Characterization of Diverse Pepper ( Capsicum spp.) Germplasms Based on Agro-Morphological Traits and Phytochemical Contents

: Pepper ( Capsicum spp.) is one of the most important crops worldwide. The fruits of Capsicum species are known to contain high amounts of vitamins and carotenoids, and they have health-promoting properties. In this study, a total of 513 pepper accessions belonging to two Capsicum species, C. annuum and C. frutescens , were investigated for their morphological characteristics and contents of phytochemicals including carotenoids, β -carotene, vitamin C, capsaicinoids, and total soluble solids. The results revealed wide variations in morphological traits and phytochemical contents between the accessions and across species. In addition, the association of fruit color and orientation with phytochemical contents was evaluated; the results indicated that germplasm with yellow-colored and pendant-oriented fruits could be important due to their high vitamin C levels. Multivariate analysis of the agro-morphological and phytochemical parameters revealed that Cap-sicum germplasm were clearly distinguished according to species. Furthermore, cluster analysis showed that germplasms belonged to three groups, and six genotypes were determined as being good genetic resources with high health-promoting phytochemical contents. Especially, vitamin C content was positively correlated with fruit diameter, weight, and wall thickness. Our ﬁndings revealed morphological and phytochemical characteristics potentially useful for breeding programs.


Introduction
Pepper (Capsicum spp.), one of the most important crops in the Solanaceae family, is cultivated and consumed worldwide [1].Fruits of the Capsicum are rich in ascorbic acid, carotenoids, phenolic compounds, and flavonoids [2].Many studies have demonstrated pepper's high nutritional value, health benefits, and medicinal properties [3,4].Among the genus Capsicum, harboring more than 35 species, 5 species (C.annuum, C. baccatum, C. chinense, C. frutescens, and C. pubescens) are domesticated [5].Health-promoting compounds have been identified in these five domesticated Capsicum species, and the proportions of these compounds depend on the genotype, maturity stage, and plant part, such as pericarps, placenta, and seed [6][7][8].
Phytochemicals contribute mainly to the organoleptic and bioactive qualities of peppers.Carotenoids and polyphenols are responsible for the visual and health-promoting characteristics, while sugars and acids determine the sensory quality of fruits in relation to flavor [9].The carotenoids are isoprenoids, which play an important role in many biological processes in plants; the major carotenoids in pepper are capsorubin and capsanthin, followed by β-carotene, lutenin, β-cryptoxanthin, zeaxanthin, violaxanthin, and antheraxanthin [10,11].Among these carotenoids, only β-carotene and β-cryptoxanthin have vitamin A activity [12].Vitamin C, which acts as a coenzyme for enzymes involved in human metabolism, accumulates in high amounts in peppers as the fruit ripens [13,14].Capsaicinoids, as alkaloids, are responsible for the pungency of Capsicum spp.Capsaicin and dihydrocapsaicin are the major capsaicinoids.Minor capsaicinoids include nordihydrocapsaicin, homocapsaicin, and homodihydrocapsaicin [15].Many of the functional properties of Capsicum species, such as dietetic and pharmacological effects, are attributed mainly to the antioxidant activity of capsaicinoids, polyphenols, and vitamin C [16,17].
Among the quality-determining traits of pepper fruits, fruit color is the most crucial since pigments are associated with flavor, nutritional, and health-promoting properties [18].Pepper fruits mature in diverse colors, including red, orange, yellow, green, and brown [19].The relative amounts of pigments such as chlorophyll, carotenoid, and anthocyanin regulate the mature color of pepper fruit [20].During the domestication process, fruit colors have been varied depending on the preferences of consumers and breeders [21].In the case of fruit orientation, there has been a transition from an ancestral erect type to a pendant type, a change that may be related to increased fruit size and reduced pedicel thickness, as well as protection from the sunlight and predation by birds [22,23].The fruit orientation is also a major horticultural trait that affects pollination, yield, and approach to harvesting [24,25].
Exploiting existing genetic variability and developing measures that preserve existing genetic resources are two of the most important plant breeding strategies [26].To develop appropriate conservation strategies, germplasms need to be properly characterized, not only for preventing diversity loss, but also for preserving potentially valuable traits and identifying the traits that contribute the most to overall diversity [27].Phytochemical characterization is an acceptable strategy for evaluating the biochemical diversity of the plant germplasm [28].Additionally, elucidating the relationships between morphological and chemical characteristics is important for agricultural management [29].
Using multivariate statistics is advantageous because the characterization and evaluation of the germplasm involves a wide range of data and a considerable number of descriptors [27].Principal component analysis (PCA) and cluster analysis (CA) are the most frequently used multivariate approaches.PCA is primarily used to reduce the number of input variables, whereas CA is used to classify genotypes [30].
Several studies investigating the morphological and chemical traits of peppers have been conducted; however, these characteristics are usually assessed separately [6,[31][32][33].Despite the usefulness of studying associations, few studies have attempted to establish correlations between morphological traits and phytochemicals [34,35].Therefore, the objectives of this study were to characterize the agro-morphological traits and phytochemicals of 513 germplasms of pepper across two species, as well as to identify the relationships between the orientation and color of mature fruits and phytochemicals.
A total of 513 Capsicum germplasms from the National Agrobiodiversity Center's (NAC) gene bank (Rural Development Administration [RDA], Jeonju, Republic of Korea) were used in this study.The plants were grown in the RDA research field (35 • 49 52.7 N 127 • 3 43.9E) using standard agronomic practices until harvest in 2019 and 2021.
Germplasms were categorized into two species, C. annuum (n = 380) and C. frutescens (n = 133).The introduction (IT) number, orientation of the fruit, and mature fruit color of 513 pepper germplasm are listed in Table S1 (Supplementary Materials).

