| Summary: | miRNA plays an important role in plant growth and development and in response to various stresses. Quantitative real-time PCR (qRT-PCR) technology is often used to detect the expression level of miRNAs and genes by comparing with reference genes. In order to screen out the optimal reference miRNAs in different tree peony varieties, the petals of 42 different early- and late-flowering tree peony varieties were used as experimental materials, and geNorm, NormFinder, Bestkeeper, and RefFinder software were used to evaluate the stability of 16 candidate reference miRNAs. The results showed that the average Ct values of all candidate reference miRNAs were between 15.34 ± 0.29 and 32.64 ± 0.38. The optimal number of reference miRNAs was four, which were <i>PsPC-5p-19095</i>, <i>PsPC-3p-51259</i>, <i>PsmiR159a</i>, and <i>PsPC-3p-6660</i> in geNorm. The stability of <i>PsPC-3p-6660</i> was the highest in the analysis results of NormFinder software. Among the analysis results of Bestkeeper software, <i>PsMIR319-p5</i> has the highest stability. Among the results of comprehensive evaluation and analysis of several software using RefFinder, the candidate reference miRNA with the highest stability was <i>PsPC-3p-6660</i>. When <i>PsPC-3p-6660</i> was used as the reference miRNA, the expression of <i>PomiR171</i> and <i>PomiR414</i> in response to different flowering times of tree peony was relatively stable in 42 tree peony varieties, indicating that <i>PsPC-3p-6660</i> was stable and reliable. The results of this study provide a reference miRNA for studying the expression changes of miRNA in different tree peony varieties and further exploring the regulatory mechanism of miRNA in different peony varieties.
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