The Grape Gene Reference Catalogue as a Standard Resource for Gene Selection and Genetic Improvement

Effective crop improvement, whether through selective breeding or biotech strategies, is largely dependent on the cumulative knowledge of a species’ pangenome and its containing genes. Acquiring this knowledge is specially challenging in grapevine, one of the oldest fruit crops grown worldwide, which is known to have more than 30,000 genes. Well-established research communities studying model organisms have created and maintained, through public and private funds, a diverse range of online tools and databases serving as repositories of genomes and gene function data. The lack of such resources for the non-model, but economically important, Vitis vinifera species has driven the need for a standardised collection of genes within the grapevine community. In an effort led by the Integrape COST Action CA17111, we have recently developed the first grape gene reference catalogue, where genes are ascribed to functional data, including their accession identifiers from different genome-annotation versions (https://integrape.eu/resources/genes-genomes/). We present and discuss this gene repository together with a validation-level scheme based on varied supporting evidence found in current literature. The catalogue structure and online submission form provided permits community curation. Finally, we present the Gene Cards tool, developed within the Vitis Visualization (VitViz) platform, to visualize the data collected in the catalogue and link gene function with tissue-specific expression derived from public transcriptomic data. This perspective article aims to present these resources to the community as well as highlight their potential use, in particular for plant-breeding applications.

Efficient Genome Editing by a Miniature CRISPR-AsCas12f1 Nuclease in Bacillus anthracis

A miniature CRISPR-Cas12f has been demonstrated to serve as an effective genome editing tool in gram negative bacteria as well as human cells. Here, we developed an alternative method to edit the genome of Bacillus anthracis based on the AsCas12f1 nuclease from Acidibacillus sulfuroxidans. When the htrA gene on the chromosome and the lef gene on the plasmid pXO1 were selected as targets, the CRISPR-AsCas12f1 system showed very high efficiency (100%). At the same time, a high efficiency was observed for large-fragment deletion. Our results also indicated that the length of the homologous arms of the donor DNA had a close relationship with the editing efficiency. Furthermore, a two-plasmid CRISPR-AsCas12f1 system was also constructed and combined with the endonuclease I-SceI for potential multi-gene modification. This represents a novel tool for mutant strain construction and gene function analyses in B. anthracis and other Bacillus cereus group bacteria.

Highly efficient multiplex genome editing in dicot plants using improved CRISPR/Cas systems

CRISPR/Cas9-mediated gene editing provides a powerful tool for dissecting gene function and improving important traits in crops. However, there are still persisting challenges to obtain high homozygous/bi-allelic (ho/bi) mutations in dicot plants. Here, we develop an improved CRISPR/Cas9 system harboring a calreticulin-like gene promoter, which can boost targeted mutations in dicots. Additionally, the pDC45_dsg construct, combining a 35Spro-tRNA_sgRNA-EU unit and PCE8pro-controlled Cas9, can achieve more than 80.0% ho/bi mutations at target sites in allotetraploid tobacco. We construct pDC45_Fast system that can simultaneously fulfill gene editing and shorten the life span of T0 generation tobacco and tomato. This study provides new tools for improving targeted gene mutagenesis in dicots, and makes manipulations of genes in Solanum more feasible.

Gene Function Prediction in Five Model Eukaryotes Exclusively Based on Gene Relative Location Through Machine Learning

The function of most genes is unknown. The best results in automated function prediction are obtained with machine learning-based methods that combine multiple data sources, typically sequence derived features, protein structure and interaction data. Even though there is ample evidence showing that a gene’s function is not independent of its location, the few available examples of gene function prediction based on gene location rely on sequence identity between genes of different organisms and are thus subjected to the limitations of the relationship between sequence and function. Here we predict thousands of gene functions in five model eukaryotes (Saccharomyces cerevisiae, Caenorhabditis elegans, Drosophila melanogaster, Mus musculus and Homo sapiens) using machine learning models exclusively trained with features derived from the location of genes in the genomes to which they belong. Our aim was not to obtain the best performing method to automated function prediction but to explore the extent to which a gene's location can predict its function in eukaryotes. We found that our models outperform BLAST when predicting terms from Biological Process and Cellular Component Ontologies, showing that, at least in some cases, gene location alone can be more useful than sequence to infer gene function.

