Variation in Fruit Traits and Seed Nutrient Compositions of Wild Camellia oleifera: Implications for Camellia oleifera Domestication

Abstract

Camellia oleifera is a woody oil crop with the highest oil yield and the largest cultivation area in China, and C. oleifera seed oil is a high-quality edible oil recommended by the Food and Agriculture Organization of the United Nations. The objectives of this study were to investigate the variation in fruit yield traits and seed chemical compositions of wild C. oleifera in China and to identify the differences between wild C. oleifera and cultivated varieties. In this study, we collected wild C. oleifera samples from 13 sites covering the main distribution areas of wild C. oleifera to comprehensively evaluate 25 quantitative traits of wild C. oleifera fruit and seed chemical compositions and collected data of 10 quantitative traits from 434 cultivated varieties for a comparative analysis of the differences between wild and cultivars. The results showed that the coefficients of variation of the 25 quantitative traits of wild C. oleifera ranged from 2.605% to 156.641%, with an average of 38.569%. The phenotypic differentiation coefficients ranged from 25.003% to 99.911%, with an average of 77.894%. The Shannon–Wiener index (H’) ranged from 0.195 to 1.681. Based on the results of principal component analysis (PCA) and phenotypic differentiation coefficients, 10 traits differed significantly between wild C. oleifera and cultivated varieties, while the differentiation coefficients (V_ST) for fresh fruit weight, oleic acid, unsaturated fatty acids, stearic acid, and saturated fatty acids were more than 95%, of which fresh fruit weight and oleic acid content were potential domestication traits of C. oleifera. The results of this study can contribute to the efficient excavation and utilization of wild C. oleifera genetic resources for C. oleifera breeding.

1. Introduction

Camellia oleifera (oil camellia) is the predominant woody oil crop in China and one of the four major woody oil crops in the world, together with oil palm (Elaeis guineensis), oil olive (Olea europaea), and coconut (Cocos nucifera) [1]. In 2022, the planting area of C. oleifera in China was about 4.67 million ha, and the C. oleifera seed oil production exceeded 1 million tons. The C. oleifera seed oil is rich in oleic acid, making up over 80% of the fatty acid compositions, known as ‘oriental olive oil’ [2]. In addition, C. oleifera seed oil also contains many functional nutrient concomitants, such as phytosterols, squalene, tocopherols, and saponin [3,4]. The nutrient components of C. oleifera seed oil have good antioxidant and anti-inflammatory activities and can help lower blood cholesterol and lipids for reducing the risks of cardiovascular and cerebrovascular diseases [5,6,7,8]. Camellia oleifera seed oil is therefore one of the healthy and high-quality edible vegetable oils recommended by the Food and Agriculture Organization of the United Nations (FAO) [5].

The first record clearly indicating C. oleifera as an oil crop was in the Ming dynasty, so the cultivation history may be less than 1000 years in China [1]. Cultivated C. oleifera was domesticated from wild C. oleifera probably in the middle reach of the Yangtze River Basin [1]. As the essential genetic resource for C. oleifera breeding, wild C. oleifera is widely distributed in the subtropical evergreen broadleaved forests of the Yangtze River Basin and South China [9]. With high-throughput sequencing-based microsatellite genotyping, rich genetic diversity and clear genetic differentiation have been found among wild C. oleifera populations from different latitudes and longitudes [10]. As a perennial woody oil crop with a short cultivation history, the domestication bottleneck of C. oleifera may be mild compared to annual herbaceous crops [11]. Nevertheless, differentiations may be expected between wild and cultivated C. oleifera leading to so-called ‘domestication traits’ in the latter, especially for fruit and seed traits under strong human selection [12,13,14,15,16]. The enlargement of C. oleifera fruits plays a pivotal role in the enhancement of C. oleifera oil production [5,6]. Nevertheless, there is a lack of systematic study on the variation in the fruit and seed traits of wild C. oleifera and the differentiations in key traits between wild and cultivated C. oleifera.

