Identification of Floral Volatile Components and Expression Analysis of Controlling Gene in Paeonia ostii ‘Fengdan’ under Different Cultivation Conditions

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.


Introduction
The tree peony (Paeonia suffruticosa Andr.) is a candidate for the national flower of China; its flowers are big, colorful, elegant, and dignified [1]. The cultivation of tree peonies has had a long history and uses a variety of systems. Tree peonies have nine flower colors and ten flower types, which have been formed after a long stage of manual selection. The flower color, flower type, flowering time, flower fragrance, and oil of the tree peony have an important economic value. There is relatively extensive research on properties of flower color [2], flower type [3], flowering time [4][5][6], and oil [7], while the research on flower fragrance is still in the initial stages. The study on the formation of flower fragrance and its regulation mechanism in tree peonies is of great significance.
Floral fragrance, a mixture of secondary metabolites released from flower and vegetative organs of plants, is composed of low-molecular-weight volatile components [8]. Each floral volatile component has individual specificity, whose type, content, and proportion is different in different kinds of plants [9]. The floral aroma components are also

Analysis of Flower Volatile Components at Different Flower Development Stages of Field-Planted Paeonia ostii 'Fengdan'
After identification and calculation, the volatile components and contents of the field-planted Paeonia ostii 'Fengdan' at different flower development stages are shown in Table S2. The results show that 28 compounds were detected in the samples from different flower development stages; these compounds could be divided into hydrocarbons, alcohols, terpenes, benzene rings, esters, phenols, and aldehydes.
The contents of aroma substances of different types vary between flower development stages of Paeonia ostii 'Fengdan' under two cultivation conditions. Figure 1 shows that the content of benzene ring compounds is the highest at the initial flowering stage of potted Paeonia ostii 'Fengdan', whereas the field-planted Paeonia ostii 'Fengdan' has the highest content at the color exposure stage. The content of ester compounds in potted and field-planted Paeonia ostii 'Fengdan'is the highest at the color exposure stage, but these compounds are not present at the flowering, full-blooming, initial decay, and decay stages in a field-planted Paeonia ostii 'Fengdan'. The content of terpenes is the highest in the half-opening stage and full-blooming stage of potted Paeonia ostii 'Fengdan', and it is also the highest at the initial flowering stage of the field-planted Paeonia ostii 'Fengdan'. Alcohols gradually increase during the seven development stages of potted plants and reach the highest level at the decay stage; however, they increase first and then decrease in field-planted Paeonia ostii 'Fengdan' and reach the highest level at the half-opening stage. The content of phenols is low, and they do not appear at half-opening and full-blooming stages of the tree peony under the two cultivation conditions. Aldehydes appear only at the color exposure and initial flowering stages of the field-planted Paeonia ostii 'Fengdan'. Hydrocarbons are the most abundant substances at the color exposure, blooming, initial flowering, and initial decay stages of potted plants, with the highest content occurring at the color exposure stage. Terpenes are the most important floral substances at the half-opening and full-blooming stages, while alcohols are the most important floral substances at the decay stage. In addition to the highest content of terpenes at the initial flowering stage, alkanes are the most important substances at each stage of the field-planted tree peonies.

Analysis of Characteristic Aroma at Different Flower Development Stages
The characteristic aroma substances common to the seven flower development stages under two cultivation conditions include (E)-β-ocimene, nerol, 1,3,5-trimethoxybenzene, and 2,4-di-tert-butylphenol. The results show that the release amounts of four

Analysis of Characteristic Aroma at Different Flower Development Stages
The characteristic aroma substances common to the seven flower development stages under two cultivation conditions include (E)-β-ocimene, nerol, 1,3,5-trimethoxybenzene, and 2,4-di-tert-butylphenol. The results show that the release amounts of four characteristic aroma substances were different at the seven flower development stages under different cultivation conditions. (E)-β-ocimene appeared in the blooming stage, initial flowering stage, half-opening stage, full-blooming stage, and decay stage of potted Paeonia ostii 'Fengdan', and the release amount is shown as follows: half-opening stage > full-blooming stage > blooming stage > initial flowering stage > decay stage. The release amount of 'Fengdan' planted in the field is as follows: the initial flowering stage > blooming stage > color exposure stage > decay stage > full-blooming stage > initial decay stage (Figure 2A). The release amount of nerol at the decay stage of potted Paeonia ostii 'Fengdan' is significantly different from that at the blooming, initial flowering, half-opening, and initial decay stages (p < 0.05). There is no significant difference in the field-planted Paeonia ostii 'Fengdan' ( Figure 2B). 1,3,5-trimethoxybenzene was detected at the seven stages of the potted tree peony, and the highest release amount occurred at the blooming stage. It was not detected at the half-opening stage of plants in the field, and the release amount was the highest at the color exposure stage ( Figure 2C). 2,4-di-tert-butylphenol released at the initial decay stage of the potted plant was significantly higher than that at other stages; the release of 2,4-di-tert-butylphenol was only detected at the color exposure, flowering, and initial flowering stages in the field-planted Paeonia ostii 'Fengdan' ( Figure 2D).

