Oxford Nanopore Full-Length Transcriptome Reveals Alternative Splicing and Its Functional Diversity in Regulating Fruit Ripening in Peach (Prunus persica)

Fruit development and ripening in peach (Prunus persica) involve complex transcriptional and post-transcriptional regulation. While short-read sequencing has advanced transcriptome studies, it often fails to accurately resolve complex transcript isoforms. This study employed Oxford Nanopore Technologies’ (ONT) full-length RNA-Seq to comprehensively characterize the transcriptomic landscape of peach fruits across three key developmental stages: the first exponential stage, the second exponential stage, and the ripening stage. Our analysis identified 44,042 non-redundant isoforms, including 1109 novel genes and 32,289 novel isoforms, significantly expanding the peach genome annotation. We further investigated alternative splicing (AS) events, revealing that intron retention (IR) and alternative 3′ splice site (A3′S) were the most prevalent types, with AS abundance peaking at the S1 stage. A total of 10,236 differentially expressed transcripts (DETs) were identified, highlighting dynamic expression patterns during fruit development. Functional characterization focused on a MADS-box gene, PpMADS6, which produced two isoforms via alternative splicing. Dual luciferase assays in tobacco leaves demonstrated that the full-length isoform, PpMADS6a, specifically activated the promoter of the fruit-softening gene PpPG1, while the truncated isoform, PpMADS6b, lost this transactivation ability. This study provides a valuable resource of full-length transcriptomes for peach and underscores the critical role of alternative splicing in generating functional diversity to fine-tune fruit development and ripening processes.