In this study, representative wild C. oleifera populations were investigated across the main distribution regions of wild C. oleifera. Fruits were collected from wild C. oleifera, and fruit traits and seed nutrient compositions of wild C. oleifera were measured and analyzed. In addition, fruit traits, seed oil contents and fatty acid compositions of 434 cultivated C. oleifera were collected from the literature. The differences in fruit traits and seed nutrient compositions were compared between wild and cultivated C. oleifera to infer the key domestication traits. This study comprehensively and systematically evaluated the variation in fruit traits and seed nutrient compositions of wild C. oleifera germplasm resources in China, providing the support for the selection of wild C. oleifera with valuable nutrient compositions. Moreover, this study can facilitate the understanding of C. oleifera domestication, especially for the formation of key domestication traits.

2. Materials and Methods

2.1. Plant Material Collection

According to the main distribution regions of wild C. oleifera in China [9], 13 representative wild C. oleifera populations within natural subtropical evergreen broadleaved forests are investigated (Supplementary Table S1). The range of samples covers the major habitats of wild C. oleifera [9]. A total of 206 wild C. oleifera sample trees and 927 wild C. oleifera sample fruits are collected (Supplementary Table S1). The wild C. oleifera populations show diverse individual plant types with obvious age structures. In each population, well-grown wild C. oleifera trees are selected and fruit samples are collected from each tree. In this study, the living C. oleifera trees in the investigated natural forests with less human interference are called wild C. oleifera. The judgment criteria of wild C. oleifera forests in this study are as follows: the habitat is a natural forest; the living C. oleifera trees are scattered in patches or sporadically, with no obvious traces of artificial cultivation, such as uniform spacing between rows and rows, continuous distribution, and the grafting of C. oleifera trees; and the C. oleifera populations have an obvious age structure, and there are a large number of young plants that can be naturally regenerated [9]. The main soil types of the various source sites include red soil and brown soil, and the landforms are mainly plains and low hills; the basal diameter of oil tea ranges from 10 to 25 cm, and the age structure is obvious, with sparse branches and leaves and fewer fruits, which are mixed with other forest trees.

Based on the Oil-Tea Camellia Genetic Resource in China, the phenotypic data of C. oleifera cultivars were counted, and the missing data for C. oleifera cultivars were excluded, so a total of 434 cultivated C. oleifera cultivars were collected and collated with quantitative trait data on fruit yield traits and seed chemical composition, from which 10 quantitative trait data such as oil yield and fatty acid content were selected. The catalog of cultivars and related trait data are shown in Supplementary Table S2.

2.2. Data Collection (or Evaluated Traits) for Wild C. oleifera

2.2.1. Fruit Trait Measurement

Fresh fruit weight and fresh seed weight were weighed using an electronic balance with an accuracy of 0.01 g. Fruit height, fruit diameter, and pericarp thickness were measured using calipers with an accuracy of 0.01 mm. The number of seeds per fruit was determined by direct counting (Figure 1). Fresh seed yield and fruit shape index were calculated as follows:

Fresh seed yield = fresh seed weight/fresh fruit weight × 100%.

Fruit shape index = fruit height/fruit diameter × 100.

2.2.2. Determination of Chemical Composition of Fruits

Determination of the Oil Content of the Kernel

The C. oleifera samples were subjected to a series of preparatory steps. First, they were hulled and subsequently crushed using a crusher. The resulting material was then passed through a 60 mesh sieve. Following this, the samples were obtained using the four-part method, as outlined in the national standard GB5491-85 “Grain and Oilseed Inspection Cuttings and Splitting Method”.

For the determination of oil content, in accordance with the national standard GB5009.6-2016 “Determination of oil content in food”, a certain quantity of prepared C. oleifera seed sample powder was accurately weighed. Soxhlet extraction was performed using a petroleum ether solution. Subsequently, the oil content of the seed kernel obtained from each C. oleifera sample was calculated based on the extracted oil [17]. The experiment was performed in triplicate.

Determination of Fatty Acid Composition

The methyl esterification of fatty acids: C. oleifera seed oil (2 mg) was aspirated with a pipette gun, followed by the addition of 1.5 mL of hexane solution. The mixture was vortexed for 30 s. Then, 40 uL methyl acetate solution and 100 μL sodium methanol/methanol solution were added, and the mixture was vortexed for another 30 s. The reaction was allowed to proceed in a water bath at 37 °C for 20 min. Upon completion, the reaction mixture was transferred to a refrigerator set at −20 °C for 10 min. Immediately thereafter, 100 μL oxalic acid–methyl acetate solution was added, and the mixture was centrifuged at 4200 rpm for 5 min. The supernatant was carefully collected and passed through anhydrous sodium sulfate to remove residual moisture. The sample was then dried under a nitrogen stream. Finally, 1 mL of n-hexane solution was added, and the mixture was vortexed for 30 s before passing through a 0.45 μm membrane. The resulting solution was then prepared for measurement.

