Identification of floral fragrance components in an F₁ population derived from Paeonia ostii 'Fengdan' × P. suffruticosa 'Chunguihuawu' Cross

Tree peony (Paeonia suffruticosa Andr.) is a unique aromatic ornamental plant in China, famous for its flowers' huge size, bright colors, and strong fragrance, and which has high ornamental, medicinal, and edible value^[1,2]. Flower shape, color, and fragrance are the most important apparent characteristics and the main indicators relied upon to evaluate the ornamental value of the flowers. Previously, people paid more attention to flower shape and color. But nowadays, flower fragrance is gradually emerging as a key factor greatly affecting customer choice in domestic and international flower markets^[3].

The fragrance released by flowers is a volatile with low molecular weight mixture emitted by plants. Almost all higher plants can emit volatile substances, which might form initially as a lipophilic liquid that passes through the cell membrane in epidermal cells for release into the local environment, thereby producing the flower fragrance^[4,5]. Aromatic components mainly include alkanes, terpenes, alcohols, aldehydes, ketones, ethers, esters, and benzene ring compounds; according to their biosynthetic pathways, these were mainly divided into the terpenoids, the alkaloids, and the phenylpropanoids and allied phenolic compounds^[6]. Being an excellent source of natural antioxidants, volatile substances emitted by tree peony flowers mainly include terpenes, alcohols, esters, aldehydes, ketones and other fragrance substances^[7−9]. These compounds can reduce the harmful effects of reactive oxygen species^[7,10]. Recently, mounting attention has been paid to characteristics of tree peony's flower fragrance, leading to numerous aromatic products being developed, such as scented tea, essential oil, flower cake, etc., underscoring the need for systematic research to elucidate the flower fragrance synthesis mechanism of tree peony.

Research to date on tree peony has mainly focused on variety research, potential germplasm resources, introduction and cultivation^[11]. Flower fragrance breeding and cultivation is now an important trend in tree peony breeding. A total of 81 scent components in the petals of 30 tree peony cultivars have been detected and classified into five fragrance patterns: a woody scent, a rose scent, a lily of the valley scent, a phenolic scent, and an unidentified scent^[8]. Cis-ocimene, D-citronellol, linalool, 1,3,5-trimethoxybenzene (TMB), and pentadecane are their major floral fragrances, respectively. Evaluation of the potential utilization of seven tree peony cultivars demonstrated that 'High Noon' might harbor a significantly high fragrance content useful for fragrant cultivar breeding^[8]. Additionally, 124 volatile components in petals of nine wild tree peony species were identified and clustered into five major chemical classes: terpenoids, alkanes, alcohols, aldehydes, and ketones, yet the main components and sensory evaluation differed starkly among species^[9]. P. ostii had herbal and waxy attributes, mainly dominated by hexanal and pentadecane; P. rockii, P. qiui, P. jishanensis, and P. decomposita featured a sweet attribute, which was positively correlated with geraniol and citronellol; P. delavayi, P. lutea, P. ludlowii, and P. potanini were characterized by an intense floral attribute dominated by linalool and trans-linalool oxide. Furthermore, that study also revealed the phylogenetic relationships of nine wild tree peony species according to the volatile components that they emitted^[9].

Studies on the genetic population construction and phenotypic trait investigation of hybrids in strawberry^[12,13], pepper^[14,15], maize^[16], and wheat^[17] have been carried out recently, which could help to lay a theoretical foundation for QTL mapping of phenotypic traits and molecular marker-assisted breeding. Most genetic populations of plants in use presently are the F₂ generation, largely because it is conducive for detecting the trait separation phenomenon according to Mendel's law of inheritance. Regarding tree peony, it is a self-incompatible plant with a highly heterozygous genome; hence, its phenotypic traits could be separated in the F₁ generation. Thus, the F₁ generation of tree peony could be considered as the hybrid F₂ generation of homozygous parents, and F₁ populations of tree peony have been constructed and used for genetic analysis based on their common phenotypic traits. For example, Li^[18] constructed an F₁ population via mixed pollination and statistically analyzed three continuous traits and six discrete traits. Correlation analysis and mixed genetic analysis of 20 phenotypic traits (such as plant height, and crown width) of the F₁ population of P. ostii 'Fengdan' and P. suffruticosa 'Xinriyuejin' uncovered 20 phenotypic traits that presented hybrid superiority and transgressive segregation, with some traits showing genetic effects controlled by major genes^[19]. However, to our knowledge, no study has yet reported on the aroma traits of F₁ genetic population in tree peony.

