miRNAs associated with auxin signaling, stress response, and cellular activities mediate adventitious root formation in apple rootstocks

Adventitious root (AR) formation is essential for the vegetative propagation of apple rootstocks. miRNAs play a significant role in regulating AR development, however, large-scale transcriptomic data on miRNA mediated AR formation in apple rootstocks is lacking. Therefore, in order to identify the molecular mechanisms underlying AR formation in 'M9-T337' apple rootstocks, transcriptomic changes occurring during key time points of AR formation (0, 3, and 16 days) were analyzed using high-throughput sequencing with a focus on miRNAs. A total of 84 known miRNAs and 56 novel miRNAs have differentially expressed were identified. Additionally, a total of 88 target genes of known miRNAs and 76 target genes of novel miRNAs were identified by degradome sequencing. The expression levels of the miRNAs and target genes were quantified by RT-qPCR. Results indicate that miRNAs and their target genes are associated with auxin signal-related (miR160 and miR390), stress response-related (miR398, miR395 and miR408), cell fate transformation-, proliferation- and enlargement-related (miR171, miR156, miR166, miR319 and miR396). These all involve pathways that participate in AR formation in 'M9-T337' apple rootstock. In addition, hormones (AUX, CTK, GA3, BR, JA, and ABA) are also involved in regulating AR formation. The candidate genes belonging to pathways associated with AR formation exhibited significantly higher expression levels, providing evidence that they may be involved in the regulation of AR development. The collective results of the present study indicate that the developmental process associated with AR formation in apple rootstock is extremely complex. The known and novel miRNAs and target genes that were identified by high-throughput and degradome sequencing, respectively, provide a framework for the future analysis of miRNAs associated with AR development in apple rootstocks, and provide new information that can be used to better understand AR development in woody plants.