The measurement conditions were as follows: the gas chromatography column was a CP-7489 capillary column (100 mm × 0.25 mm × 0.2 μm); the carrier gas was N2, and the combustion gases were H2 and air; the inlet temperature was 250 °C; the pressure was 24.52 psi; and the total flow rate was 29. The flow rate in the column was 1.8 mL/min, and the column temperature was 45 °C (4 min), increased at 13 °C/min to 175 °C (27 min), and then decreased at 4 °C/min to 135 °C (35 min). The temperature was increased to 215 °C (35 min), the detector temperature was 250 °C, and the flow rates of hydrogen, nitrogen, and air were 30.0, 30.0, and 300 mL/min, respectively. The relative fatty acid content was determined by area normalization against a fatty acid methyl ester standard [5,17]. The experiment was performed in triplicate.

Determination of the Tocopherol Content

Tocopherol standard curve: We accurately weighed 25.28 mg of α-tocopherol standard using a precision balance. We mixed the weighed α-tocopherol standard thoroughly with hexane solution to make a 10 mL solution. We transfered 1 mL of the prepared solution to a new 25 mL volumetric flask and dilute to 25 mL with hexane solution. Then, we transfered 0.5 mL, 1 mL, 1.5 mL, 2 mL, and 3 mL aliquots of the diluted solution into separate 10 mL volumetric flasks and diluted each to 10 mL with hexane solution. High-performance liquid chromatography (HPLC) was used to analyze each sample. Each sample was run three times in parallel. The standard curve for α-tocopherol was then constructed from these measurements.

Determination of tocopherol content: 0.3 g of C. oleifera seed oil was accurately weighed using a balance. The C. oleifera seed oil was then diluted to 10 mL with n-hexane solution. The diluted solution was then passed through a 0.45 μm filter membrane. The filtered solution was analyzed by high-performance liquid chromatography (HPLC), with each sample measured three times in parallel. The tocopherol content of each C. oleifera sample was calculated from the tocopherol standard curve. The liquid chromatography column used was Elite Hypersil ODS2 (5 μm, 4.6 mm × 150 mm), and the mobile phase consisted of methanol and water in a ratio of 98:2 (v/v). For each injection, 3 μL of sample was injected into the column, and the flow rate was set at 0.8 mL/min. The ultraviolet detector (DAD) was set to a maximum excitation wavelength of 295 nm. The column temperature was maintained at 25 °C and the analysis time was 10 min [5,17]. The experiment was performed in triplicate.

Measurement of Squalene

Squalene standard curve: 5.78 mg of squalene was accurately weighed and the volume was adjusted in a 25 mL volumetric flask with hexane. Then, 1 mL was aspirated and transferred to a 10 mL volumetric flask containing hexane. Aliquots of 0.5 mL, 1.0 mL, 1.5 mL, 2.0 mL, and 2.5 mL were then pipetted, and each volume was made up to 10 mL with hexane. The standard curve was generated by high-performance liquid chromatography. Chromatography was performed on a Hypersil ODS2 column (250 mm × 4.6 mm, 5 μm) with acetonitrile–methanol (60:40, v/v) as the mobile phase. The flow rate was set to 1.0 mL/min, and the sample injection volume was 10 μL.

The determination of squalene content: 0.5 g of C. oleifera seed oil was accurately weighed and dissolved in 5 mL of petroleum ether. The solution was then passed through a 160–200-mesh silica gel column. The sample solution collected after passing through the column was concentrated to dryness under a stream of N2 and then dissolved in 2.5 mL of hexane. Moreover, 1 mL of the resulting solution was passed through a 0.45 μm filter membrane before measurement by high-performance liquid chromatography. The squalene content of the sample was calculated from the squalene standard curve [5,17]. The experiment was performed in triplicate.

