Identification of Freezing-Responsive microRNAs and Their Targets in Chinese Jujube by Small RNA and Degradome Sequencing

The jujube tree fruit remains a primary fruit in northern China, yet its geographical distribution and yield are significantly constrained by freezing stress during winter. Numerous studies have highlighted the pivotal regulatory function of microRNAs (miRNAs) in plant responses to low-temperature stress. Nevertheless, the specific miRNAs involved in the response to low temperatures and their associated gene networks in Ziziphus jujuba Mill are not well understood. In this investigation, we utilized high-throughput sequencing to analyze small RNA libraries from branches subjected to temperatures of 4 °C and −30 °C. Our analysis identified a total of 342 miRNAs, comprising 123 known miRNAs and 219 novel miRNAs. The differential expression analysis revealed that under low-temperature conditions, 177 miRNAs underwent significant changes. Among them, specific upregulation of miR319 in the less cold-resistant variety and miR6483 in sensitive variety was observed. By employing degradome sequencing, we identified a total of 1551 target genes corresponding to 3059 unique miRNA target interaction pairs involving 299 miRNAs. Functional analysis using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways indicated that these target genes are primarily associated with transcriptional regulation, metabolic pathways, and genetic information processing. Through a comprehensive analysis, we pinpointed 11 genes corresponding to 9 miRNAs that are implicated in jujube tree cold stress, and 7 target genes of 7 miRNAs were confirmed by 5′-RACE analysis. These miRNAs are likely to exert crucial regulatory functions in the context of jujube tree cold stress. This study is the first to systematically identify miRNAs and their target genes in the response of Ziziphus jujuba Mill to low-temperature stress, which provides important resources for in-depth analysis of the molecular mechanism of jujube tree cold resistance and for cold-resistant breeding.