Selection and Validation of miRNA Reference Genes by Quantitative Real-Time PCR Analysis in Paeonia suffruticosa

The miRNA, a kind of endogenous non-coding small RNA, plays an essential role in regulation of gene expression in plants. Quantitative real-time PCR (qRT-PCR) assay is one of the most common methods used for quantification of miRNA expression, and levels of expression are normalized by comparing with reference genes. The present study was intended to identify the appropriate reference genes for normalizing the level of miRNA expression in various developmental stages and tissues such as the bud development process, flower development stages, and different tissues of tree peony of different flowering times. Five algorithms (Delta Ct, geNorm, NormFinder, BestKeeper, and RefFinder) were used for stability analysis. The results showed that mtr-MIR160b-p3 and gma-miR394a-5p were the most stable miRNAs expressed during the bud development process of early-flowering tree peony 'Feng dan'; PC-5p-19095 was the most stable during the bud development process of late-flowering tree peony 'Lian he', followed by gma-miR394a-5p and mtr-MIR160b-p3; the mtr-miR159a was the most stable miRNA expressed in the flower development stages of different tree peony varieties. The PC-3p-871 was the most stable miRNA expressed in different tissues of early-flowering tree peony 'Feng dan', followed by PC-5p-4, and PC-5p-4 was the most stable in late-flowering tree peony 'Lian he', followed by the mtr-miR168b. The findings of this study provide a reference for studying the changes in miRNA expression, and further exploring the regulatory mechanism of miRNA in tree peony.