The molecular mechanism of dwarfing in the apple rootstock is poorly understood, and has been attributed to low root cytokinin biosynthesis levels. Here we identified differences in root trans-zeatin content and expression of cytokinin metabolic pathway genes between dwarfing and vigorous rootstocks. Specifically, a stable low expression of IPT5b was identified in the dwarfing rootstocks. Bisulfite sequencing showed that two CpG islands (CpG2 and CpG4) in the IPT5b promoter region showed higher methylation levels in the M9 rootstock (dwarfing) compared to the Mr (vigorous) rootstock. Furthermore, IPT5b expression increased when M9 rootstocks were treated with 5-azaC, a methylation blocker, indicating that methylation levels influence IPT5b expression. In conclusion, we found low IPT5b expression with high level methylations in promoter region, leading to poor root trans-zeatin biosynthesis in the M9 rootstock, which may induce dwarfing. (C) 2016 Elsevier Inc. All rights reserved.