Collection and evaluation of thirty-seven pomegranate germplasm resources

Abstract

Pomegranates (Punica granatum L.) are gaining popularity among consumers because of their high antioxidant activity and multiple medical benefits. China is rich in pomegranate genetic resources, but how to use them effectively is a problem worthy of deep consideration. In this article, thirty-seven pomegranate varieties from seven provinces in China were collected and analyzed for twelve phenotypic traits and twelve biochemical indicators (seeds and juices). The fruit and aril fresh weight ranged between 210.5 and 576.5 g and 121.0 to 327.5 g, respectively, and the edible rate (42.58–64.80%), seed weight (1.80–3.41 g), seed number (249.1–838.9), fruit height (10.51–15.48 mm), fruit diameter (11.46–17.50 mm), skin thickness (2.14–6.98 mm), and shape index (0.82–0.96) varied among the different genotypes. The pomegranate juice total phenolic content ranged from 40.91 to 132.47 µg/mL, and the total flavonoid content (14.08–137.72 µg/mL), vitamin C content (12.80–66.63 µg/mL), pH (3.10–4.34), total soluble solids (13.13–17.50°Brix), and titratable acidity (0.26–2.71%) also varied; the pomegranate seed total phenolic content ranged from 0.62 to 1.78 mg/g, and the total flavonoid content (0.39–0.99 mg/g), vitamin C content (7.55–13.90 mg/g), DPPH radical scavenging capacity (85.98–98.24%), and ABTS scavenging ability (28.72–51%) were also measured. The coefficients of variation of the studied traits ranged from 5.62 to 54.02%, and the phenotypic traits’ Shannon–Weaver diversity indexes ranged from 0.67 to 1.53. Cluster analysis divided the 37 varieties into three categories, providing a reference for improved variety breeding. In addition, genotypic and environmental effects mainly affected the pomegranate flavor and antioxidant activity, respectively.

Introduction

Pomegranate (Punica granatum L.) is an ancient and widely cultivated fruit native to Iran, Afghanistan, and other parts of Central Asia. Besides its nutritional value, more studies established the medicinal effects of pomegranate including antibacterial, anti-inflammatory, antiviral, and benefits on cardiovascular health and obesity [1]. During the Western Han dynasty, Chinese envoy Zhang Qian introduced pomegranates into China [2]. After long-term natural hybridization, gene mutation and the implementation of varied breeding and propagation (e.g., seeding, ramets, grafting, etc.) methods, a wide collection of pomegranate varieties has been produced. Pomegranate is one of the essential fruits in China and is widely distributed and cultivated in the provinces of Henan, Shandong, Sichuan, Anhui, Shaanxi, Yunnan, and Xinjiang in China. Because of the differences in the aspects of geographical distribution, climatic environment, and genetic variation, pomegranate varieties have distinct local characteristics in different parts of China. Therefore, the recognition and measurement of such diversity and its nature and magnitude are crucial to a breeding program.

Pomegranate variety identification is based on the external and internal characteristics of the fruit. Martinez-Nicolas et al. [3] established that pomegranate fruit and seed size have a relatively strong relationship with the juice’s pH. However, the differences in pomegranate leaf and flower characteristics between varieties is not significant, which indicates a certain relationship between the phenotype and physiological status in pomegranate. Zaouay et al. [4] studied 38 Tunisian pomegranate varieties, analyzing the effects of clones, age, and their interactions on fruit quality, and demonstrated that genetics (variety) contributed the most to fruit size and skin color and thickness variation and concluded that aril and juice yields were affected by age. Li et al. [5] studied the relationship between antioxidant capacities and the planting environment of 9 pomegranate juice varieties in China; however, the studied varieties did not present a comprehensive germplasm collection. At present, pomegranate diversity studies have mainly focused on tree shape, leaves, and fruit juice [6, 7]. However, few writers have focused on extensive research into pomegranate seeds. In China, pomegranate seeds are discarded as waste, which pollutes the environment and wastes resources. Some scholars have used pomegranate seeds to develop new applications, and the addition of pomegranate seed powder to bread was shown to significantly improve its antioxidant activity [8]. Lucci et al. [9] proposed the use of a pomegranate seed ethanol extract as a nutraceutical and functional food ingredient to utilize its antihormone-dependent antioxidant and antiproliferative effects against human prostate cancer and breast cancer cells. These studies show the potential use of these fruit by-products as natural antioxidants.