Agricultural Traits
Plant height (PH), main stem length (MSL), leaf length (LL), leaf width (LW), days to 50% flowering (DF), days to 50% maturity (DM), fruit length (FL), fruit diameter (FD), fruit weight (FW), fruit wall thickness (FWT), mature fruit color, and fruit orientation of germplasms were recorded during field inspections.In each case, values are represented as the mean of triplicate determinations.

Carotenoids
All of the pepper samples from which phytochemicals were extracted were freezedried and powdered.Carotenoid contents of peppers were determined as described previously, with minor modifications [36].Briefly, 3% pyrogallol and 60% potassium hydroxide were added to 0.05 g of pepper powder, saponified in a water bath at 70 • C, and then cooled.The samples were homogenized after adding 1% sodium chloride solution and a hexane and ethyl acetate mixture, and the supernatant was collected by centrifugation.This process was repeated until all the pigment was gone.Before high-performance liquid chromatography (HPLC) analysis, the extracts were concentrated under a stream of nitrogen, resuspended in ethanol, and filtered through a 0.2 µm filter syringe.The carotenoids were separated by HPLC (Agilent 1260 Infinity II; Agilent Technologies, Inc., Santa Clara, CA, USA) on a C30 YMC column (250 mm × 4.6 mm, 5 µm; Waters Corporation, Milford, MA, USA).A 10 µL aliquot was injected into the HPLC system.Solvent A was 92:8 v/v methanol and water, and solvent B was methyl tert-butyl ether with the following gradient condition: 83% A and 17% B initially, increased to 30% B in 23 min, 41% B in 29 min, 70% B in 35 min, and returned to 17% B in 44 min at a flow rate of 1.0 mL/min.The column temperature was maintained at 40 • C, and the detection of carotenoids was performed with a photodiode array (PDA) at 450 nm.The total carotenoid content was calculated as the sum of the α-carotene, β-carotene, zeaxanthin, capsanthin, capsorubin, violaxanthin, antheraxanthin, lutein, and β-cryptoxanthin contents.

