Identification and Characterization of miRNAs in Self-Rooted and Grafted Malus Reveals Critical Networks Associated with Flowering

Grafting can improve the agricultural traits of crop plants, especially fruit trees. However, the regulatory networks and differentially expressed microRNAs (miRNAs) related to grafting in apple remain unclear. Herein, we conducted high-throughput sequencing and identified differentially expressed miRNAs among self-rooted Fuji, self-rooted M9, and grafted Fuji/M9. We analyzed the flowering rate, leaf morphology, and nutrient and carbohydrate contents in the three materials. The flowering rate, element and carbohydrate contents, and expression levels of flowering genes were higher in Fuji/M9 than in Fuji. We detected 206 known miRNAs and 976 novel miRNAs in the three materials, and identified those that were up- or downregulated in response to grafting. miR156 was most abundant in Fuji, followed by Fuji/M9, and then self-rooted M9, while miR172 was most abundant in M9, followed by Fuji/M9, and then Fuji. These expression patterns suggest that that these miRNAs were related to grafting. A Gene Ontology (GO) analysis showed that the differentially expressed miRNAs controlled genes involved in various biological processes, including cellular biosynthesis and metabolism. The expression of differentially expressed miRNAs and flowering-related genes was verified by qRT-PCR. Altogether, this comprehensive analysis of miRNAs related to grafting provides valuable information for breeding and grafting of apple and other fruit trees.