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.
In order to explore the release rule of floral volatile substances and the diurnal variation of different flower development stages of Paeonia ostii ‘Fengdan’ in potted and ground-planted conditions, dynamic headspace adsorption combined with gas chromatography-mass spectrometry(GC-MS) was used to analyze the dynamic changes in floral volatile components and contents. Quantitative real-time PCR (qRT-PCR) was used to analyze changes in flower fragrance-regulating genes PsPAL, PsTPSs, and PsbHLH at different flower development stages and a daily change process at the full-blooming stage. The results show that there were differences in aroma components and contents of Paeonia ostii ‘Fengdan’ at different flower development stages and different time quantum of every day. There were 25 and 28 aroma components identified in 7 flower development stages of tree peonies planted in pots and in the field, respectively, and 23 and 22 aroma components identified at different time quantum of the day, of which the largest and highest content was alkanes. The main characteristic aroma substances were (E)-β-ocimene, 1,3,5-trimethoxybenzene, 2,4-di-tert-butylphenol, methyl jasmonate, nerol, and cinnamyl alcohol; released amounts of the abovementioned substances varied depending on the development stage and the time of the day. The expression of flower fragrance-controlling genes (PsPAL, PsTPSs, and PsbHLH) in tree peonies varied greatly in different conditions. The results of this study provide a valuable resource to investigate floral fragrance formation in tree peonies.
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