Analysis of Daily Changes in Flower Volatile Components of Potted Paeonia ostii 'Fengdan'
Daily changes in volatile components of potted Paeonia ostii 'Fengdan' flowers were obtained by GC-MS (Table S3). A total of 23 substances were detected at four time stages, including 12 hydrocarbons, 4 benzene rings, 3 terpenes, 2 esters, 1 phenol, and 1 aldehyde.

Analysis of Daily Changes in Flower Volatile Components of Potted Paeonia ostii 'Fengdan'
Daily changes in volatile components of potted Paeonia ostii 'Fengdan' flowers were obtained by GC-MS (Table S3). A total of 23 substances were detected at four time stages, including 12 hydrocarbons, 4 benzene rings, 3 terpenes, 2 esters, 1 phenol, and 1 aldehyde.

Classification and Content Analysis of Daily Change of Flower Volatile Components
An initially increasing and then decreasing trend appeared in benzyclic substances of potted Paeonia ostii 'Fengdan' at different time stages of the day, and the content was the highest from 9:00 to 12:00 ( Figure 3A). The content of benzene ring substances in groundplanted Paeonia ostii 'Fengdan' was also the highest from 9:00 to 12:00 ( Figure 3B). Esters were the highest during the hours of 6:00-9:00 and were only detected in potted tree peonies. Terpenes showed a trend of first increasing and then decreasing levels in potted Paeonia ostii 'Fengdan', with the largest amount released from 12:00 to 15:00. However, terpenes first decreased and then increased in field-planted Paeonia ostia 'Fengdan', and the lowest content was from 12:00 to 15:00. Alcohols were only contained in the field-planted Paeonia ostii 'Fengdan', and the release amount from 9:00 to 12:00 was significantly higher than that at other time stages. Phenols and aldehydes were relatively low compared with other compounds, and their release amount was significant from 9:00 to 12:00. Alkanes were the compounds with the highest content under the two cultivation conditions, showing a trend of first decreasing and then increasing levels in potted tree peonies, with the lowest content from 9:00 to 12:00. The highest content was found from 12:00 to 15:00 under the field conditions, and the lowest content was detected from 15:00 to 18:00. low compared with other compounds, and their release amount was significant from 9:00 to 12:00. Alkanes were the compounds with the highest content under the two cultivation conditions, showing a trend of first decreasing and then increasing levels in potted tree peonies, with the lowest content from 9:00 to 12:00. The highest content was found from 12:00 to 15:00 under the field conditions, and the lowest content was detected from 15:00 to 18:00.