The environment greatly influences crop growth state and tolerance to stress. Plants grow better in a suitable environment and grow slowly or even die in harsh environments. However, the environmental impact is varied, and for pomegranates, we still do not know which aspects of the pomegranate quality are affected by the environment. In the interaction between genotype and phenotype, it is worth exploring which pomegranate parameter is mainly controlled by genotype and which parameter is closely related to the environment.

The present paper is an attempt to investigate the genetic diversity and nutrition (seed and juice) of thirty-seven pomegranate varieties from seven Chinese provinces and to propose a conceptual image and solution to the following problems: (1) analyzing phenotypic traits to determine the genetic relationship between different pomegranate cultivars; (2) screening pomegranate varieties to identify those suitable for fresh consumption, juice processing, acid extraction, and high-antioxidant seeds; and (3) revealing the effect of different environments on pomegranate phenotypic traits and nutrition. This work provides the first comprehensive assessment of pomegranate phenotypic traits and nutrition, and the relationship between them and with different environmental conditions, which has great value for breeders and processing factories.

Materials and methods

Collection of the pomegranate varieties

In 2018, thirty-seven commercial pomegranate cultivars were collected from eight cities in seven Chinese provinces (Yunnan (YN), Anhui (AH), Sichuan (SC), Henan (HN), Shandong (SD), Xinjiang (XJ), and Shaanxi (SN)) (Tables 1, 2). Climate and elevation information for the different regions were obtained from weather, elevation, and satellite positioning websites (http://haiba.ugoto.cn/, https://baike.baidu.com, http://www.gpsspg.com/maps.htm) (Table 1). From each pomegranate variety, 10 fresh fruits were randomly collected for further analyses. After determining the phenotypic traits, the seeds were cleaned, dried and used for nutrient measurements.

Table 1 Environmental information in different regions

Table 2 Origin and abbreviation of all pomegranate varieties

Characteristics of the fruit

Fruit fresh weight (FFW; g), fresh aril weight (FAW), 100-seed grain weight (SW), and skin fruit weight (SFW; g) were determined. Seed number (SN) was based on the average number of seeds from 10 fruits. Fruit height (FH; mm), fruit diameter (FD at the equator; mm), and skin thickness (ST; mm) were recorded using a digital caliper at 0.01 mm accuracy. Skin color (SC) was assessed according to a 4-point grading scale (1 = yellow-greenish; 2 = pink yellowish; 3 = red-pink; and 4 = dark-red to purple) Juice color (JC) was determined according to a 5-point grading scale (1 = light pink; 2 = pink; 3 = red-pink; 4 = red; and 5 = reddish-purple). The edible rate (ER) and shape index (SI) were calculated as FAW/FFW and FH/FD, respectively [10].

Total phenolic (TP) content determination

Total phenolic (TP) content was determined following the Folin-phenol method [11]: (1) first, 0.8 g of pomegranate seed powder was dissolved in 8 mL of 60% ethanol and sonicated for 30 min, and the mixture was then centrifuged at 12,000 rpm for 15 min; (2) then, 50 μL of the supernatant was mixed with 250 μL of Folin-Ciocalteu reagent and 750 μL of 20% sodium carbonate (Na₂CO₃), and 3 mL pure H₂O, and (3) after adequate reaction of the solution (2 h), the absorbance was read at 760 nm with a spectrophotometer (BECKMAN DU-800^®).