Vitamin C
Vitamin C was analyzed using the method by Kim et al. [37], with some modifications.Metaphosphoric acid (10%) was added to 0.05 g of pepper powder and filtered through a 0.45 µm filter syringe.Vitamin C was separated by HPLC (Dionex Ultimate 3000; Thermo Scientific, Waltham, MA, USA) in an Inno C18 column (250 mm × 4.6 mm, 5 µm; Innopia, Anyang, Republic of Korea).Solvent A was 0.05 M NH 4 H 2 PO 4 , and solvent B was methanol.The gradient elution conditions were as follows: 0 min, 100% A and 0% B; 5 min, 100% A and 0% B; 8 min, 20% A and 80% B; 13 min, 20% A and 80% B; 15 min, 100% A and 0% B; and 20 min, 100% A and 0% B at a flow rate of 0.8 mL/min.The detection of vitamin C was performed with a PDA at 264 nm.

Capsaicinoids
For the extraction of capsaicinoids, 0.2 g of pepper powder was dissolved in 20 mL of methanol.The mixture was incubated for 1 h at room temperature and 320 rpm.After centrifugation at 2800 rpm at 4 • C for 10 min, the supernatant was concentrated using a rotary evaporator.Then, the samples were resuspended in 2 mL of methanol and filtered using a 0.45 µm filter syringe.The separation of capsaicinoids was performed by HPLC (Dionex Ultimate 3000; Thermo Scientific) on a VDSpher 100 C18-E column (250 × 4.6 mm, 5 µm; VDS Optilab, Berlin, Germany).A 10 µL aliquot was injected into the HPLC system at a 1.0 mL/min flow rate; the mobile phase was 25:75 v/v water and methanol, and the column temperature was maintained at 40 • C. Capsaicinoids were detected with a PDA at 280 nm.The total capsaicinoid content was calculated as the sum of the capsaicin, dihydrocapsaicin, and capsiate contents.

Total Soluble Solids
The content of total soluble solids was measured using a PAL-3 refractometer (ATAGO, Tokyo, Japan) with three replicates and recorded in degree Brix ( • Brix).

ASTA Color Index Measurement
The ASTA value was determined as previously described with minor modifications [38].Pigment extraction from pepper was carried out by adding 100 mL of 100% acetone to 0.1 g of pepper powder and placing it at room temperature for 16 h in the dark.The absorbance of the supernatant was measured using a microplate reader (Infinite 200 PRO; Tecan, Männedorf, Switzerland) at 460 nm.The following formula was used to calculate the ASTA values: ASTA value = Absorbance of acetone extracts × 16.4 × I f /Sample weight (g) I f , the instrument correction factor, is the absorbance of the glass reference standard.The SRM 2030 or 930 glass filter (National Institute of Standards and Technology (NIST), Gaithersburg, MD, USA) [39,40] with absorbance of 0.4-0.6 at 460 nm, as determined by NIST.

Statistical Analysis
Student's t test was conducted to identify significant differences in agro-morphological and phytochemical contents between C. annuum and C. frutescens.Significant differences between mean values were computed using the Student-Newman-Keuls multiple comparison test (α = 0.05).Pearson's correlation analysis, hierarchical clustering on principal components (HCPC) analysis, and PCA were carried out using R software (version 4.3.0;R Core Team, Vienna, Austria) [41].

Morphological Variation in Pepper Germplasm
The mature fruit color and orientation of fruits presented wide phenotypic variation (Figure 1).At the mature stage, red (92.98%) was the predominant color of the fruits, with brown (0.78%) being the least.Regarding fruit orientation, the majority of germplasms showed pendant-oriented fruits (68.23%), while "declining like a pendant" (2.14%) fruits showed the fewest.Table 1 displays the range, mean, and coefficient of variation (CV) values of 10 quantitative agro-morphological features; the pepper germplasm values demonstrated wide variance.Most of the agro-morphological traits had a relatively high CV value, with eight traits having values > 20%.The highest CV was observed for FW (169.22%),followed by FD (78.93%) and FWT (74.36%), while the lowest CV was that for days to maturity (5.35%).Table 1 displays the range, mean, and coefficient of variation (CV) values of 10 quantitative agro-morphological features; the pepper germplasm values demonstrated wide variance.Most of the agro-morphological traits had a relatively high CV value, with eight traits having values > 20%.The highest CV was observed for FW (169.22%),followed by FD (78.93%) and FWT (74.36%), while the lowest CV was that for days to maturity (5.35%).
Except for PH, statistically significant differences in phenotypic traits were observed between the two Capsicum species (Table 1

