Unveiling unique alternative splicing responses to low temperature in Zoysia japonica through ZjRTD1.0, a high‐quality reference transcript dataset

Abstract Inadequate reference databases in RNA‐seq analysis can hinder data utilization and interpretation. In this study, we have successfully constructed a high‐quality reference transcript dataset, ZjRTD1.0, for Zoysia japonica , a widely‐used turfgrass with exceptional tolerance to various abiotic stress, including low temperatures and salinity. This dataset comprises 113,089 transcripts from 57,143 genes. BUSCO analysis demonstrates exceptional completeness (92.4%) in ZjRTD1.0, with reduced proportions of fragmented (3.3%) and missing (4.3%) orthologs compared to prior datasets. ZjRTD1.0 enables more precise analyses, including transcript quantification and alternative splicing assessments using public datasets, which identified a substantial number of differentially expressed transcripts (DETs) and differential alternative splicing (DAS) events, leading to several novel findings on Z. japonica 's responses to abiotic stresses. First, spliceosome gene expression influenced alternative splicing significantly under abiotic stress, with a greater impact observed during low‐temperature stress. Then, a significant positive correlation was found between the number of differentially expressed genes (DEGs) encoding protein kinases and the frequency of DAS events, suggesting the role of protein phosphorylation in regulating alternative splicing. Additionally, our results suggest possible involvement of serine/arginine‐rich (SR) proteins and heterogeneous nuclear ribonucleoproteins (hnRNPs) in generating inclusion/exclusion isoforms under low‐temperature stress. Furthermore, our investigation revealed a significantly enhanced overlap between DEGs and differentially alternatively spliced genes (DASGs) in response to low‐temperature stress, suggesting a unique co‐regulatory mechanism governing transcription and splicing in the context of low‐temperature response. In conclusion, we have proven that ZjRTD1.0 will serve as a reliable and useful resource for future transcriptomic analyses in Z. japonica .