Study on the Expression of Genes Related to Floral Fragrance Regulation
The expression patterns of flower fragrance-regulating genes PsPAL, PsTPSs, and PsbHLH were investigated by qRT-PCR. During the seven flower development stages of potted and field-planted Paeonia ostii 'Fengdan', the relative expression level of PsPAL was the highest at the full-blooming stage, followed by the blooming stage ( Figure 5A). The expression of the PsTPSs gene was significantly higher under potted conditions; it was also significantly higher at the blooming and full-blooming stages than at other stages (p < 0.05) ( Figure 5B). The relative expression of the PsbHLH gene was the highest at the decay stage (p < 0.05), followed by the full-blooming stage. The relative expression at the full-blooming stage was the highest under field-planted conditions, which was significantly different from that at other stages (p < 0.05) ( Figure 5C). highest from 15:00 to 18:00 ( Figure 4B). The highest release amount of 2,4-di-tertbutylphenol was from 6:00 to 9:00 in potted Paeonia ostii 'Fengdan', and in the field-planted 'Fengdan', from 15:00 to 18:00 ( Figure 4C). Methyl jasmonate was only detected in potted Paeonia ostii 'Fengdan' and only at three-time stages, with the highest release from 6:00 to 9:00 ( Figure 4D). The release amount of nerol and cinnamyl alcohol in the field-planted Paeonia ostii 'Fengdan' was the highest between 12:00 and 15:00 and between 9:00 and 12:00, respectively ( Figure 4E,F).  At different time stages of the day, the expression of PsPAL was the highest from 12:00 to 15:00 and from 15:00 to 18:00, which was significantly higher than that at other stages (p < 0.05) for potted Paeonia ostii 'Fengdan'. PsPAL did not change significantly throughout the day for the field-planted Paeonia ostii 'Fengdan' (Figure 6A). The expression of PsTPSs in potted tree peonies was the highest from 15:00 to 18:00, and the lowest from 9:00 to 12:00. The difference between the field-and the pot-planted peonies was detected from 9:00 to 12:00 ( Figure 6B). The expression of PsbHLH was the highest from 12:00 to 15:00 under the two cultivation conditions, and the relative expression in potted Paeonia ostii 'Fengdan' was lower than that in the field ( Figure 6C). the highest at the full-blooming stage, followed by the blooming stage ( Figure 5A). The expression of the PsTPSs gene was significantly higher under potted conditions; it was also significantly higher at the blooming and full-blooming stages than at other stages (p < 0.05) ( Figure 5B). The relative expression of the PsbHLH gene was the highest at the decay stage (p < 0.05), followed by the full-blooming stage. The relative expression at the full-blooming stage was the highest under field-planted conditions, which was significantly different from that at other stages (p < 0.05) ( Figure 5C). At different time stages of the day, the expression of PsPAL was the highest from 12:00 to 15:00 and from 15:00 to 18:00, which was significantly higher than that at other stages (p < 0.05) for potted Paeonia ostii 'Fengdan'. PsPAL did not change significantly throughout the day for the field-planted Paeonia ostii 'Fengdan' ( Figure 6A). The expression of PsTPSs in potted tree peonies was the highest from 15:00 to 18:00, and the lowest from 9:00 to 12:00. The difference between the field-and the pot-planted peonies was detected from 9:00 to 12:00 ( Figure 6B). The expression of PsbHLH was the highest from 12:00 to 15:00 under the two cultivation conditions, and the relative expression in potted Paeonia ostii 'Fengdan' was lower than that in the field ( Figure 6C).

Discussion and Conclusions
The floral fragrance is one of the important indexes used to evaluate the quality of ornamental plants. People can smell different odors due to different types and amounts of volatile components released by plants under different conditions [21]. In this study, we analyzed the volatile components of Paeonia ostii 'Fengdan' from seven flower development stages and different time stages of the day under potted and field-planted conditions. Seven volatile components were identified, including alcohols, alkanes, esters, terpenes, benzene rings, phenols, and aldehydes. Of these, the main components are (E)β-ocimene, 1,3,5-trimethoxybenzene, and geraniol, which is consistent with the previous research results [16]. Alkanes are the main volatile components of tree peonies, and the same conclusion was reached in the identification of floral components in F1 populations of Paeonia ostii 'Fengdan' and Paeonia suffruticosa 'Chunguihuawu' cross [22]. Zhang et al. [23] also found that the contents of hydrocarbons were relatively higher in the study of volatile components of tree peony varieties, such as Paeonia suffruticosa 'Lvxiangqiu' and