In this work, an F₁ segregating population was established using P. ostii 'Fengdan' (light fragrance type) as the female parent and P. suffruticosa 'Chunguihuawu' (strong fragrance type) as the male parent. Then, the flower aromatic components of this F₁ population, for both parents and progeny, were identified and analyzed. This is the first report to analyze the genetic mechanism of flower fragrance in an F₁ genetic population of tree peony. This study's findings may provide valuable information for clarifying the genetic mechanism of flower fragrance formation and help to lay a theoretical basis for flower fragrance breeding of tree peony.

Floral volatiles samples were collected from the flowers at three different flowering stages (initial flowering stage, half-opening stage, and full blooming stage) of the light fragrance-type cultivar P. ostii 'Fengdan' and the strong fragrance-type cultivar P. suffruticosa 'Chunguihuawu', as well as the half-opening stage flowers of 109 F₁ progeny. All these plants were grown at the National Tree Peony Genebank (Luoyang City, Henan Province, China) under consistent management practices and favorable growing conditions.

Floral volatiles' collection

Collection of floral volatile components was performed using the dynamic headspace sampling technique applied to tree peony flowers of moderate size at noontime on sunny and windless days in April 2020. Each sample was placed into a sampling bag connected to a Tenax TA adsorption tube and an active charcoal pump (QC-1S, Beijing Municipal Institute of Labor Protection, Xicheng District, Beijing, China); samples were collected for 3 h at a flow rate of 400 mL min^-1. In the experiment, headspace collection from ambient air served as a negative control.

After sampling, the adsorption tube was sealed with fresh-keeping film and wrapped with tin foil, then placed in an icebox and brought to the laboratory. When the collection was complete, 1,000 μL of n-hexane solution was used to elute the aroma adsorbed by the Tenax TA tube into a clean, brown injection bottle. Each injection bottle was sealed with sealing film and stored in a refrigerator at –20 °C until the samples were analyzed with the instruments below.

Floral volatiles analysis

The analysis was carried out using a gas chromatograph-mass spectrometer (Trace GC Ultra-ISQ, Thermo Fisher Scientific, 81 Wyman Street, Waltham, MA, USA) fitted with a TM-5MS capillary column (30 m × 0.25 mm, 0.25 μm). Helium was the carrier gas, delivered at a flow rate of 1.0 mL min⁻¹. The initial oven temperature was maintained at 70 °C for 1 min and raised to 142 °C at a rate of 6 °C min⁻¹, then raised to 148 °C at a rate of 1 °C min⁻¹, then raised to 180 °C at a rate of 2 °C min⁻¹, and finally raised to 250 °C at a rate of 10 °C min⁻¹ at which it was held isothermally for 20 min. The temperature of the injector, transfer line, ion source, and quadrupole were set to 250 °C, 260 °C, 230 °C, and 150 °C, respectively. The ionization potential of the mass selective detector was 70 eV and its scan range was 29–386 amu.

Data analysis

Compounds were identified by comparing their mass spectra to the NIST 2011 database. In order to fairly compare the released amounts of volatile components, 69.32 mg L⁻¹ of decanoic acid, ethyl ester served as an internal standard, with 0.4 μL of this internal standard solution added to every 80 μL of a given sample. The calculation formula is as follows:

where C is the amount of the volatile component; S_v is the peak area of the volatile compound; S_i is the peak area of the internal standard; m_i is the quality of the internal standard; m_s is the quality of the sample; and f is a correction factor of each component for the internal standard.

