## 2 Results
### Analysis of Genes Related to Monoterpene Synthesis  
Plant metabolism can be divided into primary metabolism, which contains many crucial reactions and pathways, with the main function of maintaining plant survival, and secondary metabolism, which mainly involves the interaction between plants and the environment, achieving many important functions in the growth and development [44]. Some primary metabolites, which are produced through glycolysis, the TCA cycle, or the shikimate pathway, can serve as precursors to synthesize secondary metabolites, such as terpenoids, phenols, and nitrogen/sulfur-containing compounds [45; 46]. Terpenoids are the largest family of secondary metabolites, and volatile terpenoids, such as monoterenes and sesquiterpenes, greatly contribute to the aroma of flowers and several fruits [47]. In plants, there are two pathways involved in terpene synthesis: the MEP pathway and the MVA pathway. Citronello, geraniol, and linalool were monoterenes, and they were synthesized by seven enzymatic steps in the MEP pathway [48]. Two primary metabolites, pyruvate and glyceraldehyde-3-phosphate, derived from glycolysis and the pentose phosphate pathway, were the substrates of the first enzyme (DXS) of the MEP pathway [44]. Nine related genes were detected in the upstream of MEP pathway in the transcriptome database of _Paeonia lactiflora_ (accession number SRP287892), including three DXSs, one DXR, one MCT, one CMK, one MDS, one HDS, and one HDR (Figure 7A). RNA from flowers in full bloom of cultivars '[PERSON]', 'Angel Cheeks', '[PERSON] Yu Lou', 'Gardenia', '[PERSON]', '[PERSON] Zhan Chi', '[PERSON] Xiu Qiu', 'Lollipop', 'Red Sarah Bernhardt', 'Wu Hua Long Yu', and 'Joker' were extracted. Among them, cultivars '[PERSON]', 'Angel Cheeks', and 'Fen Yu Lou' released slight monoterenoids (<1000 ng/g), the cultivars 'Gardenia', 'Hong Xiu Qiu', and 'Red Sarah Bernhardt' released medium monoterenoids (1000 ng/g-3000 ng/g), while cultivars 'Hong Cha Hua', 'Hong Feng Zhan Chi', 'Lollipop', and 'Wu Hua Long Yu' released a large amount of monoterenoids (>3000 ng/g). The cultivar 'Joker' did not release any monoterenes as the control (Figure 7B). Figure 7: MEP pathway in herbaceous peony. The numbers \(1-17\) represent 17 cultivars, and numbers assigned are described in Figure 2. (**A**) Expression pattern of genes involved in the MEP pathway of herbaceous peony. Gene expression levels in different cultivars are represented by color gradations. (**B**) The relative contents of monoterpenes, citronellol, linalool, and geraniol in 11 cultivars. The detailed data are sourced from Figures 4 and 5 and Supplementary Table S4. (**C**) Correlation heat map between monoterpenes release and gene expression levels. Correlation levels are represented by color gradations. (**D**) Correlation heat map between citronellol, linalool, and geraniol releases and TPS gene expression levels. Correlation levels are represented by color gradations. Three biological replicates of the experiments were carried out. The qRT-PCR analysis of nine monoterpene synthesis-related genes in eleven cultivars were carried out (the expression level of 'Joker' was set as 1), and it was finally found that _PIDX52_, _PIDXR1_, _PIMDS1_, and _PIHDR1_ were strongly correlated with the monoterpene release content of these 11 cultivars (Figure 7C). It is speculated that these genes may be the key genes for the monoterpene synthesis of herbaceous peony. Other compounds, such as geranyl pyrophosphate (GPP), the precursor of monoter-penoid synthesis, is generated by GPP synthase (GPPS) [49]. Four GPPSs were found in the _Paoonia lactiflora_ transcriptome, and the expression level of _PIGPPS3_ and _PIGPPS4_ was strongly correlated with monoterpene release (Figure 7C). Lastly, a series of monoterpenes are synthesized through different TPSs using substrate GPP [48]. Different TPS genes can catalyze the formation of different monoterpenes. Four TPS genes related to monoterpene synthesis were selected in the transcriptome.