Predicting family-wide consensus sequence of pre-mRNA splicing signals in legumes and their potential utility as phylogenetic markers

Recent studies have shown that an intron is not merely “junk”, but something that plays important roles in many biological processes such as gene expression regulation and alternative splicing. For purposes of studying intron structures and predicting consensus splice motifs, a total of 102 legume species were used to isolate introns across the family. Of 196 gene-targeted PCR primer pairs, we successfully amplified 118 intron-containing genes (60.2 %) and obtained a total of 1,870 introns with an average size of 143 nucleotides, ranging from 61 to 1,036. Species-based compilation of 5′- and 3′-splicing motifs showed, to some extent, lineage-specific conservation in each splicing motif. Compilation of the entire intron set permitted prediction of the consensus sequences of splicing signal motifs in legumes, AYGᵂ GTAᴮAᴮGᴴ and TⱽNC/TAGGᴴTⱽ for the 5′SS and 3′SS, respectively. Interestingly, these consensus motifs are very similar to the corresponding genome-wide splicing signals of two model systems, Arabidopsis and rice. This result suggests conservation of pre-mRNA splicing mechanism occurring in higher plants. Multiple alignments of CALTL introns demonstrated that the BP-3′SS region was relatively more conserved than the 5′SS-BP region. We speculate that length of the BP-3′SS region needs to be retained for the interaction with U2AF protein. Phylogenetic analysis demonstrates that each of three splicing motifs is not only phylogenetically informative, but also relevant to evolutionary divergence of species. This result suggests that the splice signal sequences would be a useful tool for the molecular phylogenetic analysis. We also anticipate that gene-targeted amplification in multiple genomes, described in this study, would facilitate studies on intron-located functional elements involved in gene expression regulations.