library allows the depletion of 3,674 (98.9%) annotated genes from the V. cholerae genome. To confirm its effectiveness, we screened for genes that are essential during exponential growth in rich medium and identified 369 genes for which guides were significantly depleted from the library (log2FC < -2). Remarkably, 82% of these genes had previously been described as hypothetical essential genes in V. cholerae or in a closely related bacterium, V. natriegens.ConclusionWe thus validated the robustness and accuracy of our CRISPRi-based approach for assessing gene fitness in a given condition. Our findings highlight the efficacy of the developed CRISPRi platform as a powerful tool for high-throughput functional genomics studies of V. cholerae.

The Duck 1000 Genomes Project: Achievements and perspectives

AbstractThe duck (Anas platyrhynchos) is not only a vital farm animal but also an excellent model for genetic dissection of economic traits. The integration of multiomics data provides a powerful approach to elucidate the genetic basis of domestication and phenotype variation. Since its inception in 2014, the Duck 1000 Genomes Project has aimed to uncover the genetic foundation of key economic traits in ducks by combining multiomics data including genomic, transcriptomic, and metabolomic from various natural and segregating populations. This paper summarizes the strategies and achievements of the Duck 1000 Genomes Project, highlighting the reference genome assembly, genome evolution analysis, and the identification of genes and causative mutations responsible for key economic traits in ducks. We also discuss perspectives and potential challenges in functional genomic studies that could further accelerate duck molecular breeding.

Efficient gene editing with an Arg-tRNA promoter-driven CRISPR/Cas9 in the rice blast fungus Pyricularia oryzae

CRISPR/Cas9 technology has been widely adopted for genome editing in a wide range of organisms, including many fungi. Pyricularia oryzae is a filamentous fungal pathogen that causes the devastating rice blast disease. However, an efficient and cost-effective CRISPR/Cas9 system for the rice blast fungus has yet to be established. Here, we report an 84-bp arginyl (Arg)-tRNA promoter-driven CRISPR/Cas9 system, which enables efficient and cost-effective gene editing in P. oryzae. Preliminary screening of three tRNAs from the 179 predicted tRNAs in P. oryzae showed that two Arg-tRNA CRISPR/Cas9 cassettes reproducibly generated MoB56 disruption efficiently. Further, five genes located on distinct chromosomes, including two previously uncharacterized genes, were randomly picked up to test the efficiency of the Mo_tRNAArg24-gRNA-Cas9 cassette. Ppg1 is a gene essential to the pathogenicity and important for mycelial growth and conidiation of P. oryzae, which is located at chromosome 2 and exhibited a relatively low gene replacement rate (< 1/500) by the traditional gene replacement approach. By using the Mo_tRNAArg24-gRNA-Cas9 cassette, Ppg1 gene disruption rate was increased up to 75.9%. In addition, Bip2, an uncharacterized genes located close to the centromere of chromosome 4, was disrupted at 66.7%. For all the five tested genes, our Mo_tRNAArg24-gRNA-Cas9 cassette showed high gene disruption efficiency in P. oryzae, ranging from 66.7% to 100%. Importantly, it rarely induces Cas9 toxicity to P. oryzae. The Mo_tRNAArg24-gRNA-Cas9 cassette described in this study can be adopted as an alternative for functional genomics study in P. oryzae.

A novel gene silencing strategy based on tobacco rattle virus in Hibiscus mutabilis.

Hibiscus mutabilis L. is a popular regional characteristic plant in China, cultivated for its attractive flower colors, extended bloom time, and medicinal properties. To enhance molecular breeding and gene function studies, we conducted transcriptome analysis and identified valuable genes in previous research. Nonetheless, the current inefficient and labor-intensive transformation techniques have hindered their applications. Virus-induced gene silencing (VIGS) provides a precise and effective strategy for post-transcriptional down-regulation of endogenous gene expression. We investigated the performance of tobacco rattle virus (TRV) as a tool for targeting and silencing the gene encoding the protein involved in chloroplast development, cloroplastos alterados 1 (altered chloroplast; CLA1), of H. mutabilis through Agrobacterium tumefaciens-mediated infiltration. By effectively suppressing the CLA1 gene associated with chloroplast development in H. mutabilis via the TRV-VIGS system, we have illustrated the inaugural implementation of VIGS in this species. Quantitative RT-PCR proved that HmCLA1 expression in agro-infiltrated plants was lower than in the mock-infiltrated (mock) and the control (CK) plants. Phenotypic observations corroborated the albino phenotype in leaves following successful HmCLA1 silencing. Our study showcases TRV-VIGS as a potential gene silencing tool for H. mutabilis, facilitating functional genomics studies and molecular breeding efforts in this species.

