Identification and Analysis of Graft-Responsive miRNAs in Mulberry Rootstock–Scion Interactions

Grafting profoundly influences fruit tree performance, yet the molecular mechanisms underlying rootstock–scion interactions in mulberry (Morus multicaulis) remain poorly understood. To investigate the regulatory networks linking grafting to scion physiology, we performed an integrated analysis combining small RNA sequencing, transcriptome-based KEGG pathway assessment, and qRT-PCR validation of key target genes across five rootstock–scion combinations. High-throughput miRNA profiling of phloem tissues identified 71 conserved and 156 novel miRNAs, which exhibited distinct, genotype-dependent expression patterns. Recurrent activation of nutrient- and stress-responsive families (e.g., miR399, miR397, and miR395) was observed. Target prediction and functional enrichment analyses revealed that these miRNAs likely regulate pathways related to metabolic processes, hormone signaling, cell wall modification, and stress responses. The expression trends of their predicted target genes—including TCP4, Laccase-3, and ATP sulfurylase 1—were subsequently confirmed by qRT-PCR, revealing significant rootstock-specific regulation. Collectively, this study establishes a reference framework for elucidating the molecular mechanisms underlying scion biological processes in Morus species, which is of great significance for improving fruit quality, enhancing abiotic and biotic stress resistance, and boosting long-term planting productivity.