Comparative Analysis of the Splice Sites of Different Plant Genomes

Abstract

Alternative splicing is a key mechanism that enhances transcriptome and proteome diversity in eukaryotes. While canonical GT–AG splice sites have been extensively characterized, non-canonical combinations remain poorly understood, especially in plants. Previous studies have mostly focused on model species and major crops, leaving many plant lineages unexplored. In this study, we conducted a comparative analysis of splice-site usage across 11 plant species representing diverse crops. Using high-quality RefSeq annotations, we extracted all introns within coding regions and quantified the frequencies and diversities of canonical and non-canonical splice sites. Normalized intron counts revealed that the relative proportions of non-canonical classes are stable across well-annotated genomes, while their diversity increases with the total number of introns. We further observed that non-canonical splice sites are more frequent in introns of extreme lengths, suggesting that intron architecture may influence splicing precision. A small number of recurrent non-canonical combinations (GA–AG, GT–AA, GT–AT, and GT–GG) were consistently detected across species, indicating that these motifs are evolutionarily tolerated and may arise through single-nucleotide substitutions. These results indicate that non-canonical splicing is an evolutionarily conserved feature of plant transcriptomes and provide new insights into splice-site variability beyond model species.