Statistical Program for Social Sciences 21.0 software(SPSS, Inc., Chicago, IL, USA) was used for variation analysis, Gaussian distribution test, and X² test. Origin 8.0 software (OriginLab Corporation, Northampton, MA, USA) was used to draw the figures.

Figure 1 shows the chromatographic profiles of two different tree peony cultivars in three different flowering stages, for which a significant difference was found. In total, 67 volatile compounds were identified as emitted from the flowers of these tree peonies, of which 43 volatile compounds were shared by them. These 67 volatile compounds belonged to six categories: alcohols, esters, aldehydes, terpenes, benzenes, and hydrocarbons. However, among them, 35 hydrocarbons were found, far more than in any other category; esters were another important category with 13 compounds identified; six alcohols and five aldehydes were found in the volatiles, along with benzenes and terpenes (four kinds each).

Figure 1. 

Chromatographic profiles of two tree peony cultivars at three flowering stages. (a) P. suffruticosa 'Chunguihuawu', (b) P. ostii 'Fengdan'. From top to bottom are initial flowering stage, half-opening stage, and full blooming stage. Bar = 5 cm.

Volatile components of P. suffruticosa 'Chunguihuawu' at different flowering stages

The content of each volatile component in P. suffruticosa 'Chunguihuawu' and P. ostii 'Fengdan' are shown in Table 1. A total of 56 volatile compounds were detected in three different stages of P. suffruticosa 'Chunguihuawu', consisting of three alcohols, 11 esters, three aldehydes, three terpenes, three benzenes, and 33 hydrocarbons. There were 51, 53, and 46 volatile compounds detected in the initial flowering stage, half-opening stage, and full blooming stage, respectively. At the initial flowering stage, the specific volatile components were 2-hexen-1-ol,acetate,(E)-, decanal, and (Z,E)-alpha-farnesene, while the contents of nonanal, glycerol linolenate, and nonadecane were much higher than those in the other two stages. At the half-opening stage, only α-ocimene, trans-α-ocimene, nerol, and farnesal were detected. In addition, the contents of benzene,1,4-dimethoxy-, geraniol, and pentadecane at the initial opening stage surpassed those at the other two stages, indicating these compounds may be characteristic volatile compounds at this stage. Moreover, 2,4-di-tert-butylphenol was detected at all three stages, its content exceeding that of other volatile compounds at every stage.

As shown in Fig. 2a, the six compounds were ranked in content as follows: hydrocarbons > benzenes > esters > alcohols > aldehydes > terpenes. With the opening of flowers, the total content of different compounds featured different trends. The total content of hydrocarbons and esters decreased gradually, that is, peaking at the initial flowering stage. Except for hydrocarbons and esters, the content of other volatile components in P. suffruticosa 'Chunguihuawu' reached their maximum levels at the half-opening stage.

Figure 2. 

Classification and content of volatile compounds of parents of the F₁ population in different flowering stages. (a) P. suffruticosa 'Chunguihuawu', (b) P. ostii 'Fengdan'. IF: initial flowering stage. HO: half-opening stage. FB: full blooming stage.

Volatile components of P. ostii 'Fengdan' at different flowering stages

A total of 53 volatile compounds were detected from all three flowering stages of P. ostii 'Fengdan', consisting of five kinds of alcohols, five esters, four aldehydes, three terpenes, two benzenes, and 34 hydrocarbons. Specific volatile components were detected at different flowering stages: 30 species at the initial flowering stage, 43 species at the half-opening stage, and 31 species at the full blooming stage. The unique volatile components at the initial flowering stage were trans-α-ocimene and isopropyl palmitate, whereas those at the half-opening stage were 2-hexen-1-ol,acetate,(E)-, 3-phenylpropanol, cinnamyl alcohol, cis-cinnamyl alcohol, 2-undecenal and hexadecanal, while geranyl acetate was detected only at the full blooming stage.

