China is the origin and main production area of red bayberry (Myrica rubra Sieb.). At present, there are 305 varieties of myrica rubra cultivated in China, with an area of more than 0.3 million hm² and an output of more than 1 million tons, accounting for more than 90% of the world’s area and output (Chen, 2014, Huazhong Agricultural University, pp. 4). Myrica rubra cv. Dongkui, Myrica rubra cv. Biqi, Myrica rubra cv. Dingaome, and Myrica rubra cv. Zhoushanwandaomei account for more than 85% of the output of the national cultivation area. At the same time, there are some local special variety resources, which are used to enrich the varieties and adjust the market supply to meet the consumers at different levels.

The fragrance of myrica rubra fruit is an important quality indicator. If there is a pine odor, the economic value of fresh fruit will decline significantly. The pine odor of myrica rubra is reported by the folk. Through investigation, the author found that this phenomenon exists in the myrica rubra producing areas of Zhejiang, Hunan, Hubei, Chongqing and other provinces and cities. Shangyu-tangshuang-yangmei, huangyan-baiyangmei, Yewu, Yongjia-baiyangmei, Lizhimei, Xiaoshan-tangxiang, Xianghong, Yuhang-zaoyewu, Songmaoli in Zhejiang Province all has the strong pine odor, among which, Yongjia-baiyangmei has the strongest pine odor and is very distasteful. In addition, Shangcun- and Daye waxberry in Jingzhou County, Hunan Province, and some wild waxberries in Zaojiang, Chongqing and Enshi, Hubei Province all have the pine odor (Chen, 2014, Huazhong Agricultural University, pp. 4). Therefore, many fruit growers believe that it is the result of cross between myrica rubra and Pinus Linn, but there is no experimental basis so far. Li (2004, Graduate School of Chinese Academy of Sciences, pp. 111-118) found that Juglandaceae and Rhoepteleaceae, as sister groups and myricaceae clusters, were supported by 97% and 85% BS respectively through PHYC gene pedigree analysis. It is speculated that Juglandales and Myricaceae may originate from a common tetraploid ancestor. The volatile organic compounds of Juglans regia L. are significantly different from those of myrica rubra and Pinus massoniana Lamb. (Li, 2004, Graduate School of Chinese Academy of Sciences, pp.111-118; Su, 2010, Guangxi University, pp. 24-30; Zhang et al., 2013). Sensory evaluation is often used in production, that is, olfactory perception is used to judge that the pine odor of myrica rubra is close to the pine resin odor of the secondary metabolite of Pinus Linn. However, there is no qualitative analysis on the relationship between these two. Based on this, this study analyzed the type and content of volatile organic compounds in myrica rubra fruit by GC-MS measurement, and analyzed the possible genetic relationship between myrica rubra and Pinus Linn trees by ISSR molecular markers, to explore the reason why bayberry fruit produces pine odor, so as to provide reference for breeding myrica rubra varieties without pine odor.

1 Results and Analysis

1.1 Quality and fragrance sensory evaluation of myrica rubra fruit

According to the survey, Yewu, Shangyu-baiyangmei and Yongjia-baiyangmei all have pine odor in varying degrees during their growth, but it weakens with the increase of fruit maturity, and Shangyu-baiyangmei basically has no pine odor when it is fully ripe. Compared with Biqi, the fruits of Yongjia-baiyangmei and Yewu will not change color and taste within 2.5 days under normal temperature, and the quality after 3 days is similar to that of Biqi after 12 hours of storage. At the same time, the storage resistance of Shangyu-baiyangmei is also better than that of Biqi (Table 1; Figure 1). The native varieties have a stronger pine odor than the cultivated ones. For example, Biqi and Shangyu-baiyangmei, as cultivated varieties, have no or almost no pine odor after long-term domestication and selection, while the wild varieties such as Yongjia-baiyangmei and Yewu have a strong pine odor. In addition, the color has no correlation with the pine odor, for example, the purple black Yewu and the milky white Yongjia-baiyangmei all have the pine odor.

1.2 Analysis of volatile components in Myrica Rubra fruit

GC-MS analysis results showed that 60 kinds of volatile organic compounds were detected in 4 varieties of Myrica Rubra fruits, belonging to 8 categories, including 32 kinds in Biqi, 25 kinds in Yongjia-baiyangmei, 18 kinds in both Shangyu-baiyangmei and Yewu (Table 2). As to acids and ketones, they were not detected in Yongjia-baiyangmei and Shangyu-baiyangmei, and there was only one acid in Yewu, and no other components were detected. There were 5 kinds of olefins in Yewu and 4 kinds in Biqi, but were not detected in Yongjia-baiyangmei and Shangyu-baiyangmei. There were 6 kinds of alkane component in Biqi, and were not detected i the other 3 varieties. There were 13 kinds of phenols in Yongjia-baiyangmei, 9 kinds in Shangyu-baiyangmei, 7 kinds in Yewu, and only 5 kinds in Biqi.

