Comprehensive analysis of alternative splicing and transcriptome diversity in apple using long-read sequencing

Alternative splicing (AS) is a major source of transcriptome diversity in plants, but short-read RNA sequencing (RNA-seq) has limited power for resolving full-length transcripts and complex AS patterns, particularly in perennial crops.

We integrated Oxford Nanopore Technology (ONT) long-read cDNA sequencing with Illumina RNA-seq to characterize the full-length transcriptome across seven apple (Malus domestica) tissues, including vegetative organs and three fruit developmental stages. Gene- and transcript-level expression were quantified, and AS variation was assessed using percent spliced in (PSI) and differential splicing analyses.

We identified 56,809 genes and 100,911 transcript isoforms and constructed an AS atlas comprising 29,842 events across seven AS types, involving 17,659 genes and 73,067 transcript isoforms. These analyses revealed extensive tissue-associated transcriptional and post-transcriptional regulation, including large sets of tissue-specific genes, transcript isoforms, and AS events, as well as widespread differential splicing between tissue pairs.

Together, our results show that integrating ONT long reads for isoform discovery with Illumina short reads for quantitative support improves apple transcriptome annotation and enables systematic characterization of tissue-resolved transcript and AS variation. The resulting full-length transcriptome, unified transcript annotation, and AS atlas provide a valuable resource for studies of post-transcriptional regulation, trait-associated transcript variation, and genetic improvement in apple.