The Roles of Floral Organ Genes in Regulating Rosaceae Fruit Development

The function of floral organ identity genes, APETALA1/2/3, PISTILLATA, AGAMOUS, and SEPALLATA1/2/3, in flower development is highly conserved across angiosperms. Emerging evidence shows that these genes also play important roles in the development of the fruit that originates from floral organs following pollination and fertilization. However, their roles in fruit development may vary significantly between species depending on the floral organ types contributing to the fruit tissues. Fruits of the Rosaceae family develop from different floral organ types depending on the species, for example, peach fruit flesh develops from carpellary tissues, whereas apple and strawberry fruit flesh develop from extra-carpellary tissues, the hypanthium and receptacle, respectively. In this review, we summarize recent advances in understanding floral organ gene function in Rosaceae fruit development and analyze the similarities and diversities within this family as well as between Rosaceae and the model plant species Arabidopsis and tomato. We conclude by suggesting future research opportunities using genomics resources to rapidly dissect gene function in this family of perennial plants.

Comparative pangenomics: analysis of 12 microbial pathogen pangenomes reveals conserved global structures of genetic and functional diversity

Background With the exponential growth of publicly available genome sequences, pangenome analyses have provided increasingly complete pictures of genetic diversity for many microbial species. However, relatively few studies have scaled beyond single pangenomes to compare global genetic diversity both within and across different species. We present here several methods for “comparative pangenomics” that can be used to contextualize multi-pangenome scale genetic diversity with gene function for multiple species at multiple resolutions: pangenome shape, genes, sequence variants, and positions within variants. Results Applied to 12,676 genomes across 12 microbial pathogenic species, we observed several shared resolution-specific patterns of genetic diversity: First, pangenome openness is associated with species’ phylogenetic placement. Second, relationships between gene function and frequency are conserved across species, with core genomes enriched for metabolic and ribosomal genes and accessory genomes for trafficking, secretion, and defense-associated genes. Third, genes in core genomes with the highest sequence diversity are functionally diverse. Finally, certain protein domains are consistently mutation enriched across multiple species, especially among aminoacyl-tRNA synthetases where the extent of a domain’s mutation enrichment is strongly function-dependent. Conclusions These results illustrate the value of each resolution at uncovering distinct aspects in the relationship between genetic and functional diversity across multiple species. With the continued growth of the number of sequenced genomes, these methods will reveal additional universal patterns of genetic diversity at the pangenome scale.

Discovery of a MUC3B gene reconstructs the membrane mucin gene cluster on human chromosome 7

Human tissue surfaces are coated with mucins, a family of macromolecular sugar-laden proteins serving diverse functions from lubrication to formation of selective biochemical barriers against harmful microorganisms and molecules. Membrane mucins are a distinct group of mucins that are attached to epithelial cell surfaces where they create a dense glycocalyx facing the extracellular environment. All mucin proteins carry long stretches of tandemly repeated sequences that undergo extensive O-linked glycosylation to form linear mucin domains. However, the repetitive nature of mucin domains makes them prone to recombination and render their genetic sequences particularly difficult to read with standard sequencing technologies. As a result, human mucin genes suffer from significant sequence gaps that have hampered investigation of gene function in health and disease. Here we leveraged a recent human genome assembly to identify a previously unmapped MUC3B gene located within a cluster of four structurally related membrane mucin genes that we entitle the MUC3 cluster at q22 locus in chromosome 7. We found that MUC3B shares high sequence identity with the known MUC3A gene, and that the two genes are governed by evolutionarily conserved regulatory elements. Furthermore, we show that MUC3A, MUC3B, MUC12 and MUC17 in the human MUC3 cluster are exclusively expressed in intestinal epithelial cells. Our results complete existing genetic gaps in the MUC3 cluster that is a conserved genetic unit during primate evolution. We anticipate our results to be the starting point for detection of new polymorphisms in the MUC3 cluster associated with human diseases. Moreover, our study provides the basis for exploration of intestinal mucin gene function in widely used experimental models such as human intestinal organoids and genetic mouse models.