Discussion and Conclusions
The floral fragrance is one of the important indexes used to evaluate the quality of ornamental plants. People can smell different odors due to different types and amounts of volatile components released by plants under different conditions [21]. In this study, we analyzed the volatile components of Paeonia ostii 'Fengdan' from seven flower development stages and different time stages of the day under potted and field-planted conditions. Seven volatile components were identified, including alcohols, alkanes, esters, terpenes, benzene rings, phenols, and aldehydes. Of these, the main components are (E)-β-ocimene, 1,3,5-trimethoxybenzene, and geraniol, which is consistent with the previous research results [16]. Alkanes are the main volatile components of tree peonies, and the same conclusion was reached in the identification of floral components in F 1 populations of Paeonia ostii 'Fengdan' and Paeonia suffruticosa 'Chunguihuawu' cross [22]. Zhang et al. [23] also found that the contents of hydrocarbons were relatively higher in the study of volatile components of tree peony varieties, such as Paeonia suffruticosa 'Lvxiangqiu' and Paeonia suffruticosa 'Shouanhong'. N-alkane is one of the main components of plant leaf wax. Besides appearing on leaves, plant wax also appears on the surface of flowers or fruits of plants. Therefore, it is speculated that the flower surface of Paeonia ostii 'Fengdan' may be covered with a wax layer. In addition, terpenes, benzene rings, and alcohols are also volatile components with a high content found in this study, which is consistent with the results obtained by Li et al. [15] in the research of 30 tree peony varieties. 1,3,5-trimethoxybenzene is the most typical among benzene ring substances, which is consistent with the research results of Song et al. [24].
The content of volatile components is easily affected by the cultivation environment, flower development stage, and aroma collection method [25]. This study found that characteristic aroma substances were (E)-β-ocimene, nerol, geraniol, etc. at different cultivation conditions, different development stages, and different times of the day, which is consistent with the research results of Zhang et al. [23]. Changes occurred in volatile aroma compounds with daily rhythm, flowering stage, and different flower parts for Oncidium. Major aroma volatile compounds were linalool, benzaldehyde, β-myrcene, tiglaldehyde, benzyl alcohol, and nerolidol [26]. (E)-β-ocimene is a kind of acyclic monoterpene compound, which has a grass fragrance, flower fragrance, and orange flower oil smell. It is a common floral substance in many flowers and plants [27]. In the present study, the content of (E)-βocimene was the highest at the half-opening stage of potted Paeonia ostii 'Fengdan' and the initial flowering stage of the field-planted Paeonia ostii 'Fengdan'. 1,3,5-trimethoxybenzene is a kind of benzene ring compound, which has a fresh and elegant fragrance [28] and 2,4-di-tert-butylphenol has a carbonaceous acid taste [29]. Methyl jasmonate widely exists in plants and exogenous spraying of methyl jasmonate can significantly promote the release of floral components of Styrax japonicus [30] and Echinacea purpurea L. Moench [31]. Nerol is one of the important components of flower aroma. Its release amount was the highest at the decay stage of potted Paeonia ostii 'Fengdan'; however, there was no significant difference at the flower development stage of the field-planted Paeonia ostii 'Fengdan', but nerol's content is affected by daily changes.
Terpenoid Synthase (TPS) is a key enzyme for the synthesis of terpenoids. Magnard et al. [32] identified the genes on the pathway related to the synthesis of terpenes and phenylpropanoid/benzene ring compounds from the genomic level and found that the TPSs gene family has the function of catalyzing the synthesis of aromatic substances, such as linalool, nerolidol, etc. In the study of volatile substances in Jasminum sambac, it was found that the release of linalool reached its peak after the opening of petals and was consistent with the maximum expression of the JsTPS gene, which may be related to the formation of jasmine fragrance [33]. Overexpression TPS1 gene in Magnolia champaca indicated that McTPS1 is linalool synthase, responsible for producing the most monoterpenes in cedar flowers [34]. Across tree peony cultivars, the expressions of three TPS genes PdTPS1, PdTPS2 and PdTPS4 were significantly positively correlated with linalool emissions [35]. Phenylalanine ammonia lyase (PAL) is the key rate-limiting enzyme of the phenylpropanoid metabolic pathway and is involved in regulating 1,3,5-trimethoxybenzone, 2,4-di-tertbutylphenol, and other benzene ring compounds. The expression of the PAL gene is regulated by the feedback of phenylpropanoid metabolic pathway products [36]. Jiang et al. [37] found that the expression of LrPAL in the Lycoris radiata flower was higher at the bud and withering stages than in the blooming stage. Aslam screened two bHLH family transcription factors (CpMYC2 and CpbHLH13) from the transcriptome library of Chimonanthus praecox constructed at different flower development stages. Two transcription factors were the most expressed at the full-blooming stage, and overexpression of CpMYC2 and CpbHLH13 in Arabidopsis and tobacco both promoted the synthesis of monoterpene (linalool) and sesquiterpene (β-caryophyllene), indicating that they play an important role in the synthesis of volatile components at the flowering stage [20]. The specific regulatory mechanisms of these genes need to be studied further.
As one of the important indexes to evaluate the quality of tree peonies, the fragrance of different tree peonies has different intensity and characteristics due to the difference in aroma components and release amounts and has attracted more and more attention in recent years. In this study, 25 and 28 volatile components were identified during the seven flower development stages of potted and field-planted Paeonia ostii 'Fengdan', and 23 and 22 volatile components were identified at different time stages of a single day, with alkanes identified as the main components. The main aroma components were (E)-β-ocimene, 1,3,5-trimethoxybenzene, 2,4-di-tert-butylphenol, methyl jasmonate, nerol, and cinnamyl alcohol. The releasing trend of the main aroma components was different under different cultivation conditions and at different times of the day. The expression of floral fragrancecontrolling genes in tree peonies varied greatly in different cultivation modes, at different development stages, and during daily changes. The relative expression level of PsPAL was the highest at the full-blooming stage, and the highest expression level was found in potted Paeonia ostii 'Fengdan' from 12:00 to 15:00 and from 15:00 to 18:00, while there was no significant difference in the field-planted Paeonia ostii 'Fengdan'. PsTPSs gene was significantly higher at the blooming and full-blooming stages than in other stages. The highest expression level was found in potted Paeonia ostii 'Fengdan' from 15:00 to 18:00, while in the field-planted Paeonia ostii 'Fengdan', the highest expression level was found from 9:00 to 12:00. The relative expression of PsbHLH in potted plants was lower than that in the field, and the highest expression was from 12:00 to 15:00. Under different cultivation methods, the volatile components and contents of Paeonia ostii 'Fengdan' are different at different development stages and different time stages in a single day. This study provides a theoretical basis for revealing the formation and regulation mechanism of Paeonia ostii 'Fengdan' fragrance characteristics and also provides technical support for accelerating the industrial development and utilization of the tree peony fragrance.