Phytochemical Variation in Pepper Germplasm
The distributions of ASTA values and carotenoid, β-carotene, vitamin C, capsaicinoid, and total soluble solid contents in the 513 germplasms are presented in Table 2.The ASTA value ranged from 16.00 to 729.31, with a mean of 224.35.Carotenoid and β-carotene contents varied from 52.50 to 3495.98 and 5.97 to 392.74 µg/g, with mean values of 951.18 and 76.90 µg/g, respectively.The vitamin C content ranged from 0.10 to 18.46 mg/g, with a mean value of 4.95 mg/g.Moreover, capsaicinoid content ranged from 0.00 to 1219.90 mg/100 g, with a mean value of 227.81 mg/100 g.The total soluble solid content also ranged from 4.40 to 18.60 • Brix, with a mean value of 9.74 • Brix.

Associations of Fruit Color and Orientation Traits with Phytochemical Contents
The influence of the mature fruit color and orientation of fruit on phytochemicals was also investigated across species (Figure 2 and Table S2) and within species (Figures S1 and S2).The ASTA value and carotenoid content tended to increase with darker fruit color.Specifically, germplasms with brown-or red-colored mature fruits had higher levels of ASTA and carotenoids than those of orange-or yellow-colored mature fruits.This trend was more pronounced in C. annuum, which did not contain the germplasms with brown-colored mature fruit, while in C. frutescens, germplasms with brown-colored mature fruit showed significantly higher ASTA and carotenoid levels than those of others.In contrast, β-carotene content did not show a significant difference among germplasms possessing differentcolored mature fruits both across and within species.The germplasms were divided into two groups based on the orientation of fruits.The germplasms with intermediate-oriented fruits had similar levels of ASTA and carotenoids to those with pendant-oriented fruits, and they had higher levels than the other two types.In the case of C. annuum, germplasms with "declining like a pendant"-oriented fruits had the lowest levels of ASTA and carotenoid, while in C. frutescens, germplasms with pendant-oriented fruits showed higher levels of ASTA and carotenoid than the other two types.Germplasms with intermediate-oriented fruits had higher β-carotene content than the other three types, whereas germplasms with "declining like a pendant"-oriented fruits had lower β-carotene content than the other three types.Similar to the result of comparison according to the mature colors of fruits, no significant difference was shown in β-carotene content among germplasms possessing different fruit orientations within species.Germplasms with yellow-colored mature fruits had higher levels of vitamin C than the other three types, which all had comparable levels of vitamin C.Among the three mature color types of C. annuum, germplasms possessing orange-or yellow-colored mature fruits showed higher levels of vitamin C than those of red-colored mature fruits.In C. frutescens, which had a lower average vitamin C content than C. annuum, germplasms with brown-colored mature fruits showed much higher vitamin C levels than the other three.On the other hand, the content of capsaicinoids was lowest in germplasms with yellow-colored mature fruits and highest in germplasms with orange-colored mature fruits.C. frutescens also showed the lowest capsaicinoid content in germplasms with yellow-colored mature fruits, whereas C. annuum showed no significant difference in capsaicinoid content depending on the color of mature fruit.Regarding the orientation of fruits, germplasms with erect fruits had the lowest level of vitamin C but the highest level of capsaicinoids, with the reverse being true for pendant-oriented fruits.Likewise, in the intraspecific comparison of the vitamin C content of C. frutescens and the capsaicinoid content of C. annuum, contrasting levels of erect type and pendant type were observed.The total soluble solid level did not differ significantly between germplasms with different-colored mature fruits and was slightly lower in germplasms with "declining like a pendant"-oriented fruits.In the comparison within species, germplasms with yellowand "declining like a pendant"-oriented fruits in C. annuum had the lowest level of total soluble solids, whereas, no significant difference was revealed in C. frutescens.

