Analysis of DNA methylation during spontaneous rooting from the stem apex in <Emphasis Type="Italic">Rubus idaeus</Emphasis>

Rubus idaeus L. is of great economic value. Some varieties of Rubus idaeus have a unique feature of spontaneous rooting from the stem apex. To determine whether DNA methylation is associated with the spontaneous rooting process, variations in the methylation at stem apex during four root developmental stages were investigated, using the methylation-sensitive amplification polymorphism (MSAP) technique and the bisulfite sequencing analysis (BSA). The results showed that the DNA methylation levels and patterns were significantly different between the four developmental stages. A total of 824 CCGG amplified sites were detected by MSAP. MSAP screening revealed that the level of DNA methylation at stages I to IV was 34.95, 36.04, 36.29, and 37.50%, respectively. The number of methylated sites and their methylation levels tended to decrease at stages III and IV (root differentiation and elongation) compared with those at stage I (stem elongation). After cloning and sequencing of the 16 polymorphic differentially methylated DNA fragments, BLAST search results indicated that they might be involved in differentiation of the lateral root primordium, plant defense, signal transduction, and energy metabolism. Results of the qRT-PCR and BSA analyses confirmed that methylation of some key genes was closely associated with their expression at the different developmental stages. These findings could be useful for future studies on the potential role of DNA methylation in spontaneous rooting from the stem apex, implying its importance in rooting regulation and rapid expansion of raspberry populations.