Characterisation of early fruit development-specific miRNAs and their targets in peach using small RNA and degradome sequencing

Peach (Prunus persica L.) is a key drupe fruit crop characterized by a double-sigmoid growth pattern during fruit development. MicroRNAs (miRNAs) play pivotal regulatory roles in this process; however, their functions in early peach fruit development remain poorly understood. To address this, we conducted high-throughput small RNA (sRNA) sequencing at three critical developmental stages: 20, 50, and 75 d post-anthesis (DPA20, DPA50, DPA75), representing exponential growth, lag phase, and rapid regrowth, respectively. We identified 124 known miRNAs from 70 families and 86 novel miRNAs, with 97 and 79 miRNAs differentially expressed between DPA50 vs DPA20 and DPA75 vs DPA50, respectively. Degradome sequencing revealed 216 target genes for 74 known and eight novel miRNAs, with targets of ppe-miR160a and ppe-miR393b validated using RNA ligase-mediated rapid amplification of 5' cDNA ends (RLM-5'RACE). Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) confirmed the expression patterns of five miRNAs and their target genes, consistent with sequencing data. Network analysis revealed the involvement of the auxin signaling pathway and a complex miRNA regulatory network in fruit enlargement. Key miRNA-target interactions, such as ppe-miR160a-ARFs, ppe-miR172a/c/d-AP2s, ppe-miR166a -ATHBs, and ppe-miR319c/e-TCPs were implicated in cell proliferation and differentiation, with novel findings including ppe-miR171b/f/h-SCL6s in cell division. In summary, peach miRNAs primarily regulate early fruit growth and development by modulating auxin signaling and mediating cell division and differentiation. These findings provide critical insights into miRNA-mediated mechanisms underlying peach fruit development and establish a foundation for future research on drupe fruit crops.