Olefin content in Biqi was the highest, which was 73.2 μg/L, followed by Yewu (67.6 μg/L), and were not detected in two white Myrica Rubra varieties. The highest phenol content was in Yongjia-baiyangmei (108.0 μg/L), followed by Shangyu-baiyangmei (100.0 μg/L). The highest alcohol content was in Shangyu-baiyangmei (78.3 μg/L), followed by Biqi (72.0 μg/L) and Yongjia-baiyangmei (53.87 μg/L), the lowest was in Yewu (only 25.5 μg/L). The highest alkane content was in Biqi (37.0 μg/L), followed by Yongjia-baiyangmei (12.0 μg/L), while were not detected in the other two varieties. 45.0 μg/L aldehydes were detected in Shangyu-baiyangmei, 3 times as much as Biqi, and were not detected in the other 2 varieties. The acid substance of Yewu was nearly 3 times as much as that of Biqi, while were not detected in the other two varieties (Table 2).

For the main volatile organic compounds in the first three positions (Table 3), there is no obvious regularity in composition and content among the four varieties. Caryophyllene, o-cymene and ethyl acetate were the main volatile organic compounds of Biqi, accounting for 73.22% of the total volatile organic compounds; 1-methyl-4-(1-methylethylidene)cyclohexene, terpinen-4-ol, terpinene were the main volatile organic compounds of Yongjia-baiyangmei, accounting for 53.30%; Terpinene-4-ol, terpinene, 1,3,8-p-mentha triene were the main volatile organic compounds of Shangyu-baiyangmei, accounting for 61.37%; Caryophyllene, L-terpineol, D-limonene were the main volatile organic compounds of Yewu, accounting for 53.77%. The α-pinene, β-pinene, β-caryophylione were the main volatile organic compounds of Pinus massoniana, accounting for 51.70%; The main substances of Pinus massoniana through qualitative analysis were α-pinene, camphene, β-pinene. The volatile organic compounds of pine needles and turpentine of Pinus massoniana were different from those of four Myrica rubra varieties. The SS * data in the table refer to the Master’s thesis of Su (2010, Guangxi University, pp. 4-10).

1.3 The results of ISSR analysis on the genetic relationship between four Myrica Rubra varieties and Pinus Linn trees

After 7 primers amplification (Figure 2), the detection results of 5 samples can be divided into 2 categories and 3 sub categories. Among the four Myrica Rubra varieties, Biqi and Yewu have the closest genetic relationship, and there was no difference between them from 500 to 2000 bp; Shangyu-baiyangmei and Yongjia-baiyangmei were closely related, and there was no difference between them from 750 to 2000 bp; However, there was no genetic relationship between the Myrica Rubra varieties and Pinus Linn trees.

2 Discussion

Volatile organic compounds in fruits can be perceived by human olfactory organs, thus affecting consumers’ choices. The volatile organic compounds of different fruits are obviously different, such as strawberry (Fragaia ananassa Duchesne), pomegranate (Punica granatum L.), red bayberry (myrica rubra), cherry (Cerasus pseudocerasus (Lindl.) G. Don), Putao (syzygium jambos), and guava (Psidium guajava L.guava) (Mariana et al., 2014; Chen et al., 2015; Ola et al., 2015), There are also great differences in the types and contents of volatile organic compounds among different varieties of the same fruit. At present, GC-O (Gaschromatogra phy olfactometry) technology is usually used to evaluate the main volatile organic compounds or active components of volatile organic compounds (Jelen et al., 2012; Chen et al., 2016). However, GC-O method usually requires a lot of work and time, and requires higher evaluators. However, GC-MS method has better separation effect and higher sensitivity, which is more suitable for the determination of volatile gaseous substances.