Total flavonoid (TF) content determination

Total flavonoid (TF) content was determined using the method of Viuda-Martos et al. [12] as follows: (1) first, 0.2 g of pomegranate seeds were dissolved in 4 mL of 60% ethanol and thoroughly ground to obtain an extract; (2) the phenolic extract was centrifuged at 12,000 rpm for 15 min; (3) then, 1 mL of supernatant was mixed with 0.3 mL of 5% NaNO₂ and 0.3 mL of a 10% AlCl₃ solution; (4) after 5 min, 2 mL of 1 M NaOH was added, the total volume was brought to 10 mL with ddH₂O, and (5) the absorbance was measured at 510 nm with a spectrophotometer (BECKMAN DU-800^®).

Vitamin C (VC) content determination

The VC content was determined using the method of Kampfenkel et al. [13] with slight modifications as follows: (1) first, 1 g of pomegranate seed powder was mixed with 5 mL of a trichloroacetic acid solution and then sonicated for 30 min; (2) after centrifugation at 12,000 rpm for 15 min, the supernatant was filtered through a filter to obtain an extract, and (3) 1 mL pomegranate juice or pomegranate seed powder extract solution was mixed with 1 mL of a 0.5% trichloroacetic acid solution, 1 mL of ethanol, and 0.5 mL of a 0.4% phosphoric acid solution; (4) after 5 min, 1 mL of a 5% 2,2′-bipyridine solution and 0.5 mL iron trichloride were added, the mixture was incubated in a water bath at 37 °C for 30 min, and (5) the absorbance was measured at a wavelength of 525 nm with a spectrophotometer (BECKMAN DU-800^®).

DPPH radical scavenging capacity estimation

To make the DPPH solution, 3.98 mg of DPPH was accurately weighed and mixed well with 100 mL of 80% ethanol and kept in the dark at 4 °C in a refrigerator. The sample extract was mixed with 2 mL of DPPH solution and allowed to stand for 30 min. The absorbance at 517 nm was measured for triplicate samples. The following formula was used:

$$K = \left( {1 - \frac{{A_{i} - A_{j} }}{{A_{c} }}} \right) \times 100\%$$

(1)

where K is the sample’s clearance rate of DPPH free radicals, Ai is the absorbance of 2 mL of DPPH solution + 2 mL of sample extract, Aj is the absorbance of 2 mL of sample extract + 2 mL of an ethanol solution, and Ac is the absorbance of 2 mL of DPPH solution + 2 mL of an ethanol solution [14].

ABTS scavenging ability

The ABTS radical cation is generated by a reaction of 7 mM ABTS and 2.45 mM potassium persulfate with 12 h of incubation at room temperature in the dark. The ABTS + solution was diluted with phosphate buffer saline (PBS) at pH 7.4 to an absorbance of 0.70 ± 0.02 at 734 nm before analysis. The ABTS solution (3.9 mL) was added to 0.1 mL of the tested sample and mixed thoroughly. The mixture was incubated in a water bath at 37 °C for 30 min, and the absorbance was read at 760 nm by BECKMAN DU-800. We used water as a control. The experiment was repeated three times, and the result was calculated using the following formula:

$$K = \left( {\frac{{A_{control} - A_{test} }}{{A_{control} }}} \right) \times 100\%$$

(2)

where A control is distilled water (0.1 mL) mixed the ABTS solution (3.9 mL) and A test is the tested sample (0.1 mL) mixed the ABTS solution (3.9 mL) [15].

Total soluble solids (TSS), pH, and titratable acidity (TA)

Total soluble solids (°Brix) and pH were determined on juice samples using a handheld refractometer and digital pH meter, respectively. Titratable acidity (TA) was measured colorimetrically by titration with 0.1 N NaOH using the pH indicator phenolphthalein[16].