As seen in Fig. 2b, the content of hydrocarbons was always the greatest at all three stages, followed by benzenes and terpenes, being least for the other three compound categories. The content of terpenes and alcohols during various flower stages was ranked as follows: full blooming stage > half-opening stage > initial flowering stage; for aldehydes, the ranking differed, as follows: half-opening stage > initial flowering stage > full blooming stage; the same ranking was found for other three compounds: half-opening stage > full blooming stage > initial flowering stage. Across all three stages, α-ocimene, 1,3,5-trimethoxybenzene, pentadecane, and heptadecane were detected, whose contents were much higher than those of other volatile compounds. The total relative content of those four compounds was 19.66%, 38.81%, and 32.83% respectively at the three stages, showing a trend of increasing initially and then decreasing. Our experimental results show that these four compounds are the main volatile components of P. ostii 'Fengdan'.

Comparing the volatile components of P. suffruticosa 'Chunguihuawu' and P. ostii 'Fengdan'

The two tree peony cultivars had 43 components in common, mainly 2,4-di-tert-butylphenol and various hydrocarbons such as dodecane, tetradecane, and pentadecane. It was worth noting that 14 volatile components were exclusive to P. suffruticosa 'Chunguihuawu', most of them being ester volatile oils. Similarly, 11 kinds of compounds, such as myroxide, 1,3,5-trimethoxybenzene and octyl p-methoxycinnamate, were only detected in the flowers of P. ostii 'Fengdan', but most of these were oxygenated derivatives of hydrocarbons. These results confirmed there were significant differences in volatile components between the two cultivars studied here.

Analysis of volatile components in the F₁ population

Types and content of volatile components in F₁ population

Due to differences in flower color, flower type, and floral fragrance between the parents, the hybrids exhibited clear segregation for these traits. After the volatile components of hybrids were counted, it was found that the hybrids had more complex profiles of volatiles than did their corresponding parent accessions. For 109 progeny, they featured 73 volatile compounds, namely six alcohols, 18 esters, five aldehydes, six terpenes, four benzenes, and 35 hydrocarbons, while their parents harbored only 56 and 53 volatiles, respectively. Shared by both parents and progeny were 22 compounds, such as pentadecane, 2,4-di-tert-butylphenol, cetene, hexadecane, 8-heptadecene, and heptadecane, but most of them were hydrocarbons. However, seven compounds—α-pinene, alloocimene, phenylethyl acetate, citronellyl acetate, neryl acetate, (Z,E)-α-farnesene and tetradecanoic acid—were detected only in progeny (i.e., absent from parents altogether). After analyzing the data, α-ocimene and benzene,1,3,5-trimethoxy were found present in 104 and 69 progeny, these compounds attaining their highest content for progeny F₁-1, at 37.39 μg g⁻¹ and 10.81 μg g⁻¹, respectively. Nerol existed in 104 progeny, and its content was the highest for progeny F₁-2 and F₁-6. Finally, 2,4-di-tert-butylphenol was the only compound that presented in all progeny, whose content was maximal for progeny F₁-8 and F₁-10.

Genetic variation analysis of volatile components in the F₁ population

The inheritance of these 73 components can be broadly divided into two cases: (i) 50 components separated in the progeny, which showed the inheritance of quality traits, and (ii) 23 components not segregated in the progeny, which showed the inheritance of quantitative traits. Variation analysis was performed for these 23 components, whose statistical results are shown in Table 2. The CV (coefficient of variation) of the 23 components in the F₁ population ranged from 0.618% to 3.207%, indicating small differences. The CV of octadecane was the lowest, and that of of tridecane the highest. Distribution testing of these 23 components showed that only cetene, hexadecane, octadecane, and docosane conformed to a Gaussian (normal) distribution. The frequency distribution analysis of these four components (Fig. 3) showed they had relatively prominent unimodal distributions, thus conforming to the characteristics of quantitative traits. However, the absolute values of skewness and kurtosis of the four components were greater than 1 or close to 1, demonstrating the presence of positive skewness and extremeness.

Figure 3. 

