Leaf patterning of Clivia miniata var. variegata is associated with differential DNA methylation

Leaf patterns (yellow, green and striped) of Clivia miniata var. variegata might be caused by differential DNA methylation in CCGG sites in response to heterogeneous environmental pressure. Clivia miniata is an important ornamental plant.Clivia miniata var. variegata (Cmvv) is a variegated leaf mutant of C. miniata. Typical Cmvv has attractive green and yellow-stripped leaves. The study has revealed that an explant of Cmvv, even a full-green explant, could regenerate plants of three different types: yellow, green, ands triped; normal-appearing chloroplasts were found in guard cells but not in mesophyll cells of all the three types of Cmvv using confocal laser scanning microscopy (CLSM).Thus, we speculated that cells of the three types of Cmvv had an identical mutation and the mutation might disturb mesophyll cell chloroplast biogenesis after symplastic isolation of guard cells. Using CLSM and methylation sensitive amplification polymorphism (MSAP), we found that (a) striped leaves of Cmvv are due to sectorial decreases in chlorophyll levels and the decreases are associated with CG hypermethylation; (b) extent of epigenetic divergence among the three types of Cmvv leaves is positively correlated with intensity of leaf-color difference; and (c) green stripes of two plants are clustered in one group based on the MSAP profiles, but green and yellow stripes of a plant are not. Sequencing analysis indicated that CG hypermethylation in gene bodies of CPSAR1 and ycf2 might lead to gene silencing and yellow leaves/stripes of Cmvv. All together, it is possible that cytosine methylation involved regulating leaf color of Cmvv, also striped pattern of Cmvv might be caused by differential DNA methylation in response to heterogeneous environmental pressure. Furthermore, a novel leaf-color epigenetic hypothesis was proposed in this article.