Lineage-specific sequence evolution and exon edge conservation partially explain the relationship between evolutionary rate and expression level in A. thaliana

Abstract

Rapidly evolving proteins can aid the identification of genes underlying phenotypic adaptation across taxa, but functional and structural elements of genes can also affect evolutionary rates. In plants, the 'edges' of exons, flanking intron junctions, are known to contain splice enhancers and to have a higher degree of conservation compared to the remainder of the coding region. However, the extent to which these regions may be masking indicators of positive selection or account for the relationship between dN/dS and other genomic parameters is unclear. We investigate the effects of exon edge conservation on the relationship of dN/dS to various sequence characteristics and gene expression parameters in the model plant Arabidopsis thaliana. We also obtain lineage-specific dN/dS estimates, making use of the recently sequenced genome of Thellungiella parvula, the second closest sequenced relative after the sister species Arabidopsis lyrata. Overall, we find that the effect of exon edge conservation, as well as the use of lineage-specific substitution estimates, upon dN/dS ratios partly explains the relationship between the rates of protein evolution and expression level. Furthermore, the removal of exon edges shifts dN/dS estimates upwards, increasing the proportion of genes potentially under adaptive selection. We conclude that lineage-specific substitutions and exon edge conservation have an important effect on dN/dS ratios and should be considered when assessing their relationship with other genomic parameters.

Keywords: Arabidopsis thaliana; dN/dS; lineage-specific evolution; splice enhancer.

Fig. 1

dN, dS, dN/dS and NI after exon edge removal. dN/dS (a), dN (b), dS (c) and NI (d) for a sample of 1443 genes with at least one fully alignable exon between A. thaliana and A. lyrata, after removing one codon at a time from exon edges (black), to a maximum of 30. The effects of random codon removal are shown in red. Distributions significantly differ when 30 codons are removed sequentially, but not randomly, compared to when no codons are removed. For sequential removal vs. no removal, Kruskal–Wallis P = 0.02 (dN/dS) and < 2.2 × 10⁻¹⁶ (NI). For random removal vs. no removal, Kruskal–Wallis P = 0.08 (dN/dS) and 0.49 (NI).

Fig. 2

Variables that have a significantly different correlation with dN/dS after the sequential removal of 30 codons from exon edges, compared to random codon removal. The four variables shown – expression breadth, expression level, tau and GC content – are those which have significantly different estimates of rho for their correlation with dN/dS before and after codon removal. Two criteria are met for each variable: that rho is significantly different after sequential, compared to random codon removal, and that rho is significantly different after sequential, compared to no codon removal. Estimates of dN/dS are made using alignments of A. thaliana against A.lyrata. Data for this figure, including P-values and sample sizes, are shown in Table S6 (Supporting information).