Figure 2. Variations in phytochemicals according to mature fruit color (A) and fruit orientation (B).
Different letters indicate significant differences between groups (p < 0.05).

Principal Component and Clustering Analysis
PCA was performed on the entire dataset of 513 pepper germplasms.Four principal components with eigenvalues > 1 explained 77.13% of the total variance in the germplasms (Table S3).The first principal component (PC1) explained 32.33% of the total variance, with an eigenvalue of 5.17, in agro-morphological and phytochemical variables.The second principal component (PC2) explained 25.61% of the total variance, with an eigenvalue of 4.10.The third (PC3) and fourth (PC4) principal components explained 12.09% and

Principal Component and Clustering Analysis
PCA was performed on the entire dataset of 513 pepper germplasms.Four principal components with eigenvalues > 1 explained 77.13% of the total variance in the germplasms (Table S3).The first principal component (PC1) explained 32.33% of the total variance, with an eigenvalue of 5.17, in agro-morphological and phytochemical variables.The second principal component (PC2) explained 25.61% of the total variance, with an eigenvalue of 4.10.The third (PC3) and fourth (PC4) principal components explained 12.09% and 7.10% of the total variance, respectively.Hence, PC1 and PC2 were used to construct score and loading plots for the analysis of distributions and associations among the agromorphological traits and phytochemical contents of pepper germplasm.Vitamin C (14.89%), FWT (13.23%),FD (10.38%),MSL (10.23%), and capsaicinoids (9.96%) made the largest contributions to the variance along PC1.The ASTA value (17.11%) and carotenoids (16.69%) were the main contributors to the variance along PC2.In Figure 3, the score and loading plots depict the distribution of pepper germplasms according to species; most C. annuum species were on the positive side of PC1, whereas most C. frutescens species were on the negative side.Furthermore, distinct aggregation was observed between the two species.
7.10% of the total variance, respectively.Hence, PC1 and PC2 were used to construct score and loading plots for the analysis of distributions and associations among the agro-morphological traits and phytochemical contents of pepper germplasm.Vitamin C (14.89%), FWT (13.23%),FD (10.38%),MSL (10.23%), and capsaicinoids (9.96%) made the largest contributions to the variance along PC1.The ASTA value (17.11%) and carotenoids (16.69%) were the main contributors to the variance along PC2.In Figure 3, the score and loading plots depict the distribution of pepper germplasms according to species; most C. annuum species were on the positive side of PC1, whereas most C. frutescens species were on the negative side.Furthermore, distinct aggregation was observed between the two species.Based on HCPC analysis, 513 pepper germplasms were divided into three clusters (Figure 4 and Table S4).Based on HCPC analysis, 513 pepper germplasms were divided into three clusters (Figure 4 and Table S4).

