The genome sequence of the diploid and highly homozygous Vitis vinifera genotype PN40024 serves as the reference for many grapevine studies. Despite several improvements to the PN40024 genome assembly, its current version PN12X.v2 is quite fragmented and only represents the haploid state of the genome with mixed haplotypes. In fact, being nearly homozygous, this genome contains several heterozygous regions that are yet to be resolved. Taking the opportunity of improvements that long-read sequencing technologies offer to fully discriminate haplotype sequences, an improved version of the reference, called PN40024.v4, was generated. Through incorporating long genomic sequencing reads to the assembly, the continuity of the 12X.v2 scaffolds was highly increased with a total number decreasing from 2,059 to 640 and a reduction in N bases of 88%. Additionally, the full alternative haplotype sequence was built for the first time, the chromosome anchoring was improved and the number of unplaced scaffolds was reduced by half. To obtain a high-quality gene annotation that outperforms previous versions, a liftover approach was complemented with an optimized annotation workflow for Vitis. Integration of the gene reference catalogue and its manual curation have also assisted in improving the annotation, while defining the most reliable estimation of 35,230 genes to date. Finally, we demonstrated that PN40024 resulted from 9 selfings of cv. “Helfensteiner” (cross of cv. “Pinot noir” and “Schiava grossa”) instead of a single “Pinot noir”. These advances will help maintain the PN40024 genome as a gold-standard reference, also contributing toward the eventual elaboration of the grapevine pangenome.

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.

The abundance of transcriptomic data and the development of causal inference methods have paved the way for gene network analyses in grapevine. Vitis OneGenE is a transcriptomic data mining tool that finds direct correlations between genes, thus producing association networks. As a proof of concept, the stilbene synthase gene regulatory network obtained with OneGenE has been compared with published co-expression analysis and experimental data, including cistrome data for MYB stilbenoid regulators. As a case study, the two secondary metabolism pathways of stilbenoids and lignin synthesis were explored. Several isoforms of laccase, peroxidase, and dirigent protein genes, putatively involved in the final oxidative oligomerization steps, were identified as specifically belonging to either one of these pathways. Manual curation of the predicted sequences exploiting the last available genome assembly, and the integration of phylogenetic and OneGenE analyses, identified a group of laccases exclusively present in grapevine and related to stilbenoids. Here we show how network analysis by OneGenE can accelerate knowledge discovery by suggesting new candidates for functional characterization and application in breeding programs.

Aedes aegypti Act4 is a paralog of the Drosophila melanogaster indirect flight muscle actin gene Act88F. Act88F has been shown to be haploinsufficient for flight in both males and females (amorphic mutants are dominant). Whereas Act88F is expressed in indirect flight muscles of both males and females, expression of Act4 is substantially female-specific. We therefore used CRISPR/Cas9 and homology directed repair to examine the phenotype of Act4 mutants in two Culicine mosquitoes, Aedes aegypti and Culex quinquefasciatus. A screen for dominant female-flightless mutants in Cx. quinquefasciatus identified one such mutant associated with a six base pair deletion in the CxAct4 coding region. A similar screen in Ae. aegypti identified no dominant mutants. Disruption of the AeAct4 gene by homology-dependent insertion of a fluorescent protein marker cassette gave a recessive female-flightless phenotype in Ae. aegypti. Reproducing the six-base deletion from Cx. quinquefasciatus in Ae. aegypti using oligo-directed mutagenesis generated dominant female-flightless mutants and identified additional dominant female-flightless mutants with other in-frame insertions or deletions. Our data indicate that loss of function mutations in the AeAct4 gene are recessive but that short in-frame deletions produce dominant-negative versions of the AeAct4 protein that interfere with flight muscle function. This makes Act4 an interesting candidate for genetic control methods, particularly population-suppression gene drives targeting female viability/fertility.

The increasing availability of omics data from non-model plant species has created a pressing need for centralized, user-friendly platforms that maximize the utility of these datasets in a FAIR manner. Here, we introduce PlantaeViz, a web-based tool designed for the integration, visualization, and analysis of multi-omics data across a wide range of plant species. PlantaeViz offers advanced functionalities, including gene catalogues built from curated literature, transcriptomic meta-analyses presented as gene expression atlases, gene co-expression and regulatory networks with on-the-fly ontology analyses, cistrome visualization and metabolomics-transcriptomics integration, among other tools, providing a robust framework for hypothesis generation and biological interpretation. Gene Cards applications, tailored to each plant species, provide both community-curated and automatically generated functional annotation information. One of the platform's core features is its big data approach: over 58,000 publicly available SRA transcriptomic samples have been processed and visualized to date. Significant efforts have been made in orthology assessment using multiple layers of evidence, as well as in the automatic classification and standardization of omics metadata through regular expressions and data mining. As a result, around 90% of transcriptomic runs have been successfully classified according to sample tissue. These data have been used to construct gene networks via computationally intensive methods based on diverse algorithms. We present a study case to illustrate the platform's integration and exploratory capabilities. PlantaeViz bridges genomics and functional knowledge between model and non-model plant species and aims to expand its species catalogue of species in the future, democratizing access to large-scale plant omics data. Further developments will include the incorporation of additional data types, and the implementation of new tools to further support plant research across diverse biological contexts.

Water stress challenges global crop productivity, particularly for perennial species such as grapevines, where effective water management is crucial for berry quality and yield. Aquaporins, a family of water channel proteins, play a key role in regulating water transport within plant cells, affecting water uptake and redistribution. Although the transcriptional response of aquaporin genes to water stress in grapevines has been documented, their translational regulation remains less explored. This study investigates the transcriptional and translational dynamics of three Plasma Membrane Intrinsic Proteins and three Tonoplast Intrinsic Proteins in leaves and roots of a grafted 'Pinot Noir' on 'Kober 5BB' rootstock during water deficit conditions and recovery. Aquaporin translation analyzed by polysome profiling and co-sedimentation analysis of their transcripts highlighted that water stress had a general negative effect, although significant only for VviTIP1-3. Conversely, recovery measured at 6 h after rewatering was characterized by a boost of translation reactivation for all but one aquaporins. Transcriptional profiling of the same aquaporins revealed significant down-regulation at prolonged stress in roots, highlighting the contribution of aquaporins to osmoregulation and drought tolerance. Moreover, transcriptional modulation resembles a long-term adaptative response to limit water loss. In the leaf, only two specific genes, VviPIP2-5 and VviTIP2-1, were modulated during water deficit and even more during recovery and positively correlated with stomatal conductance and leaf water potential. They represent important regulators of water homeostasis and good candidates for breeding programs. This study uncovered an additional level of aquaporin post-transcriptional control finely tuning vines to changing external conditions.