Histograms (frequency distribution) for traits of the F₁ population from the cross of P. ostii 'Fengdan' and P. suffruticosa 'Chunguihuawu'. (a) Histogram for cetene, (b) histogram for hexadecane, (c) histogram for octadecane, (d) histogram for eicosane.

Genetic analysis of 29 aromatic components in the F₁ population

Most terpenes, alcohols, esters, aldehydes, and benzenes have special odor characteristics. Among the 73 kinds of volatile compounds detected in the genetic population, 29 were aroma compounds. Their genetic analysis revealed that four of these 29 were shared by the parents, while another 18 were belonged to a single parent, with another seven kinds not found in either parent.

Genetic analysis of aromatic components shared by parents but separated in progeny

Among the four aromatic components shared by the parents, three got separated in progeny. The X² test results showed that this agreed with theoretical separation ratio of 1:1, 1:7, or 15:1 according to Mendelian's law of genetic separation (Table 3). This suggested the three components were controlled by single or multiple genes. Notably, there was a generally higher content of α-ocimene in progeny than the parent, exhibiting transgressive inheritance.

Genetic analysis of aromatic components shared by parents and not separated in progeny

Among the four aromatic components shared by the parents, only one was not separated in the progeny, in that 2,4-di-tert-butylphenol was detected in all progeny (Table 4). It was the characteristic aromatic component of this tree peony genetic population and had the potential for stable inheritance. According to the relevant parameters of the progeny distribution, the average value was less than mid-parent value and the variation was continuous, demonstrating typical quantitative trait inheritance.

Genetic analysis of aromatic components in one of the parents

There were 18 kinds of components in line with this phenomenon, mainly including esters, terpenes, alcohols, and so on (Table 5). Among them, 14 components agreed with the theoretical separation ratio by the X² test. In the case of separation ratios of 1:1 and 3:1, the components could be controlled by one pair of dominant genes; while a separation ratio of 1:7 indicated possible control by two pairs of recessive genes; however, the separation ratio of 15:1 suggested they might be controlled by multiple pairs of dominant genes. These correspond to the inheritance of quality traits. On the contrary, for trans-α-ocimene, benzene,1,4-dimethoxy-, nerol, and geranyl acetate, the X² test was significant (P < 0.05), which did not obey the test. Phenylethyl alcohol, benzene,1,4-dimethoxy-, nerol, and geranyl acetate were the characteristic aromatic components of this tree peony genetic population, these inherited from the male parent P. suffruticosa 'Chunguihuawu'.

Genetic analysis of de novo aroma compounds

Some de novo aroma compounds (those present in the hybrid, but absent in the parents, such as α-pinene, neryl acetate, etc.) were detected exclusively in the progeny. The capacity to produce these compounds might have been inherited from the parents in an additive genetic fashion. Seven kinds of components were line with this phenomenon, all of which were terpenes and esters, and they got separated in the progeny. The first four components in Table 6, namely α-pinene, alloocimene, phenylethyl acetate, and citronellyl acetate, agreed with the theoretical separation ratio of 1:3 or 1:15 according to the X² test; hence, we speculated they could be respectively controlled by a pair, or multiple pairs, of recessive genes, showing the inheritance of quality traits. Nevertheless, for neryl acetate, (Z,E)-α-farnesene, and tetradecanoic acid, ethyl ester, the X² tests were significant (P < 0.05), which did not obey the test.

The release of a flower's aroma occurs with the unfolding of its petals. The formation of aromatic components is a dynamic process during the development of flowers from the initial flowering stage to the full blooming stage^[20]. According to the analysis of volatile components of two peony cultivars at different flowering stages, it can be seen that the types of volatile compounds showed a trend of first increasing and then decreasing, that is, they peaked at the half-opening stage. However, the trends of the six volatile compounds were quite different between the two cultivars. The total content of volatile compounds in P. suffruticosa 'Chunguihuawu' reached its maximum at the initial flowering stage, and then decreased gradually, and vice versa for P. ostii 'Fengdan'. When tree peony flowers transition from the half-opening stage to the full blooming stage, their pollen sac will burst open, which can interfere with detection of volatile components in living plants. Hence, when studying the volatile components of different tree peony cultivars (or varieties) at the same flowering stage, the flowers at the half-opening stage are the best choice. This is because the types of volatile compounds are most abundant and their total content is relatively moderate during this pivotal stage.