Chromosome-level genome assembly of American sweetgum (Liquidambar styraciflua, Altingiaceae)

The deciduous American sweetgum (Liquidambar styraciflua, Altingiaceae) is a popular ornamental and economically valuable tree renowned for its sweet-smelling bark resin, abundant volatile substances, and spectacular fall leaf color. However, the absence of a reference genome hinders thorough investigations into the mechanisms underlying phenotypic variation, secondary metabolite synthesis and adaptation, both in this species and other Liquidambar members. In this study, we sequenced and constructed a chromosome-level assembly of the L. styraciflua genome, covering 662.48 Mb with a scaffold N50 of 39.54 Mb, by integrating PacBio, Illumina and chromosome conformation capture data. We identified 58.83% of the genome sequences as repetitive elements and 25,713 protein-coding genes, 97.28% of which were functionally annotated. The genome sequencing reads, assembly and annotation data have been deposited in publicly available repositories. This high-quality genome assembly provides valuable resources for further evolutionary and functional genomic studies in American sweetgum and other Liquidambar species.

Chromosome-scale genome assembly of Astragalus membranaceus using PacBio and Hi-C technologies

Astragalus membranaceus (Fisch.) Bge (AM) is a medicinal herb plant belonging to the Leguminosae family. In this study, we present a chromosome-scale genome assembly of AM, aiming to enhance the molecular biology and functional studies of Astragali Radix. The genome size of AM is about 1.43 Gb, with a contig N50 value of 1.67 Mb. A total of 98.16% of the assembly anchored to 9 pseudochromosomes using Hi-C technology. The assembly completeness was estimated to be 97.27% using BUSCO with the long terminal repeat assembly index (LAI) of 16.22 and quality value (QV) of 48.58. Additionally, the genome contained 67.98% repetitive sequences. Genome annotation predicted 29,914 protein-coding genes, including 73 genes involved in the flavonoid biosynthetic pathway and 2,048 transcription factors. The high-quality genome assembly and gene annotation resources will greatly facilitate future functional genomic studies in Leguminosae species.

FEAtl: a comprehensive web-based expression atlas for functional genomics in tropical and subtropical fruit crops

BackgroundFruit crops, including tropical and subtropical fruits like Avocado (Persea americana), Fig (Ficus carica), Date Palm (Phoenix dactylifera), Mango (Mangifera indica), Guava (Psidium guajava), Papaya (Carica papaya), Pineapple (Ananas comosus), and Banana (Musa acuminata) are economically vital, contributing significantly to global agricultural output, as classified by the FAO’s World Programme for the Census of Agriculture. Advancements in next-generation sequencing, have transformed fruit crop breeding by providing in-depth genomic and transcriptomic data. RNA sequencing enables high-throughput analysis of gene expression, and functional genomics, crucial for addressing horticultural challenges and enhancing fruit production. The genomic and expression data for key tropical and sub-tropical fruit crops is currently lacking a comprehensive expression atlas, revealing a significant gap in resources for horticulturists who require a unified platform with diverse datasets across various conditions and cultivars.ResultsThe Fruit Expression Atlas (FEAtl), available at http://backlin.cabgrid.res.in/FEAtl/, is a first-ever extensive and unified expression atlas for tropical and subtropical fruit crops developed using 3-tier architecture. The expressivity of coding and non-coding genes, encompassing 2,060 RNA-Seq samples across 91 tissue types and 177 BioProjects, it provides a comprehensive view of gene expression patterns for different tissues under various conditions. FEAtl features multiple tabs that cater to different aspects of the dataset, namely, Home, About, Analyze, Statistics, and Team and contains seven central functional modules: Transcript Information,Sample Information, Expression Profiles in FPKM and TPM, Functional Analysis, Genes Based on Tau Score, and Search for Specific Gene. The expression of a transcript of interest can be easily queried by searching by tissue ID and transcript type. Expression data can be displayed as a heat map, along with functional descriptions as well as Gene Ontology and Kyoto Encyclopedia of Genes and Genomes.ConclusionsThis atlas represents a groundbreaking compilation of a wide array of information pertaining to eight distinct fruit crops and serves as a fundamental resource for comparative analysis among different fruit species and is a catalyst for functional genomic studies. Database availability: http://backlin.cabgrid.res.in/FEAtl/.