Materials
Potted and field-planted Paeonia ostii 'Fengdan' were used as experimental materials in this study. On 1 November 2020, the plants were transplanted into the pots and were cultured in the greenhouse (17 ± 1 • C) for early flowering. The volatile components of potted Paeonia ostii 'Fengdan' were collected in the greenhouse (23 ± 1 • C) in January 2021. The potted containers were black plastic flowerpots with a height of 40 cm and a diameter of 35 cm. In April 2021, the volatile components of the flowers of Paeonia ostii 'Fengdan' planted in the same field were collected from the peony resource nursery of Luoyang Academy of Agriculture and Forestry Sciences. The fragrance of potted and field-planted Paeonia ostii 'Fengdan' was collected at different flower development stages (CE: color exposure stage; BS: blooming stage; IF: initial flowering stage; HO: half-opening stage; FB: full-blooming stage; ID: initial decay stage; DE: decay stage) and different time stages of the day (6:00-9:00, 9:00-12:00, 12:00-15:00 and 15:00-18:00) at the full-blooming stage, and the air around the tree peony was taken as a contrast. After collection, the adsorption tubes were sealed with fresh film and wrapped with tin foil, marked, and placed in a low-temperature ice box for storage in the refrigerator. Each treatment was repeated 5 times. Petal samples were collected at different flower development stages and different time stages of a single day, and three biological repeats were taken from each sample. The samples were immediately frozen in liquid nitrogen and stored at −80 • C until RNA extraction.

Collection of Volatile Components
The volatile components of potted and field-planted Paeonia ostii 'Fengdan' flowers were collected by dynamic headspace bagging adsorption collection method according to Li et al. [7]. In order to reduce the impact of environmental factors during the experiment, the sampling time for different flower development stages was set to start at 9:00 a.m. The flowers with the same size and opening degree were placed in the tasteless and transparent sampling bags with openings at both ends. The activated-carbon tube (outer diameter 6 mm, length 75 mm) was inserted at the upper end of the sampling bag, and the Tenax TA adsorption tube (outer diameter 6 mm, length 100 mm) was inserted at the lower end, and both ends were sealed with plastic strips. The odorless and transparent silica gel tube (5 × 7 mm) was used to connect the Tenax TA adsorption tube with the QC-1S air sampler (Beijing Institute of Labor Protection Sciences, Beijing, China). The uniform flow rate of the air sampler was 400 mL/min and the sampling time was 3 h. After sampling, 1000 µL n-hexane was added into the adsorption tube to get the eluent into a 2 mL brown injection bottle (Agilent, Santa Clara, CA, USA). Samples were stored in the refrigerator at −20 • C until they were taken out for instrument analysis.

