Identification and characterization of microRNAs involved in double-color formation in Paeonia suffruticosa ‘Shima Nishiki’ by high-throughput sequencing

Paeonia suffruticosa ‘Shima Nishiki’ is a valuable double-color cultivar, but the mechanism of its double-color formation is still unknown at the post-translational level. Moreover, many studies have shown that microRNAs (miRNAs) play a key role in regulating anthocyanin biosynthesis. In this study, small RNA sequencing was performed to identify and characterize miRNAs in six libraries of the red (LH) and pink (LF) petals. A total of 591 conserved and 53 novel miRNAs were identified, among which 68 conserved and 2 novel miRNAs were differentially expressed between the LH and LF libraries. Meanwhile, the corresponding target genes for these differentially expressed miRNAs (DEMs) were successfully predicted and annotated. Among them, two DEMs (miR858 and miR156a-5p) and their corresponding target genes possibly related to anthocyanin biosynthesis were screened. We further confirmed the relative expression of 15 candidate miRNAs (2 DEMs and other 13 miRNAs) and the 4 corresponding target genes of these DEMs by quantitative real-time PCR (qRT-PCR). Among these four target genes, only CL2846.Contig2_All [Annotation: transcription factor MYB12] showed a substantially higher differential expression between the red and pink petals. Based on the above results, we concluded that miR858 and miR156a-5p (especially miR858) are possibly involved in mediating double-color formation. These results provide a new understanding about the mechanism of double-color formation in P. suffruticosa ‘Shima Nishiki’.