Chromosome-level genome assembly of Tritrichomonas foetus, the causative agent of Bovine Trichomonosis

Tritrichomonas foetus is a parasitic protist responsible for bovine trichomonosis, a reproductive disease associated with significant economic burden to the livestock industry throughout the world. Here, we present a chromosome-level reference genome of T. foetus -KV-1 (ATCC 30924) using short-read (Illumina Miseq), long-read (Oxford Nanopore) and chromatin-linked (Hi-C) sequencing. This is the first chromosome-level genome of a parasitic protist of the order Tritrichomonadida and the second within the Parabasalia lineage, after Trichomonas vaginalis, the human-associated causative agent of the sexually transmitted infection in humans. Our constructed genome is 148 Mb in size, with a N50 length of the scaffolds of 22.9 Mb. The contigs are anchored in five super-scaffolds, corresponding to the expected five chromosomes of the species and covering 78% of the genome assembly. We predict 41,341 protein-coding genes, of which 95.10% have been functionally annotated. This high-quality genome assembly serves as a valuable reference genome for T. foetus to support future studies in functional genomics, genetic conservation and taxonomy.

Identification of Transposon Insertion Sites in MaizeMu-Tagged Mutants Using Mu-Seq

Mutator(Mu) transposons facilitate untargeted insertional mutagenesis in maize by moving within the genome and disrupting genes. Such an approach has been used to generate collections such as theBonnMuresource, aMu-tagged maize population for functional genomics studies. Mutant-Seq (Mu-Seq) is a sequencing-based method for the high-throughput identification and mapping ofMuinsertion sites. The approach involves the construction of multiplexed sequencing libraries (known as Mu-Seq libraries) fromMu-tagged populations, followed by high-throughput sequencing and data processing using the Mu-Seq Workflow Utility (MuWU) tool, to determine the location ofMuinsertions. Here, we provide a detailed protocol for Mu-Seq, from the generation of the maizeMu-tagged mutant population to data analysis. Researchers can use this approach to develop mutant collections customized to specific genetic backgrounds of interest, which can aid in characterizing genotype-specific mutations and identifying candidate genes linked to visible mutant phenotypes.

Use of Maize (Zea maysL.) Mutator Transposon-Induced Mutants of theBonnMuResource for Forward and Reverse Genetics Studies

TheBonnMuresource represents a tagged collection of maize (Zea maysL.)Mutator(Mu) transposon-induced mutants, designed for functional genomics studies. Here, we describe the use of theBonnMucollection for identifying and characterizing mutations. Specifically, we describe workflows for use in both reverse and forward genetics strategies in maize. For reverse genetics, users first acquire aBonnMuF2stock of interest based on data accessible at the Maize Genetics and Genomics Database (MaizeGDB). We provide details here for their subsequent propagation and for the confirmation ofMuinsertions by genotyping via PCR, with the ultimate goal of establishing genotype–phenotype relationships of interest. For forward genetics studies, we describe a workflow that involves a combined approach of Mutant-Seq (Mu-Seq) and bulked segregant RNA-seq (BSR-Seq), to identify the causal gene underlying a mutant phenotype of interest.

BonnMu: A Resource for Functional Genomics in Maize (Zea mays L.).

The BonnMu resource is a public transposon-tagged population designed for reverse and forward genetics studies in maize (Zea mays L.). The resource was created by crossing an active Mutator (Mu) transposon line into different inbred lines to induce insertional mutations. The resulting F1 generation was self-pollinated to generate segregating BonnMu F2 stocks. The Mu-tagged BonnMu F2 stocks have insertions in 83% of all annotated maize gene models, and Mu insertion positions and photos of the seedling phenotypes of the segregating BonnMu F2 stocks are deposited in the Maize Genetics and Genomics Database (MaizeGDB), with seeds available to the community. Here, we discuss the creation, expansion, and application of the BonnMu resource for identifying and characterizing mutations induced by Mu transposons, which represents a useful tool for functional genomics studies in maize.

