ABSTRACT: Background: MicroRNAs (miRNAs), a class of small non-coding endogenous RNAs that regulate gene expression posttranscriptionally, play multiple key roles in plant growth and development and the stress response. Knowledge of and the roles of miRNAs in pomegranate fruit development have not been explored. Results: Pomegranate, which accumulates a large amount of anthocyanins in skin and arils, is valuable to human health, mainly because of antioxidant properties. In this study, we developed a small RNA library from pooled RNA samples from young seedling to matured fruits and identified both conserved and pomegranate-specific miRNA from 29,948,480 high-quality reads. For the pool of 15- to 30-nt small RNAs, ~50% were 24 nt. The miR157 family was the most abundant, followed by miR156, miR160, and miR159, with many variants within each family. The base bias at the first position from the 5’ end has a strong preference for U for most 18- to 26-nt miRNAs but a preference for A for 18-nt miRNAs. In addition, for all 24-nt miRNAs, the nucleotide U is preferred (97%) in the first position. RT-qPCR was used to validate the expression of the predominant miRNA families in leaves, male and female flowers, and multiple fruit developmental stages; miR156, miR156-v1, miR159, miR159-v1, and miR319 were upregulated during the later stages of fruit development. Gene ontology and KEGG pathway analyses revealed predominant metabolic processes and catalytic activities, important for fruit development. In addition, KEGG pathway analyses revealed the involvement of miRNAs in ascorbate and linolenic acid, starch and sucrose metabolism; RNA transport; plant hormone signaling pathways; and circadian clock. Conclusion: Pomegranate largely contains anthocyanin, flavonoids, and antioxidants, which play critical roles in treating cancer, Alzheimer disease, and preventing heart attacks. Our first and preliminary report of novel miRNAs provides information on the biochemical compounds of pomegranate for future research. The functions of the targets of these novel miRNAs need further investigation.
