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

Area of Science:

• Plant genomics
• Transcriptomics
• Molecular biology

Background:

• Alternative splicing (AS) generates transcriptome diversity in plants.
• Short-read RNA sequencing (RNA-seq) has limitations in resolving full-length transcripts and complex AS patterns, especially in perennial crops like apple (Malus domestica).

Purpose of the Study:

• To comprehensively characterize the full-length apple transcriptome across multiple tissues.
• To identify and analyze alternative splicing variations and their tissue-specific regulation.
• To create a valuable resource for understanding post-transcriptional regulation and genetic improvement in apple.

Main Methods:

• Integration of Oxford Nanopore Technology (ONT) long-read cDNA sequencing with Illumina RNA-seq.
• Characterization of the full-length transcriptome in seven apple tissues, including vegetative organs and fruit developmental stages.
• Quantification of gene and transcript expression, and assessment of AS variation using percent spliced in (PSI) and differential splicing analyses.

Main Results:

• Identification of 56,809 genes and 100,911 transcript isoforms.
• Construction of an AS atlas with 29,842 events across seven AS types, involving 17,659 genes.
• Discovery of extensive tissue-associated transcriptional and post-transcriptional regulation, including tissue-specific genes, isoforms, and widespread differential splicing.

Conclusions:

• Integrating ONT long reads with Illumina short reads enhances apple transcriptome annotation and AS characterization.
• The study provides a systematic characterization of tissue-resolved transcript and AS variation.
• The generated full-length transcriptome, unified annotation, and AS atlas serve as a crucial resource for apple research and breeding.