Analysis Conditions of GC-MS
(1) GC conditions: The temperature at the injection port was 250 • C, and the injection volume was 3 µL. The carrier gas was high-purity helium (99.999%). The split injection method was adopted, and the flow rate was 1.0 mL/min. The capillary column was an elastic quartz capillary column HP-5MS (0.25 mm × 30 m, 0.25 µm). The heating program was as follows: the initial temperature was 70 • C; it was kept for 1 min and raised to 142 • C at the rate of 6 • C/min; then it was raised to 148 • C at the rate of 1 • C/min, and then raised again to 180 • C at the rate of 2 • C/min; finally, it was raised it to 250 • C at the rate of 10 • C/min and kept at that temperature for 20 min.
(2) MS conditions: The temperature of the ion source was 230 • C, the ionization source was EI, the ionization energy of EI was 70 eV, the temperature of the fourth pole was 260 • C, and the temperature of the transmission line was 250 • C. The scanning mode was full scanning, and the scanning range was 29-386 amu.

Qualitative and Quantitative Analysis of Volatile Components
The ethyl acetate solution with the concentration of 69.32 mg/L ethyl caproate was selected as the internal standard solution, and 0.4 µL of the solution was added to the 80 µL sample. GC-MS (Agilent 8890/6973A, USA) was used to detect the sample with added internal standard solution. The volatile components were separated by gas chromatography and identified by mass spectrometry, and the total ion current chromatograms consisting of chromatographic peaks of each component were obtained. The volatile components were identified through NIST 17 standard spectrum library retrieval and the related literature. The content of each component was calculated by using the peak area ratio of volatile matter to the internal standard. The following calculation formula was used: Content of aroma substances (µg/g) = [peak area of each volatile substance/peak area of the internal standard] × Internal standard concentration (mg/L) × Internal standard volume (µL)/Sample weight (g) × F (f is the correction factor of each component to the internal standard, f = 1).

Analysis of Gene Expression Pattern of Floral Fragrance Regulation
Total RNA was extracted from collected petals using an RNAprep Pure Plant Kit (Polysaccharides and Polyphenolics-rich, TIANGEN). RNA quality and purity were confirmed by 1% agarose gel electrophoresis, using the DL2000 DNA Marker (TaKaRa, Dalian, China) as a size indicator. RNA concentrations and ratios of absorbance at 260 nm to that at 280 nm (260/280) were determined using a NanoDrop 1000 spectrophotometer (Implen, Munich, Germany). Total RNA was reverse transcribed into cDNA according to the steps of PrimeScript RT reagent Kit with gDNA Eraser (TaKaRa).
Based on the full-length transcriptome data of the third generation of the tree peony in the early laboratory, DNAMAN 8 and Primer Premier 5.0 were used to design specific amplification primers in the ORF frame sequence of candidate genes, and primers without primer dimer, hairpin structure, mismatch, and other information were selected. The primer sequence shown in Table 1 was synthesized by Bioengineering (Shanghai, China) Co., Ltd. EF1-α gene was used as an internal control to normalize gene expression. The TaKaRa TB Green Premium Ex Taq II kit was used for qRT-PCR experiment, and the reaction system was as follows: 10 µL TB Green Premix Ex Taq II, 0.8 µL For-primer and Rev-primer, respectively, 0.4 µL ROX Reference Dye II, 6 µL RNase free water, and 2 µL cDNA solution. The cycle procedure was as follows: pre-denaturation 95 • C, 30 s; denaturation at 95 • C for 5 s; annealing and extension at 60 • C for 3 s, 39 cycles. Results were calculated using the formula of relative expression = 2 −∆∆Ct .

Data Analysis
Excel 2013 and SPSS 21 software were used for statistical analysis, and Origin 2018 software was used for generating figures.  Data Availability Statement: The datasets generated during and/or analyzed during the current study are listed in the text and its additional files.

Conflicts of Interest:
We declare that we have no known competing financial interest or personal relationship that could have appeared to influence the work reported in this paper.