The maturity index (MI)

The fruit maturity index (MI) was determined as TSS/TA according to Martinez et al. [17]. The classification of MI values for sweet = 31–98, sour–sweet to sweet = 25–30, sour–sweet: 17–24, sour–sweet to sour = 9–16 and sour = 5–8.

Analysis of phenotypic diversity

The mean (x) and standard deviation (δ) of quantitative traits were calculated using the Python3.7 language (Numpy library and Pandas library). Assuming all data metrics follow the normal distribution probability, the quantitative characterization of all materials was divided into 5 levels based on the mean and standard deviation data. The standard normal distribution coefficients (X-1.2816δ), (X-0.5244δ), (X-0.5244δ), (X-1.2816δ) were divided into 5 levels, and the probability of occurrence of levels 1 to 5 was 10%, 20%, 40%, 20%, and 10%, respectively. Nonnumerical characteristics were represented by assignment. The diversity of a characteristic was determined using the Shannon–Weaver diversity index (H’) as follows:

$$H^{\prime } = - \sum\limits_{i = 1}^{n} {\left( {P_{i} \times \ln P_{i} } \right)\,} (i = 1,2,3 \ldots )$$

(3)

where H’ is the diversity index and Pi is the effective percentage of the distribution frequency for the Nth rank of a trait [18].

Statistical analysis

The data were analyzed by one-way analysis of variance (ANOVA), and sample means were compared by Tukey’s test. P < 0.05 was considered significant in all cases. Python scripts were used for the Pearson Correlation Analysis and Cluster analysis.

Results and discussion

Phenotypic diversity of the pomegranate varieties

The Shannon–Weaver diversity index (SHDI) was calculated to compare the phenotypic diversity among characters and regions [19]. The larger the SHDI value, the richer the diversity of the community. As shown in Table 3, the coefficient of variation (VCo) between twelve quantitative traits of the 37 local pomegranate varieties ranged from 5.62 to 54.02%, and the SHDI varied from 0.67 to 1.53, which was higher than that reported by Polyzos et al. [20] in a study on thirty-four garlic genotypes of greek garlic (0.37 to 0.99). This variation also indicates rich diversity within the Chinese pomegranate germplasm resources. Through analyzing the fruit picking date, all of the early-maturing varieties (YN-TLZ, YN-GY, YN-HMN, YN-HZZ, YN-RZ, and SC-QPRZ) originated from the Yunnan and Sichuan provinces, while the late-maturing varieties (AH-EBZ, AH-DBZ, and SN-DBW) came from Anhui and Shaanxi provinces. Yunnan and Sichuan are located in areas with a high average temperature, abundant precipitation and high altitude (> 1000 m), while Anhui and Shaanxi are located in low-altitude areas. Accordingly, the ripening time of pomegranate planted in higher altitude regions is remarkably earlier than that for the fruits of the lower altitude regions, and the maturing time of pomegranates is closely associated with the climate and altitude of cultivated areas.

Table 3 Phenotypic parameters of thirty-seven pomegranate varieties

The fresh fruit weight (FFW) of the 37 varieties ranged between 210.5 and 576.5 g, with 86.5% of the fruit weighing more than 500 g (SC-QPRZ: 576.5 g, SD-DHP: 568.0 g, and SD-DQP: 558.5 g) and with four varieties weighing less than 300 g (AN-HBEH: 255.0 g, SD-QY: 295.0 g, YN-GY: 291.5 g, and SD-ZPT: 210.5 g) (Table 3). Pomegranate aril yield is one of the most important industrial production parameters [21]. Fresh aril weight (FAW) varied between 121.0 and 327.5 g, with the SD-DQP, SC-QPRZ and SD-DHP varieties suitable for fruit juice processing. Skin fruit weight (SFW) varied from 89.5 to 277.2 g, with SC-QPRZ, SD-DHP, AH-BYSZ, SD-DHP, and AH-HMN as top varieties and YN-TLZ, SD-QY, AH-HBEH, AH-QPRZ, and SD-ZPT as the bottom. This pattern largely overlapped with the FFW variety ranking. Remarkably, the fruit weight of the same variety planted in different areas (Sichuan and Anhui) was obviously different. The FFW and SFW values of QPRZ in Sichuan Province were much higher than those of the same variety grown in Anhui province. Based on the FAW and FFW ratio, the YN-TLZ showed the highest edible rate (ER) (64.83%), while SN-DHT had the lowest edible rate (42.58%).