Reagents and Equipment

Main reagents: methyl acetate and ethyl acetate (all analytical reagents), Shanghai Chemical Reagent Company (Shanghai, China); distilled water, sodium methanol/methanol solution, petroleum ether, oxalic acid–methyl acetate, and anhydrous sodium sulfate (all analytical reagents), Shanghai Xilong Chemical Company (Shanghai, China); methanol, acetonitrile, and n-hexane (all chromatography pure reagents), Tedia Company (Columbus, OH, USA); and silica gel powder, Qingdao Ocean Chemical Factory (Qingdao, China). The other reagents were analytical reagents.

The main equipment used in this study is as follows: The laboratory is equipped with an Anke TDL-5-A low-speed centrifuge manufactured by Shanghai Anting Scientific Instrument Factory (Shanghai, China); we use a HH-4 digital thermostatic water bath manufactured by Changzhou Guohua Electric Appliances Company, Ltd (Changzhou, China); and a 1100 high-performance liquid chromatograph and 6890 N gas chromatograph manufactured by the Agilent Company (Palo Alto, CA, USA) is used. The AR1140 electronic analytical balance is manufactured by the OHAUS trading company (Parsippany, NJ, USA). The QL-861 vortex machine is manufactured by Qilimbeier Instrument Manufacturing Company of Haimen (Haimen, China).

2.3. Data Collection (or Evaluated Traits) of the Cultivated C. oleifera

To facilitate the comparative analysis with wild C. oleifera, the following 10 quantitative traits were selected from the collected data set of cultivated C. oleifera: fresh fruit weight, fresh seed yield, oil rate of kernel, stearic acid, palmitic acid, saturated fatty acid, oleic acid, linoleic acid, linolenic acid, and unsaturated fatty acid. The catalog of cultivars and related trait data are shown in Supplementary Table S2. The range of origin of the cultivars is a representation of the main C. oleifera production areas. The 19 bioenvironmental climate factors were obtained from the World Climate Database website (http://www.worldclim.org, accessed on 15 January 2024) based on the coordinate information of the sample plots.

2.4. Statistical Analysis

Data statistics were performed using Microsoft Office 2021 software (Home and Student 2021 version); multiple comparisons, analysis of variance, nested ANOVA, and correlation analysis between wild C. oleifera fruit yield traits and seed chemical composition were performed using SPSS 27.0 software. The correlation analysis between environmental climatic factors and wild C. oleifera fruit yield traits with seed chemical composition was carried out using SPSS 27.0 software. Ten traits of wild and cultivated C. oleifera were resampled to estimate their means (30 at a time, with 10,000 replications). The 99.9% confidence interval (99.9% CI) of the estimate was inferred by resampling means positions (10,000 bootstrap samples). Correction for significance was performed using the Bonferroni method [18]. Principal component analysis and cluster analysis were performed on 444 germplasm resources using Origin 2021 software (Version 2021b). TOPSIS was achieved by SPSSPRO (https://www.spsspro.com, accessed on 22 January 2024).

V_ST is the coefficient of phenotypic differentiation, which indicates the percentage of interpopulation variation to total genetic variation, V_ST(%) = [δ²_t/S/(δ²_t/S + δ²_S)] × 100, where δ²_t/S is the between-population variance component and δ²_S is the within-population variance component.

The Shannon–Wiener index is a quantitative expression describing the degree of variability in trait diversity, and the formula is as follows: H′ = −$\sum _{i=1}^{n}PilnPi$, where H′ is the diversity index, and Pi is the effective percentage of the distribution frequency within the material at level i of a trait.

The degree of trait dispersion is expressed by the coefficient of variation (CV) of trait characteristics, CV(%)= s/$\overline{\mathrm{x}}$ × 100, where $\overline{\mathrm{x}}$ is the trait mean, and s is the standard deviation.

3. Results

3.1. Comprehensive Evaluation of Yield Traits and Seed Chemical Composition of Wild C. oleifera Fruits from Different Seed Sources

