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Article|30 Jan 2023|OPEN
The role of DNA methylation in the maintenance of phenotypic variation induced by grafting chimerism in Brassica 
Ke Liu1 , Tingjin Wang1 , Duohong Xiao1 , Bin Liu1 , Yang Yang1 , Kexin Xu1 , Zhenyu Qi2 , Yan Wang1 , Junxing Li1 , Xun Xiang1 , Lu Yuan1 , and Liping Chen,1 ,
1Department of Horticulture, College of Agriculture and Biotechnology, Zhejiang University, Hangzhou 310058, China
2Agricultural Experiment Station, Zhejiang University, Hangzhou 310058, China
*Corresponding author. E-mail:,

Horticulture Research 10,
Article number: uhad008 (2023)
Views: 69

Received: 13 Sep 2022
Accepted: 18 Jan 2023
Published online: 30 Jan 2023


Grafting facilitates the interaction between heterologous cells with different genomes, resulting in abundant phenotypic variation, which provides opportunities for crop improvement. However, how grafting-induced variation occurs and is transmitted to progeny remains elusive. A graft chimera, especially a periclinal chimera, which has genetically distinct cell layers throughout the plant, is an excellent model to probe the molecular mechanisms of grafting-induced variation maintenance. Here we regenerated a plant from the T-cell layer of a periclinal chimera, TCC (where the apical meristem was artificially divided into three cell layers – from outside to inside, L1, L2, and L3; T = Tuber mustard, C = red Cabbage), named rTTT0 (r = regenerated). Compared with the control (rsTTT, s = self-grafted), rTTT0 had multiple phenotypic variations, especially leaf shape variation, which could be maintained in sexual progeny. Transcriptomes were analyzed and 58 phenotypic variation-associated genes were identified. Whole-genome bisulfite sequencing analyses revealed that the methylome of rTTT0 was changed, and the CG methylation level was significantly increased by 8.74%. In rTTT0, the coding gene bodies are hypermethylated in the CG context, while their promoter regions are hypomethylated in the non-CG context. DNA methylation changes in the leaf shape variation-associated coding genes, ARF10IAA20ROF1, and TPR2, were maintained for five generations of rTTT0. Interestingly, grafting chimerism also affected transcription of the microRNA gene (MIR), among which the DNA methylation levels of the promoters of three MIRs associated with leaf shape variation were changed in rTTT0, and the DNA methylation modification of MIR319 was maintained to the fifth generation of selfed progeny of rTTT0 (rTTT5). These findings demonstrate that DNA methylation of coding and non-coding genes plays an important role in heterologous cell interaction-induced variation formation and its transgenerational inheritance.