Ordinarily, the harder the seed, the greater the impact of taste. The main reason why soft-seeded pomegranate is popular in the market is that its taste and flavor are better than those of other varieties. The seed number (SN) was between 253.5 and 838.9 for all varieties. An increase in seed number will affect the taste of pomegranate, but it helps improve the nutritional value of the pomegranate seeds. The fruit height (FH) and diameter (FD) reflect the size of the pomegranate, and their ratio is defined as the shape index (SI), which can be effectively used to evaluate the shape of the fruit. The fruit SI of all varieties except for that of SN-SBT and YN-GY were above 0.85, and among those, AH-HBYH and AH-HBEH had the highest, reaching 0.95 and 0.96, respectively. Skin thickness (ST) significantly differed among the varieties and ranged between 2.14 and 6.98 mm. More significantly, all varieties with the thinnest skin, smallest seed number, shape index, and edible rate originated from Yunnan Province (Table 3).

Skin color is a critical quality attribute in pomegranate marketing. The attractive, red color is an important parameter for commercial quality classification, which influences consumer behaviors [22]. The color for peels and arils of 37 pomegranate varieties from China varied from white to deep red. Most of them had pink yellowish peels and red-pink aril. The SD-ZPT, SD-DHP, SC-ZM, and SN-DHT varieties exhibited a beautiful red color and a very attractive appearance.

Correlation analysis of phenotypic parameters

We can compose some conclusions through correlation analysis of the phenotypic parameters of pomegranate (Fig. 1). Fresh fruit weight (FFW), fresh aril weight (FAW), skin fruit weight (SFW), fruit height (FH), and fruit diameter (FD) were significantly positively correlated with each other. A significantly negative correlation was found between edible rate (ER) and skin thickness (ST). Skin color (SC) and juice color (JC) were weakly positively correlated.

Fig. 1

Pearson’s correlation plot based on the correlation of phenotypic traits. Red and green represent positive and negative correlation, respectively. The darker the color, the larger the correlation coefficient value

Genetic relationship of different pomegranate varieties

To effectively compare the difference in pomegranate cultivars, all phenotypic characteristics were normalized before conducting the cluster analysis (Fig. 2). Cluster analysis indicated that the pomegranate varieties were not simply affected by geographical location. In addition, the genetic distance of some varieties from different provinces (such as XJ-HT, SN-DHT and SD-QL) was close (Fig. 2). The 37 pomegranate varieties were divided into three subcategories according to genetic distance (Fig. 2). The first category featured the bright color of the peel and aril, a large number of seeds, and a moderate skin thickness (XJ-KST, XJ-HT, SN-DHT, SN-YCL, SD-QL, YN-HZZ, YN-HMN, SC-ZM, YN-RZ, HN-RZ, YN-TLZ, YN-GY, and SD-ZPT). The features of the second category were medium fruit size, light skin but brightly colored aril, and heavy seed weight (HN-YDZ, AH-HBYH, SN-SBS, SN-BRZ, AH-EBZ, SD-DMY, SN-DZSL, SN-DBW, SN-LXYH, SN-LXEH, SD-QY, AH-HYSZ, AH-QPRZ, SD-TSSB, and AH-HBEH). The third category was characterized by large fruits and a thick fruit skin (AH-HMN, AH-FYSZ, SN-SBT, AH-DBZ, SN-JPT, AH-BYSZ, SD-DQP, SC-QPRZ, and SD-DHP). The fact that AH-HMN and YN-HMN were not in the same group was due to the significant differences in fruit size and skin thickness.