Telomere-to-telomere, gap-free assembly of the Rosa rugosa reference genome

Rosa, a genus esteemed worldwide for its ornamental plants, has encountered barriers in functional genomic studies and further genetic enhancement due to incomplete sequences and floating regions in previously sequenced genomes. Our groundbreaking study introduced a meticulously assembled, continuous, and fully bridged reference genome for Rosa rugosa, constructed through a sophisticated combination of PacBio High-Fidelity, ONT ultra-long reads, and Hi-C data. This robust assembly spanned 444.55 Mb and encompassed 34 109 protein-coding genes. We have uniquely assembled each chromosome into single, gap-free structures, successfully identifying all 14 telomeres and seven centromeres, a feat not achieved previously. The centromeric regions were distinguished by tandem repeats, primarily composed of centromere-specific 159-bp monomers, and a significant enrichment of ATHILA/Gypsy long terminal repeat retrotransposons in proximal regions. Our research highlighted recent tandem duplications as instrumental in bolstering R. rugosa's stress tolerance, environmental adaptability, and enhanced anthocyanin synthesis. Furthermore, our study ventured into uncharted territory by predicting transcription factors potentially regulating anthocyanin biosynthesis through the employment of gene co-expression networks, providing new avenues for research. This comprehensive reference genome not only serves as a cornerstone for in-depth exploration of genomic architecture and functionalities in R. rugosa but also acts as a catalyst for innovative breeding strategies and genetic refinement within the genus.

Accelerating wheat improvement through trait characterization: advances and perspectives.

Wheat (Triticum spp.) is a primary dietary staple food for humanity. Many wheat genetic resources with variable genomes have a record of domestication history and are widespread throughout the world. To develop elite wheat varieties, agronomical and stress-responsive trait characterization is foremost for evaluating existing germplasm to promote breeding. However, genomic complexity is one of the primary impediments to trait mining and characterization. Multiple reference genomes and cutting-edge technologies like haplotype mapping, genomic selection, precise gene editing tools, high-throughput phenotyping platforms, high-efficiency genetic transformation systems, and speed-breeding facilities are transforming wheat functional genomics research to understand the genomic diversity of polyploidy. This review focuses on the research achievements in wheat genomics, the available omics approaches,and bioinformatic resources developed in the past decades. Advances in genomics and system biology approaches are highlighted to circumvent bottlenecks in genomic and phenotypic selection, as well as gene transfer. In addition, we propose conducting precise functional genomic studies and developing sustainable breeding strategies for wheat. These developments in understanding wheat traits have speed up the creation of high-yielding, stress-resistant, and nutritionally enhanced wheat varieties, which will help in addressing global food security and agricultural sustainability in the era of climate change.

Highly contiguous genome assembly and gene annotation of the short-finned eel (Anguilla bicolor pacifica)

In East Asia, anguillid eels are commercially important. However, unlike other species, they have not been successfully cultivated throughout their lifecycle. Facing population decline due to overharvesting and environmental pressures, the industry is turning to alternatives, such as Anguilla bicolor pacifica (short-finned eel). However, genomic data for short-finned eels are unavailable. Here, we present in-depth whole-genome sequencing results for short-finned eel obtained using two sequencing platforms (PacBio Revio, and Illumina). In this study, we achieved a highly contiguous genome assembly of the short-finned eel, comprising 19 pseudochromosomes encompassing 99.76% of the 1.087 Gb genome sequence with an N50 of 16.88 and 61.07 Mb from contig and scaffold, respectively. Transcripts from four different tissues led to the annotation of 23,095 protein-coding genes in the eel genome, 98.66% of which were functionally annotated. This high-quality genome assembly, along with the annotation data, provides a foundation for future functional genomic studies of short-finned eels.

The Genetics and Functional Genomics of Osteoarthritis.

Osteoarthritis is the most prevalent whole-joint degenerative disorder, and is characterized by the degradation of articular cartilage and the underlying bone structures. Almost 600 million people are affected by osteoarthritis worldwide. No curative treatments are available, and management strategies focus mostly on pain relief. Here, we provide a comprehensive overview of the available human genetic and functional genomics studies for osteoarthritis to date and delineate how these studies have helped shed light on disease etiopathology. We highlight genetic discoveries from genome-wide association studies and provide a detailed overview of molecular-level investigations in osteoarthritis tissues, including methylation-, transcriptomics-, and proteomics-level analyses. We review how functional genomics data from different molecular levels have helped to prioritize effector genes that can be used as drug targets or drug-repurposing opportunities. Finally, we discuss future directions with the potential to drive a step change in osteoarthritis research.