Bioinformatic identification of putative intergenic small open-reading frames in the grapevine genome and their roles in responding to biotic and abiotic stress

• The intergenic small open reading frames of grape genome were comprehensive identified. • Most nsORFs were transcriptionally active under stress. • This study fills the gap of neglected intergenic sORFs in grape. High-throughput sequencing has greatly deepened our understanding of genomic information. However, due to the limitation of software threshold setting, many existing short fragment genes that actually exist are missed. For example, the intergenic region, which is regarded as a ‘black box’ of the genome, contains vast amounts of information and is worthy of greater efforts to explore. This study presents a comprehensive identification of 3357,289 small open reading frames (sORFs) within the intergenic regions of grape genome, which resulted in 2884,287 novel sORFs (nsORFs) after excluding UniProt-matched sORFs. To further investigate these functions, we assessed the Codon Adaptation Index (CAI) of the nsORFs’ and selected 2433,873 with CAI ≥ 0.7 for subsequent analysis. Using the Rfam database, we identified 1312 nsORFs matching known non-coding RNA (ncRNA) families.A total of 781 nsORFs were jointly identified by both SignalP-5.0 and NLStradamus and were classified as signal peptides. Using transcriptome data from grape powdery mildew and salt stress studies, we found 236,843 and 145,938 transcriptionally active nsORFs with CAI ≥ 0.7, respectively. Gene Ontology (GO) annotation of six powdery mildew-associated nsORFs indicated their involvement in stress response. Our research focused on previously overlooked intergenic sORFs in the grape genome, enhancing genomic annotations and providing a foundation for discovering novel gene regulatory mechanisms.