Discussion
Characterizing morphological traits and quantifying variability are essential for understanding the genetic diversity of germplasm resources [42].In this study, the agromorphological traits of pepper all showed considerable variability among the germplasms (Table 1).Previous studies have also shown wide variations in morphological traits among Capsicum germplasms [43][44][45].The CV is used to determine the variability in a population.Germplasms with CV values > 20% are considered to possess considerable genetic diversity [46].The agro-morphological traits showed high variability in this study, as stated above, with CV values ranging from 5.35% to 169.22%.Phenotypic diversity among plant germplasms could be due to variations in local environmental factors, such as terrain, soil conditions, weather, sunlight, and rainfall, among others [27].Common garden experiments minimize the impact of environmental factors on morphological traits [47].
In the present study, therefore, it is reasonable to infer that genetic differences between germplasms were the major factors contributing to the morphological diversity among the 513 Capsicum germplasms.
Significant differences were found in germplasms within and among species.Most of the traits differed significantly between the two Capsicum species.Among the six phytochemical parameters, the levels of capsaicinoids varied the most.Previous works have claimed that C. chinense is the most pungent [48,49], while C. baccatum is the least pungent domesticated Capsicum species [50][51][52].In the present study, C. annuum was less pungent than C. frutescens, with an average value of 86.25 mg/100 g.This was because there were more nonpungent C. annuum germplasms than C. frutescens germplasms.
We found that the phytochemical traits varied in relation to mature fruit color and fruit orientation (Figure 2).Most peppers exhibit red color in the mature stage; however, some cultivars' fruits remain orange color in maturity, which is associated with the nonexpression of carotenoid synthetase or mutation of the gene [20,53].The ASTA value, which is strongly correlated with carotenoid content [54], is also related to the red color of pepper fruits [55].Consistent with this, values of ASTA and carotenoids tended to increase with a darker red color of fruits.Germplasms with yellow-colored mature fruits had higher vitamin C levels than the other types.This result was in line with previous work that compared red-colored Brazilian cultivars with yellow-colored ones [56].Although the domestication-related traits of Capsicum have not been fully elucidated, some traits, such as various fruit colors, low pungency, and nondeciduous habit, may have been selected for in pepper [22,57].The orientation of fruits, from erect to pendant, has also been described as a trait selected for during the domestication of pepper [58].Previous genetic studies have identified single recessive genes and QTLs involved in the regulation of fruit orientation; however, the genetic mechanism underlying this trait has not been fully understood [59][60][61].The straight or curved growth of the pedicel determines the direction of the fruit, with the former becoming erect-and the latter becoming pendant-type.It is known to be regulated by a series of cell proliferation, differentiation, and elongation [62].In the comparison between pendant and erect types, we found that the former had higher vitamin C content and lower capsaicinoid content.Significant positive correlations were found between fruit size traits, including weight, wall thickness, diameter, and length, and vitamin C content (Table 3).Hence, the pendant orientation of fruits and associated traits could be selected for to produce peppers with high vitamin C content [22,23].
In this study, PCA was used to reduce 16 quantitative parameters to four principal components with eigenvalues > 1; these components were able to explain 77.13% of the variance in the germplasms (Table S3).C. annuum germplasms displayed greater diversity than C. frutescens germplasms, likely reflecting more intensive human selection, followed by evolutionary and domestication processes, driving the worldwide expansion of C. annuum [63,64].Among the morphological variables, fruit-related traits are the ones that contributed the most to study the diversity of 513 germplasms.The FWT (13.23%) and FD (10.38%) stand out.These results support previous studies showing that fruit-related traits have great discriminatory power [65].Our CA divided the 513 germplasms into three groups (Figure 4).Unlike previous studies, both the PCA and CA of agro-morphological and phytochemical traits grouped the pepper germplasms according to species [66,67].

Conclusions
This study demonstrated the diversity in a large population of pepper germplasms, across two species, in terms of agro-morphological traits and phytochemical contents.Most of the quantitative traits showed significant differences (p < 0.05).The findings suggest that some morphological traits, including the mature fruit color and orientation of fruits, could indicate the phytochemical contents of pepper fruits.In particular, genotypes with yellowcolored and pendant-oriented fruit had higher levels of vitamin C. The interconnectedness of quantitative traits was summarized in a PCA scatter plot, validating the differential performance of pepper germplasms of the two species.The fruit-related traits were those that showed greater relevance in analyzing the diversity of germplasms and correlation among several crucial fruit-related traits and phytochemicals.Overall, six accessions of pepper, IT305411, IT158764, IT330733, IT324816, IT319736, and IT319741, could be good resources, with high value of beneficial phytochemicals in the fruits of these plants.Furthermore, vitamin C content was positively correlated with FD, FW, and FWT.The findings of this study could provide information on agro-morphological traits, associated with high phytochemical contents, that could be usefully facilitated in the selection process of breeding programs.

Supplementary Materials:
The following supporting information can be downloaded at https: //www.mdpi.com/article/10.3390/agronomy13102665/s1: Figure S1: Variations in phytochemicals of C. annuum according to mature fruit color (A) and fruit orientation (B).Different letters indicate significant differences between groups (p < 0.05); Figure S2: Variations in phytochemicals of C. frutescens according to mature fruit color (A) and fruit orientation (B).Different letters indicate significant differences between groups (p < 0.05); Table S1: List of 513 Capsicum germplasms investigated in this study; Table S2: Variations of phytochemicals in 513 pepper germplasms according to mature fruit color and fruit orientation; Table S3: Eigenvalues, individual and cumulative contributions of variables in the first four principal components; Table S4: Average cluster values of agricultural traits and phytochemical contents of 513 accessions of Capsicum spp.

