In this project we will use a cmt3a DNA (methyl transferase) mutant to study epigenetic effects on gene expression and heritability in tomato. We will use cmt3a because other DNA methylation mutants (nrpd1, nrpe1, met1) are completely sterile and they cannot be used for studies of heritability. Approximately 5% of the hypomethylated regions in cmt3a persist in backcrossed progeny with a wild type CMT3a - these regions lack the genomic information for reestablishment of the DNA methylation. The other 95% regain their methylation indicating that they have genomic features that guide the recruitment of CMT3a. In the first subproject we will characterize the heritability and gene expression characteristics of CMT3a targets using bisulphite sequencing and RNAseq. The methylation patterns will be followed over at least five generations and in controlled environment and glasshouse environments. The second subproject will involve a multiple correspondence analysis of epigenetic properties (heritability, gene expression) with intrinsic (repetition, base composition etc ) and extrinsic (gene, type of transposon) genomic features. From this activity we expect to identify different classes of DNA methylation loci. The robustness of this classification model will be tested in mutant lines for mutants other than cmt3a that will influence the pattern of DNA methylation or demethylation. Finally we will develop a dCas9 SunTag system in which the TET1 DNA hydroxylase will target DNA demethylation to specific regions of the genomes so that we can further test the classification model and explore the extent to which heritable epigenetic changes to gene can be engineered into the tomato genome. The project is basic science addressing a fundamental question in epigenetics: why is DNA methylation attracted to some regions of the genome and not others. In the longer term the project will inform approaches to crop improvement by epigenetic modification.