Fig. 2

Cluster analysis of 37 Chinese pomegranate varieties based on phenotypic traits. Each small square reflects the phenotypic characteristics of pomegranate varieties. The color represents the normalized value, with red representing the larger value and blue representing the lower value. Each row represents the normalized content of different phenotypic characteristics from one variety. Each column represents the difference in the normalized results of different varieties in a single specific phenotype. Seven colors distinguish the seven provinces

Additionally, the phenotypic indicators of pomegranate could be classified into three categories through cluster analysis (Fig. 2). The skin color (SC) and juice color (JC) showed a close relationship. Seed number (SN), fresh fruit weight (FFW), fresh aril weight (FAW), skin fruit weight (SFW), fruit height (FH), and fruit diameter (FD) were clustered into one group, reflecting fruit weight and size. Generally, the size of fruits was proportional to the weight, i.e., larger fruits were generally heavier. Edible rate (ER), shape index (SI), 100-seed weight (SW) and skin thickness (ST) were grouped together, which is in agreement with the correlation analysis of the phenotypic parameters.

Nutrition and flavor analysis of pomegranate juice

The total phenolic (TP) content in the pomegranate juice ranged from 40.91 to 132.47 μg/mL (Table 4), which was lower than that reported in a previous study (2380–9300 mg/L) of eight Iranian cultivars [23]. Total flavonoid (TF) content values ranged from 14.08 to 137.72 μg/mL. The results show that the highest pomegranate juice total flavonoid content was that of SN-JPT, followed by that of SN-LXYH, SN-YCL, SN-DHT, and SN-SBT. In addition, all the above mentioned varieties were planted in Shaanxi Province. The pomegranate juice VC content ranged from 12.80 to 66.63 μg/mL, with the top values observed for XJ-KST, SD-QL, AH-HBYH, SN-JPT and YN-RZ (Table 4). The results were lower than those reported for five Pakistani (10.5 to 12.6 mg/100 mL) [24] and Spanish (80 to 190 μg/mL) [25] cultivars.

Table 4 Juice nutrition and flavor analysis of thirty-seven pomegranate cultivars grown in China

Nutrition analysis showed the difference in the same variety (RZ, HMN or QPRX) from different growing areas. The RZ cultivar grown in Yunnan Province had a higher TP and VC content but a lower TF content than those of the RZ cultivar grown in Henan Province. Similarly, the TP and VC contents in YN-HMN were higher than those in AH-HMN, but the TF content was significantly lower. Differences in soluble solids content between YN-RZ and HN-RZ were not significant, but QPRZ and HMN showed significant differences when planted in different areas, which showed that the geographical locations and climatic conditions are important factors affecting the antioxidant activity and soluble solids content.

The AH-HBEH and SC-ZM pomegranate juice exhibited the highest (4.34) and lowest (3.10) pH, respectively, with a similar range as that of Spanish pomegranate (pH = 2.56–4.31) [26] and a maximum value higher than that of Moroccan pomegranate (pH = 2.76–4.03) [27]. Among the 37 pomegranate cultivars, the total soluble solids (TSS) in the pomegranate juice of SC-ZM (17.50°Brix) and YN-TLZ (13.13°Brix) exhibited the highest and lowest values, respectively. The range was lower than that reported by Fernandes et al. (14.87–18.04°Brix) [26] and Ferrara et al. (14.7–18°Brix) [28].