The results of the study showed (Supplementary Table S3) that there was a wide variation in yield characteristics and seed chemical composition of wild C. oleifera fruits collected from 13 wild C. oleifera sample plots. The fresh fruit weight ranged from 0.939 to 11.477 g, the peel thickness ranged from 0.128 to 0.293 cm, the number of seeds per fruit ranged from 1.697 to 3.836, the fresh seed weight ranged from 0.530 to 4.453, the fresh seed yield ranged from 25.851 to 50.276%, the α-tocopherol content ranged from 0.057 to 0.286 mg/g, squalene content ranged from 0.035 to 0.255 mg/g, the oil rate of kernel ranged from 32.860 to 55.725%, palmitic acid content ranged from 7.784 to 10.782%, stearic acid content ranged from 1.625 to 3.097%, saturated fatty acid content ranged from 10.524 to 13.466%, palmitoleic acid content ranged from 0.067 to 0.173%, oleic acid content ranged from 71.156 to 80.164%, monounsaturated fatty acid content ranged from 73.324 to 81.660%, linoleic acid content ranged from 4.531 to 10.829%, linolenic acid content ranged from 0.264 to 0.552%, polyunsaturated fatty acid content ranged from 4.990 to 11.277%, and unsaturated fatty acid content ranged from 83.949 to 89.746% (Supplementary Table S3).

The coefficients of variation of the 13 seed sources of wild C. oleifera ranged from 9.592 to 27.374%, with a mean of 16.977. The coefficients of variation among seed sources for the 25 quantitative traits ranged from 2.605 to 156.641%, with a mean of 38.569%, with higher variation (CV value > 50%) for nervonic acid, myristic acid, margaric acid, squalene, α-tocopherol content, fresh seed weight, and fresh fruit weight (Supplemental Table S4).

The phenotypic differentiation coefficients of 25 quantitative traits for the fruit yield traits and seed chemical composition of wild C. oleifera ranged from 25.003 to 99.911%, with a mean of 77.894% (Supplementary Table S5). The phenotypic differentiation coefficient for fruit size-related traits (86.602%) was higher than that for seed chemotaxonomy (73.796%). In addition, the lowest phenotypic differentiation coefficient for saturated fatty acid content (25.003%) was found in wild C. oleifera from different seed sources, while the higher phenotypic differentiation coefficient for unsaturated fatty acid content (87.185%) was found in wild C. oleifera (Supplementary Table S5). In addition, phenotypic differentiation coefficients were lower for saturated fatty acid content and higher for unsaturated fatty acid content in different seed sources of wild C. oleifera (Supplementary Table S5). The 25 quantitative traits H’ varied in the range of 0.195 to 1.681, indicating that different seed sources of wild C. oleifera exhibited a high level of phenotypic diversity (Supplementary Table S3).

3.2. Correlations between 25 Quantitative Traits of Wild C. oleifera and Meteorological Factors

Correlation analyses using Pearson’s correlation coefficients were performed on 13 wild C. oleifera populations, and complex relationships among 25 quantitative traits were estimated (Supplemental Table S6). Significant positive correlations were found between fresh fruit weight, fruit height, fruit diameter, and fresh seed weight, with coefficients ranging from 0.872 to 0.963. α-Tocopherol showed a significant negative correlation with saturated fatty acid (r = −0.616) and cis-11-vaccenic acid (r = −0.759) and a significant positive correlation with the oil rate of kernels (r = 0.639), linoleic acid (r = 0.558), and unsaturated fatty acid (r = 0.849). Palmitic acid showed a significant positive correlation with saturated fatty acid content (r = 0.907) and a significant negative correlation with oleic acid (r = −0.596) and unsaturated fatty acid content (r = −0.687). Stearic acid showed a significant negative correlation with linoleic acid content (r = −0.612). Oleic acid showed a significant negative correlation with palmitic acid (r = −0.596) and palmitoleic acid (r = −0.603) content. Unsaturated fatty acids showed a significant negative correlation (r = −0.756) with saturated fatty acids content (Supplementary Table S6).

Based on the correlation between environmental variables (correlation coefficient > 0.800), only six environmental climatic factors were selected, namely, annual mean temperature (Bio1), mean diurnal range (mean of monthly (max temp–min temp)) (Bio2), temperature annual range (Bio7), annual precipitation (Bio12), precipitation seasonality (Bio15), and the precipitation of the warmest quarter (Bio18) (Supplementary Table S7). The results of the correlation between environmental climatic factors and the chemical composition of wild C. oleifera seeds showed that Bio1 and stearic acid content showed a significant negative correlation (r = −0.610), Bio2 showed a significant positive correlation with palmitic acid (r = 0.559) and saturated fatty acids (r = 0.570), and Bio7 and squalene content (r = −0.615) showed significant negative correlation. Bio12 showed a significant positive correlation with the number of seeds per fruit (r = 0.585), Bio15 showed a significant positive correlation with neuronic acid content (r = 0.578), and Bio18 showed a significant positive correlation with squalene content (r = 0.745) (Supplementary Table S8). The α-tocopherol content of wild C. oleifera showed a significant decreasing trend with increasing latitude (p < 0.05), with a linear regression equation of y = −0.01559x + 0.58673 (R² = 0.25) (Supplementary Figure S1).

