Species-specific gene duplication inArabidopsis thalianaevolved novel phenotypic effects on morphological traits under strong positive selection

AbstractGene duplication is increasingly recognized as an important mechanism for the origination of new genes, as revealed by comparative genomic analysis. However, the ways in which new duplicate genes contribute to phenotypic evolution remain largely unknown, especially in plants, owing to a lack of experimental and phenotypic data. In this study, we identified the new geneExov,derived from a partial gene region duplication of its parental geneExov-L, which is a member of an exonuclease family, into a different chromosome inArabidopsis thaliana. We experimentally investigated the phenotypic effects ofExovandExov-Lin an attempt to understand how the new gene diverged from the parental copy and contributes to phenotypic evolution. Evolutionary analysis demonstrated thatExovis a species-specific gene that originated within the last 3.5 million years and shows strong signals of positive selection. Unexpectedly, RNAseq analyses reveal that the new gene, despite its young age, has acquired a large number of novel direct and indirect interactions in which the parental gene does not engage. This is consistent with a high, selection-driven substitution rate in the protein sequence encoded byExovin contrast to the slowly evolvingExov-L, suggesting an important role forExovin phenotypic evolution. We analyzed phenotypic effects ofexovandexov-lsingle T-DNA-insertion mutants;doubleexov, exov-lT-DNA insertion mutants; and CRISPR/Cas9-mediatedexovcrpandexov-lcrpknockouts on seven morphological traits in both the new and parental genes. We detected significant segregation of morphological changes for all seven traits when assessed in terms of single mutants, as well as morphological changes for seven traits associated with segregation of doubleexov, exov-lmutants. Substantial divergence of phenotypic effects between new and parental genes was revealed by principal component analyses, suggesting neofunctionalization in the new gene. These results reveal a young gene that plays critical roles in biological processes that underlie morphological and developmental evolution inArabidopsis thaliana.