The titratable acidity (TA, expressed as citric acid percentage) and the maturity index (MI, ration of TSS toTA) are critical for the juice flavor and palatability of pomegranates and can be used for classification. The MI values for the tested genotypes were in the range of 6.46–57.81, which can be further grouped as sour (SC-ZM), sour–sweet to sour (YN-HMN, AH-HMN, and YN-HZZ), sour–sweet (SN-SBS), sour–sweet to sweet (XJ-KST, HN-YDZ, SD-DMY, XJ-HT, SD-DQP, and SD-ZPT) and sweet (all other varieties) (Table 4). In addition, pomegranate cultivars are also classified by TA as sweet, sour–sweet, and sour [26, 28]. Sweet pomegranate varieties have an acidity lower than 0.9% and are mainly destined for fresh consumption. Sour–sweet cultivars have an acidity between 1 and 2% and are used for soft drink production. Sour varieties have an acidity higher than 2% and are used in the food industry for acid extraction [29]. According to the above classification criteria, the AH-HMN, YN-HZZ, and YN-HMN varieties can be used for juice production, and SC-ZM is ideal for industry acid extraction criteria; the others are suitable for fresh consumption. Each person has a different definition of sweetness and acidity, which depends on their diet preferences and habits. Therefore, the result of MI classification is different from that of TA classification, and the MI classification is more detailed than TA classification.

Nutrition and antioxidant activity analysis of pomegranate seeds

Pomegranate seeds are rich in oil and have antioxidant activity that differs greatly between varieties [30]. In the present study, the seed TP content ranged from 0.57 to 1.78 mg/g (Table 5). The total phenolic content in HN-RZ was the highest, while SN-DBW exhibited the lowest total phenolic content. The TF content in pomegranate seeds ranged from 0.39 to 1 mg/g (Table 5). The VC content in the seeds ranged from 7.55 to 13.90 mg/g, with the highest and lowest values observed for SN-BRZ and AH-HYSZ seeds, respectively. Our results showed that lower TP and VC contents were not entirely consistent with those of previous studies [30], which could be attributed to discrepancies in experimental methods, reference materials, and pomegranate varieties. This research used the same experimental methods to compare multiple pomegranate varieties, which can mitigate experimental errors and increase the reliability of the data. Two in vitro assays (DPPH and ABTS) were used to evaluate the potential antioxidant activity of the pomegranate seeds. The DPPH and ABTS scavenging ability of the 37 Chinese pomegranate juices varied from 83.39 to 98.24% and from 28.72 to 51%, respectively. Among them, YN-RZ showed the strongest antioxidant ability.

Table 5 Seed nutrition and antioxidant activity analysis of thirty-seven pomegranate cultivars grown in China

Correlation analysis of pomegranate juice and seeds

Figure 3 shows the correlations among the characters studied. Significant correlations were found between TSS and TA, MI and TA, and pH and TA. Therefore, pH can also be used as a reference index for pomegranate juice flavor. Furthermore, the above indicators can reflect pomegranate juice taste and provide references for customers and factories. Additionally, TP and TF were positively correlated with each other (Fig. 3), which is consistent with published data [30]. With respect to the antioxidant activity, there is a strong positive relationship between TP and DPPH, as well as between TP and ATBS.

Fig. 3

Pearson’s correlation plot based on the correlation of physico-chemical traits of pomegranate juice (a) and seed (b). Red and green represent positive and negative correlation, respectively. The darker the color, the larger the correlation coefficient value

Principal component analysis (PCA)

In this study, we collected multiple data from 37 pomegranate varieties for analysis. If each indicator was analyzed separately, the results would likely be isolated rather than integrated. Additionally, blindly reducing indicators can lose a great deal of information and is prone to producing erroneous conclusions. The advantages and uses of different pomegranate varieties are distinct. Therefore, it is essential to find a reasonable method for reducing the loss of information contained in the original indicator while reducing the indicators that need to be analyzed to achieve a comprehensive analysis of the collected data. Principal component analysis (PCA) is one such method of dimensionality reduction. An Eigen value provides a measure of the significance of the factor; thus, the factors with the highest eigenvalues are the most significant, and eigenvalues ≥ 1 are considered significant [31].

The 24 indexes of the 37 varieties were subjected to principal component analysis in this study. The eigenvalues of the top six principal components were all greater than 1. Among them, the contribution values of the first and second principal components were 18.9% and 15.1%, respectively, and the cumulative contribution rate was 30% (Fig. 4).