Our results for the analyzed volatile components of P. suffruticosa 'Chunguihuawu' and P. ostii 'Fengdan' at different flowering stages showed that the types of n-alkanes were the most common and basically consistent, with the relative content of n-alkanes being the highest found, a result is in line with findings reported by Zhou et al.^[21]. Another study found that the main components of plant leaf wax are n-alkanes^[22]. Plant wax will not only appear on the leaves, but may also appear on the surface of other vegetative organs, such as flowers and fruits^[23]. Therefore, we speculated that the flower surface of the two tree peony cultivars studied here may be covered with a wax layer. Moreover, pentadecane accounted for many peak areas in P. suffruticosa 'Chunguihuawu' and the content of heptadecane in the n-alkanes detected for P. ostii 'Fengdan' was the highest overall. Zhou et al.^[24] indicated that n-alkanes had a high threshold value and thus their contribution to floral fragrance was limited. Whether n-alkanes constitute floral compounds is still an open question, but it is not in doubt that a high level of n-alkanes may lighten the fragrance of a flower. Further, P. suffruticosa 'Chunguihuawu' is a purplish red-flowered cultivar, and P. ostii 'Fengdan' is a white-flowered cultivar. The aromatic components and contents of different tree peony cultivars are directly related to flower color, which may partly explain the pronounced differences in floral fragrance and fragrance pattern between these two cultivars in this study. In addition, benzene,1,3,5-trimethoxy-, phenylethyl alcohol, benzene,1,4-dimethoxy-, and geraniol are the common characteristic aromatic components of tree peony flowers; the contents of these four compounds in either cultivar are high, which may be closely linked to the strong scent of tree peony when it blooms. Benzene,1,3,5-trimethoxy- has high medicinal value, phenylethyl alcohol can make derived food products such as tree peony tea or tree peony wine taste better^[25], benzene,1,4-dimethoxy- can be used as a fragrance-fixing agent, and geraniol is often used as essence. Therefore, studying the aromatic components of different tree peony varieties provides a certain theoretical basis for improving the economic value of tree peony.

The inheritance of traits in hybrids is not entirely an additive process, and some volatile compounds can be overexpressed or underexpressed in hybrids^[26]. The hybrids in this experiment basically displayed intermediate or transgressive inheritance for the trait of floral fragrance. Likewise, both propensities were also documented in Capsicum by Moreno et al.^[14]. A portion of the progeny presented volatiles whose amount and content was in between the range of the corresponding parents, as expected, but another portion of the progeny surpassed either parent in certain volatiles. This suggests that hybridization may help to enhance the scent of a particular cultivar or developing hybrids with a more diverse spectrum of volatiles than that possessed by their parents. In addition, some progeny produced volatiles that did not appear in the parents, indicating that the two genomes may interact during hybridization, leading to the convergence of genetic factors that are silenced in both parents, thereby increasing the overall complexity and potential of the volatile profiles in progeny. This view is consistent with the findings of Moreno et al.^[14] as well as Delort et al.^[26].

Crosses of P. suffruticosa 'Chunguihuawu' × P. ostii 'Fengdan' could be used to obtain novel aroma types with more intense combinations of floral and fruity aromas. Hybrids of this population presented the following: (i) higher levels of α-ocimene than the parents; (ii) more fruity esters, including the combination of traces of alloocimene, phenylethyl acetate, and citronellyl acetate, which the parents did not present; and (iii) α-pinene (increased woody aromas), also lacking in the parents. Genetic analysis of the aromatic components demonstrated that the volatiles differed among hybrids, which might provide fundamental information for a better understanding of tree peony's genetics and its aroma improvement. Future crosses using ideal volatile-complementary parental material could be selected based on hybrids having the best scents.