Alternative Splicing Is Predominantly Stochastic in Mungbean (Vigna radiata)

Alternative splicing (AS) can produce multiple mature mRNAs from the same primary transcript, thereby generating diverse proteins and phenotypes from the same gene. To assess the prevalence of AS in mungbean (Vigna radiata), we analyzed whole-genome RNA sequencing data from root, leaf, flower, and pod tissues, and found that at least 37.9% of mungbean genes are subjected to AS. The number of AS transcripts consistently exhibited a strong correlation with gene and exon number on all chromosomes, and thus resembled a uniform probabilistic event rather than a specific regulatory function. The proportion of missense splicing was close to the expected frequency of random splicing. However, alternative donor and acceptor AS events tended to occur at multiples of three nucleotides (i.e., the codon length) from the main splice site. Genes with high exon number and expression level, which should have the most AS if splicing is purely stochastic, exhibited less AS, implying the existence of negative selection against excessive random AS. Functional AS is probably rare: a large proportion of AS isoforms exist at fewer than one copy per cell on average, or are expressed at much lower levels than default transcripts. Conserved AS was only detected in 349 genes (1.5% of all genes in the genome) out of 1503 genes (6.7% of all genes) with conserved introns in Vigna angularis, and in 16 genes in more distant species like soybean. These observations highlight the challenges of finding and cataloging candidates for experimentally proven AS isoforms in a crop genome.