Enhancing nature's palette through the epigenetic breeding of flower color in chrysanthemum

Summary Flower color is an important character of ornamental plants and one of the main target traits for variety innovation. We previously identified a CmMYB6 epigenetic allele that affects the flower color in chrysanthemum, and changes in flower color are caused by the DNA methylation level of this gene. However, it is still unknown which DNA methyltransferases are involved in modifying the DNA methylation levels of this gene. Here, we used dead Cas9 (dCas9) together with DNA methyltransferases that methylate cytosine residues in the CHH context to target the CmMYB6 promoter through transient and stable transformation methods. We found that CmDRM2a increased the DNA methylation level of the CmMYB6 promoter, the expression of CmMYB6 decreased and a lighter flower color resulted. By contrast, both CmDRM2b and CmCMT2 enhanced DNA methylation levels of the CmMYB6 promoter, the expression of CmMYB6 increased and a deeper flower color resulted. Furthermore, the regulatory mechanism of DNA methyltransferase in the formation of chrysanthemum flower color was investigated, pointing to a new strategy for silencing or activating CmMYB6 epiallele to regulate anthocyanin synthesis. This lays a solid foundation for regulating flower color in chrysanthemum through epigenetic breeding.