In this study, 60 volatile organic compounds in the Myrica rubra fruit were detected by GC-MS, belonging to 8 categories, which was significantly different from the results of Zhang et al. (2013) and Kang et al. (2009) that “alkanes and olefins are the most important volatile components of most Myrica rubra varieties”. This study found that olefins may be a typical characteristic marker in the varieties of purple Myrica rubra and white Myrica rubra. The former has the highest content in the top two, while the latter has not been detected. A total of 19 ingredients were detected in Biqi, 8 more than those studied by Zhang et al. (2014), which may be related to differences in planting area, management level and extraction and detection methods. Chen et al. (2015) used solid phase microextraction and other three methods to analyze the odor composition of three varieties of Biqi, Dongkui and Fenhong. Among the 36 aromatic components detected, Chen found that “Biqi” has a strong vanilla smell, and the main component is methyl benzoate; “Dongkui” has a strong grass smell, and its main component is 2,6-dimethyl-2,4,6-octriene; The “Fenhong” has a strong fragrance, and its main components are α- pinene. This is similar to the results of this study on characteristic volatile organic compounds, and the main volatile organic compounds of Pinus massoniana (pine needles and turpentine) were the same as the main components of the pine odor of Fenhong species, both of which were α-pinene. Chen et al. (2016) used PCA analysis to classify the odor of 11 Myrica rubra varieties, such as Biqi, Dongkui and Fenhong, into three categories: α-pinene, β-caryophyllene and ethyl acetate. Kang et al. (2009) showed that the most important volatile organic compounds of Myrica rubra are caryophyllene, menthol, 4-terpineol, linalool oxide, benzyl alcohol, β-phenylethanol, α-methyl benzyl alcohol, 3-methylbutyric acid, acetic acid, etc. In the four Myrica rubra tested this time, the highest caryophyllene content  was only in the two purple red varieties of Biqi and Yewu, while the top three volatile organic compounds in the other two white varieties were all free of caryophyllene, which is different from the result of Kang et al. (2009) that “extremely high caryophyllene content is a typical feature of Myrica rubra”.

However, among the volatile organic compounds of Dongkui and Biqi in different years and habitats, the content of caryophyllene is the highest in the same year, but the absolute value is very different. For example, Kang et al. (2009) measured the content of 1 069.8 μg/L in Xianju-dongkui, while the author only measured 688.3 μg/L in the materials sampled in Huangyan. Lin et al. (2015) studied 3 local varieties of Myrica rubra in Fujian Province, and found that the relative content of hydrocarbons accounted for 89.26%~95.99% of the volatile organic compounds respectively, but the volatile organic compounds and their contents in the flesh of different varieties of Myrica rubra were very different. The volatile organic compounds of Fugong 1 were mainly composed of octadecane, heptane, 1-caryophyllene, etc., accounting for 76.69% in total; The volatile organic compounds in Ruansi-anhaibian are mainly composed of 1-caryophyllene and epoxy caryophyllene, accounting for 83.30%. Three main volatile organic compounds of Pinus massoniana were compared in this study, α- Pinene, camphene, β-pinene, was different from Biqi, Yongjia-Baiyangmei, Shangyu-Baiyangmei and Yewu.

From the analysis of flowering period, it seems that there is possibility of hybridization between Myrica rubra and Pinus Linn trees. The flowering period of male flowers of Myrica rubra and Pinus Linn trees is late February to early April, and that of female flowers is early March to early April. It was inferred that Pinus Linn trees (♂) × Myrica rubra (♀) are likely to cross pollinate naturally within a certain period of time. The main volatile components of Pinus massoniana are α-pinene, camphene, β-pinene, which is similar to some components of some Myrica rubra varieties mentioned above. In this way, considering the possibility of genetic relationship, for this reason, artificial pollination, spot pollination, twig shaking and other cross pollination tests were carried out in Biqi, Shangyu-baiyangmei, Yongjia-baiyangme and Yewu respectively for two consecutive years. The results showed that they were not fertilized, so it can be ruled out that Pinus massoniana, as a gymnosperm, may cross with the angiosperm Myrica rubra. Why there are pine odor components in some Myrica rubra varieties remains to be further studied.

It can be inferred that the occurrence of pine odor in some Myrica rubra has its genetic evolution process, leading to changes in the content of inherent volatile organic compounds. Therefore, the pine odor components can be reduced and the fruit quality can be improved through cultivation, hybridization and other techniques in variety breeding. For example, Shangyu-Baiyangmei is an example of the gradual reduction and even disappearance of pine odor. Compared with Yongjia-Baiyangmei, Shangyu-Baiyangmei has experienced a long period of folk breeding, and in Erdu Town of Shangyu, we selected Erdu-Baiyangmei without pine odor; The Yongjia-Baiyangmei has not been selected, so the inherent characteristics of pine odor have not changed. The similarity of color and smell can not infer the genetic evolution of a variety. For example, the presence of lycopene in sweet orange does not mean that it is related to tomato varieties (Xu et al, 2009). The author found that the Myrica rubra varieties with pine odor have strong storage tolerance, and the storage time under natural conditions is 2 times or more than that of the control, which may also be one of the directions of Myrica rubra breeding in the future.

