Hypomethylation in plants is associated with improved stress responses and is therefore a favourable trait. However, when methylation mutants are produced there is often a transgenerational decrease in fitness, meaning this is not a viable avenue to explore in crops.

Utilising a set of epigenetics recombinant inbred lines (epiRILs), where met1-3 was crossed with Col-0 to produce a ‘mosaic pattern’ in DNA methylation, we found that a region of chromosome 3 where was inheritance solely from the Col-0 parent. This region contains the gene IBM1, for which transgenerational deleterious effects has been previously associated with change in intronic methylation, leading to alternative splicing of the IBM1 transcript and ectopic genomic hypermethylation occurring at gene bodies. In the same regions we also found another gene, a vacuolar ATPase (VHA), had a similar methylation profile to IBM1 and complementary expression within the flower. We hypothesized that the interaction of IBM1 and VHA could explain the transgenerational decrease in fitness observed in met1-3 plants. Existing RNA-seq data was assessed for this possibility, and a range of genetic approaches were applied including crossing of IBM1 and VHA knockout mutants and CRISPR-Cas9 to generate double mutants. We propose there is incompatibility between the pollen and carpels of these methylation mutants, ultimately resulting in decreased fitness and viability