Fig. 4

Principal Component Analysis of 37 pomegranate varieties and 24 characters. The longer the arrow, the greater the contribution

As shown in Fig. 4, FFW, FAW, and FD were the maximum positive values of the PCA1 eigenvector, and these indicators reflect the size of the fruit’s appearance, which is one of the key indicators that attracts consumer purchasing. Higher PCA1 values indicated larger fruit, and SD-DHP, SC-QPRZ, SD-DHP, YN-RZ, and YN-HZZ were representative varieties. In the PCA1 eigenvector, the indexes with the most significant negative values were TSS/TA (MI) and pH, which can reflect the fruit flavor. The representative varieties in the negative direction of PCA1 were AN-HYSZ, AH-HBEH, YN-GY, SD-TSSB, and YN-TLZ. These pomegranate varieties are sweet and have a low acid content. The most distinctive features of PC2 positive values were ER, STP, and ABTS, which can fully reflect the nutritional status of the pomegranate seeds. The higher the positive value of the pomegranate variety in PCA2, the stronger its seed antioxidant capacity. The main negative contributors to PCA2 were STH, PW, and TA, whose absolute values of the eigenvector were more than 2. These values mainly reflected the comprehensive information (size, flavor, and weight) of fruit. Representative varieties of the PCA2 negative axis area were AH-HMN, SN-SBS, and AN-BYSZ.

The effect of environmental factors on phenotypic and antioxidant physicochemical traits

We analyzed the correlation of environmental factors with twelve phenotypes and the physicochemical traits of three pomegranate varieties from different regions (Table 6). Since the number of phenotypic samples was ten and the number of repetitions of physiochemical experiments was three, we evaluated their correlation with the environment separately. The environment influences the weight and size of the pomegranate, which also affects the coloration of the fruit (Table 6). The higher the rainfall and altitude, the brighter color of the fruit skin, while too much temperature and precipitation will cause the juice to be lighter. In addition, the contents of total phenols, flavonoids, and VC in the pomegranate were strongly correlated with temperature, precipitation and altitude. In addition, the TSS and TA of the pomegranate did not differ significantly in different environments. Pomegranate flavor was mainly affected by genotype, which is supported by earlier work [32]. This research shows an important effect of altitude on the pomegranate VC content (Table 5). This finding was also reported by Mphahlele et al. [33]. In addition, he proposed that the VC content of pomegranate at middle altitudes was apparently higher than that at other altitudes.

Table 6 Correlation between phenotypic traits, physicochemical traits and environmental factors

Our study provided a detailed report on the phenotypic diversity of 37 Chinese pomegranate varieties grown in seven provinces. The phenotypic diversity of the pomegranate coefficient variation was between 5.62 and 54.02%, and the Shannon–weaver diversity index was between 0.67 and 1.53. The cluster analysis clearly showed phenotypic differences and genetic relationships among the studied cultivars, which laid the foundation for cross-breeding and selection breeding. The determination of pomegranate flavor can be used as a reference for selecting varieties for fresh consumption use or industrial acid treatment. Additionally, this paper comprehensively assesses the antioxidant activities of different pomegranate cultivar juices and seeds to provide a reference for the production of functional beverages and the development of pomegranate seed products.

The phenotype of pomegranate is the ultimate manifestation of both the environment and the genotype, and the different pomegranate varieties showed differences associated with climate change. After observing and comparing pomegranate varieties in different regions, we found that genetics mainly influenced pomegranate flavor. Furthermore, the effect of different cultivation environments on the fruit size was diverse. Most noteworthy, the environment had a significant impact on the antioxidant capacity of the pomegranate juice and seeds. Moreover, the genotype × environment interaction effects showed a variable influence on the pomegranate varieties. More detailed research is needed to determine how environmental factors affect the nutritional changes of pomegranate and their mechanisms of action.