3 Materials and Methods

3.1 Test materials

1 kg of Yongjia-baiyangmei (YJBYM), Shangyu-baiyangmei (SYBYM), Yewu (YW), and Biqi (BJ) fruits of similar size and maturity were randomly selected and stored in a freezer at -18℃ for future use. Pinus massoniana needles and turpentine were collected from the Experimental Base of Citrus Research Institute of Zhejiang Province.

Main instruments and equipment include: Finnigan Trace MSGC-MS (Finnigan, USA), manual extraction head (model 75 μm Carboxen/PDMS), SPME manual injector (Superco, USA), etc.

3.2 Test method

3.2.1 Investigation

The method of combining general survey with resource nursery survey was adopted. According to the general survey, the main production area of Yongjia-baiyangmei is Yongjia County; Shangyu-baiyangmei is in Shangyu City and Yuyao City; Yewu is in Huangyan County and Linhai City; Biqi is in Yuyao and Huangyan counties. Three orchards were randomly selected for each production area, and 10 trees were selected for field investigation and fresh fruit tasting. The spot survey was conducted in the Myrica rubra Germplasm Resource Nursery of Citrus Research Institute of Zhejiang Province (Huangyan District, Taizhou City, Zhejiang Province, covering an area of 4 hm²). Five trees were selected for each variety, and 10 fruits were randomly selected for tasting for each tree.

3.2.2 Detection of volatile organic compounds

3.2.2.1 Sample treatment

Add 2.4 g CaCl₂ and the seedless Myrica rubra pulp, and put them into a juicer to extract juice. Quickly put 8 mL red Myrica rubra juice into a 15 mL extraction bottle, and add 5 μL 1×10^-6 internal standard 3-nonone. Add magnetic rotor and seal it with aluminum foil and sealing film; Under the aging temperature of 250℃, put the solid phase microextraction head at the gas chromatography sample inlet for aging for 40 min. After the extraction head was aged, insert it into the headspace of the extraction bottle, carefully push out the fiber head, and then put the extraction bottle in a 40℃ water bath for extraction for 30 min; After the fiber head was pushed back, pull out the extraction head from the extraction bottle, insert it into the GC-MS sample inlet with the set conditions, push out the fiber head, and then desorb it at 220℃ for 3 minutes. After the fiber head was pushed back, pull out the extraction head to collect data.

3.2.2.2 GC-MS analysis

Chromatographic conditions: carrier gas was high-purity helium; The chromatographic column was PEG-20M capillary column (30 m×0.25 mm×0.25 μm); Volume flow 1 ml/min; The column temperature rise mode was programmed: 60 ℃ (5 min), 240 ℃ (5 min), 5 ℃ per minute; Temperature setting of gasification chamber: 250 ℃.

Mass spectrum conditions: electron impact (EI) ionization mode was adopted, and the electron energy was set to 70 eV; The temperature of ion source was 230 ℃, and the temperature of gas spectrum/mass spectrum connecting tube was 250 ℃; The scanning range was 50~300 amu.

3.2.2.3 Method

Qualitative analysis: Agilent7890-5975GC-MS qualitative analysis method was used for turpentine.

Quantitative analysis: (1) calculate the relative content of each compound component with the peak area normalization method; (2) Choose a concentration of 1.0×10^-6 3-nonone as internal standard, which is quantified by SIM (selective ion detection). The calculation formula was based on the method of Tian (2010, Shandong Agricultural University, pp. 20-25) and slightly modified. Volatile organic matter content (μg/g)=[component peak area/internal standard peak area × Internal standard concentration (μg/g) × Internal standard volume/sample weight (g)].

3.3 ISSR molecular marker detection

3.3.1 Material preparation and DNA extraction

One hour before the test, the young leaves on the upper crown of the four Myrica rubra varieties and the young tip leaves of Pinus massoniana (CK) were collected from the resource nursery, and washed and dried with purified water.

3.3.2 ISSR molecular marker experiment and consumables

The procurement of materials and computer experiments were organized according to the methods of Luo et al. (2015).

Authors’ Contributions

WY participated in the determination of pine odor components and the detection of genetic relationship; NHZ participated in data sorting and writing the first draft of the thesis; YBG participated in some experiments; CFY was the project leader, guiding experimental design, data statistics, thesis writing and revision. All authors read and approved the final manuscript.

Acknowledgments

This study was jointly funded by Discipline Construction Project for Characteristic Fruit Trees in Eastern Zhejiang of Zhejiang Academy of Agricultural Sciences, the 13th Five-Year Bayberry Breeding Project of Zhejiang Province (2016C02052-2) and Forestry Science and Technology Project Cooperated by Province and Academy in Zhejiang Province (2019SY05-1).

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