Agronomy 2023 ,Figure 1 .
Figure 1.Frequency distribution of qualitative traits among the 513 accessions of Capsicum.

Figure 1 .
Figure 1.Frequency distribution of qualitative traits among the 513 accessions of Capsicum.

Figure 2 .
Figure 2. Variations in phytochemicals according to mature fruit color (A) and fruit orientation (B).Different letters indicate significant differences between groups (p < 0.05).

Figure 3 .
Figure 3. Principal component analysis biplot for the agro-morphological traits and phytochemical contents of Capsicum species.
Cluster 1, which included 123 germplasms and most of the C. frutescens species, had the highest values for capsaicinoids, MSL, LL, LW, and days to 50% flowering.Accessions C. frutescens IT305411 and C. annuum IT158764 in cluster 1 had the highest value of capsaicinoids.C. annuum species were largely divided into two clusters.The majority of C. annuum species were classified into cluster 2, which consisted of 331 germplasms and had the highest values for ASTA, carotenoids, β-carotene, PH, days to 50% maturity, and FL.In cluster 2, accessions C. annuum IT330733 and IT324816 had the highest carotenoid contents.Cluster 3 contained approximately one-seventh of the C. annuum germplasms and had the highest values for vitamin C, FD, FW, and FWT.Accessions C. annuum IT319736 and IT319741 in cluster 3 had the highest value of vitamin C.

Figure 3 .
Figure 3. Principal component analysis biplot for the agro-morphological traits and phytochemical contents of Capsicum species.
Cluster 1, which included 123 germplasms and most of the C. frutescens species, had the highest values for capsaicinoids, MSL, LL, LW, and days to 50% flowering.Accessions C. frutescens IT305411 and C. annuum IT158764 in cluster 1 had the highest value of capsaicinoids.C. annuum species were largely divided into two clusters.The majority of C. annuum species were classified into cluster 2, which consisted of 331 germplasms and had the highest values for ASTA, carotenoids, β-carotene, PH, days to 50% maturity, and FL.In cluster 2, accessions C. annuum IT330733 and IT324816 had the highest carotenoid contents.Cluster 3 contained approximately one-seventh of the C. annuum germplasms and had the highest values for vitamin C, FD, FW, and FWT.Accessions C. annuum IT319736 and IT319741 in cluster 3 had the highest value of vitamin C.

Figure 4 .
Figure 4. Hierarchical clustering on principal components.The Capsicum accessions within each cluster are shown.

Figure 4 .
Figure 4. Hierarchical clustering on principal components.The Capsicum accessions within each cluster are shown.
).The average PH was similar between C. annuum (138.93 cm) and C. frutescens (137.55 cm), with the latter having a broader range (72.30-388.30cm) compared to the former (45.70-225.00cm).The average MSL of C. frutescens (33.44 cm) was greater than that of C. annuum (22.06 cm), as were the average LL (20.59 vs. 14.28 cm) and LW (10.41 vs. 6.58 cm), respectively.In terms of days to flowering and maturity, C. annuum flowered earlier (82.14 days) than C. frutescens (92.04 days) but matured later (136.10 vs. 133.56days).Moreover, C. annuum displayed a larger average FL and FD (10.06 and 2.44 cm, respectively) than C. frutescens (3.96 and 1.20 cm, respectively).Both the weight and wall thickness of fruits were also higher in C. annuum than C. frutescens, with average values of 26.93 and 3.47 g, and 2.23 and 0.69 mm, respectively.

Table 1 .
Descriptive statistics of agro-morphological traits in 513 germplasm of two Capsicum species.

Table 2 .
Descriptive statistics of phytochemical contents in 513 germplasms of four Capsicum species.

Table 3 .
Pearson's correlation coefficients of agricultural traits and phytochemical contents.