The Role of RNA-directed DNA Methylation in Genome Dominance and Imprinting in Brassicaceae

During seed development, DNA methylation is partially controlled by the small RNA-directed DNA methylation (RdDM) pathway. In RdDM, small interfering (si)RNAs are produced by RNA Polymerase IV, which are abundant in gametophytes and developing seeds. The siRNA binds Argonaut 4 to target methylation machinery to regions of the genome via interaction with Pol V transcripts. Methylation is critical for proper expression of imprinted genes, and thus the role of Pol IV- dependent siRNAs and RdDM are of interest. Arabidopsis thaliana RdDM mutants show no obvious reproductive defects. However, Brassica rapa RdDM mutants show a reduction in seed set and seed weight. While A. thaliana is a primarily selfing species, B. rapa is a traditional outcrosser whose genome reflects a mesohexaploid expansion. To determine why these two species differ in seed development in RdDM mutants, we will use a comparative evolutionary genomics approach. We hypothesize that RdDM may be more critical in B. rapa because methylation is mediating 1) contributions from parental genomes during outcrossing (imprinting), and/or 2) contributions from the three subgenomes present in this species (genome dominance). To distinguish between these alternatives, we are characterizing RdDM mutants in three additional Brassicaceae species that vary in breeding system and genome copy number. Once mutant lines are established via CRISPR-Cas9, we will compare seed set and weight between species 1) differing in breeding system to test the extent to which methylation affects imprinting, or 2) differing in genome copy number to test the role of methylation in genome dominance.