Integrated DNA methylation, transcriptome and physiological analyses reveal new insights into superiority of poplars formed by interspecific grafting

Plant grafting has a long history and it is extensively employed to improve plant performance. In our previous research, reciprocal grafts of Populus cathayana Rehder (C) and Populus deltoides Bart. Ex Marsh (D) were generated. The results showed that interspecific grafting combinations (scion/rootstock: C/D and D/C) grew better than intraspecific grafting combinations (C/C and D/D). To further understand differences in molecular mechanisms between interspecific and intraspecific grafting, we performed an integrated analysis, including bisulfite sequencing, RNA sequencing and measurements of physiological indicators, to investigate leaves of different grafting combinations. We found that the difference at the genome-wide methylation level was greater in D/C vs D/D than in C/D vs C/C, but no difference was detected at the transcription level in D/C vs D/D. Furthermore, the grafting superiority of D/C vs D/D was not as strong as that of C/D vs C/C. These results may be associated with the different methylation forms, mCHH (71.76%) and mCG (57.16%), that accounted for the highest percentages in C/D vs C/C and D/C vs D/D, respectively. In addition, the interspecific grafting superiority was found mainly related to the process of photosynthesis, phytohormone signal transduction, biosynthesis of secondary metabolites, cell wall and transcriptional regulation based on both physiological and molecular results. Overall, the results indicated that the physiological and molecular phenotypes of grafted plants are affected by the interaction between scion and rootstock. Thus, our study provides a theoretical basis for developing suitable scion-rootstock combinations for grafted plants.