3.3. Trait Characteristics of 434 Cultivated C. oleifera Varieties

The statistical analysis of 434 C. oleifera cultivars revealed that the fresh fruit weight was 1.390 g~83.210 g, fresh seed yield was 6.180~79.380%, the oil rate of kernels was 11.800~70.630%, stearic acid content was 0.300~6.040%, palmitic acid content ranged from 0.300% to 12.020%, saturated fatty acid content ranged from 2.800% to 15.130%, oleic acid content ranged from 70.100% to 87.200%, linoleic acid content ranged from 0.480% to 17.200%, linolenic acid content ranged from 0.000% to 1.400%, and unsaturated fatty acid content ranged from 81.980% to 91.400% (Supplementary Table S9). The CV values of the 10 quantitative traits of cultivated C. oleifera ranged from 1.307% to 49.423%, with the largest variation in linolenic acid content and the smallest variation in unsaturated fatty acid content. The H’ values of 10 quantitative traits ranged from 0.267 to 1.626, indicating that cultivated C. oleifera varieties are also characterized by rich diversity (Supplementary Table S9).

The oil content, fresh fruit weight, and saturated fatty acid content of cultivated C. oleifera showed a significant decreasing trend with increasing latitude (p < 0.01), and the linear regression equations were y = −0.55076x + 58.92634 (R² = 0.05337), y = −1.55853x + 67.56822 (R² = 0.15494), and y = −0.07872x + 12.60323 (R² = 0.03587). The unsaturated fatty acid content showed a significant increasing trend with increasing latitude (p < 0.01), and the linear regression equation was y = 0.10644x + 85.8808 (R² = 0.05979) (Figure 2).

3.4. Principal Component Analysis

In this study, PCA analysis was performed on wild C. oleifera and C. oleifera cultivars. The comparative analyses of fruit traits (fresh fruit weight and fresh seed yield) revealed a clear differentiation between wild C. oleifera and the cultivars in terms of fresh fruit weight (Figure 3A). The principal components of seed chemical composition revealed that the dimensions implied by the eight quantitative traits could be simplified into two significant components, with a cumulative contribution of 91.914% (Figure 3C; Supplementary Table S10). The first factor was kernel oil rate with a contribution of 70.088% and can be called kernel oil rate factor. The second factor was oleic acid content with a contribution of 21.827% and can be referred to as the oleic acid factor. The differentiation between wild C. oleifera and the cultivars on PC2 (oleic acid factor) was more pronounced (Figure 3B). In this study, the phenotypic differentiation coefficients (V_ST) between wild C. oleifera and the cultivars were calculated. The results showed that the phenotypic differentiation coefficients of fresh fruit weight, stearic acid, saturated fatty acid, oleic acidm and unsaturated fatty acid content were all greater than 95%, and the degree of differentiation between wild C. oleifera and the cultivars was high (

Table 1. Comparison of nested ANOVA and phenotypic differentiation coefficients between wild and cultivated C. oleifera ⁽¹⁾.

Fruit Characters and Nutrients	Among Provenances			Within Provenances			Random Error		Phenotypic Differentiation Coefficient/%
	Mean Square	F Value	Component/%	Mean Square	F Value	Component/%	Mean Square	Component/%
Fresh fruit weight	36604.016	475.468 **	95.637	1592.856	20.69 **	4.162	76.985	0.201	95.830
Fresh seed yield	1754.040	19.957 **	82.193	292.123	3.324	13.689	87.893	4.119	85.723
Oil rate of kernel	214.558	5.062 *	45.778	211.747	4.996 *	45.179	42.384	9.043	50.330
Palmitic acid (C16:0)	5.015	14.588 **	84.272	0.592	1.723	9.948	0.344	5.781	89.442
Stearic acid (C18:0)	18.061	14.402 **	91.383	0.449	0.358	2.272	1.254	6.345	97.574
Saturated fatty acid (SFA)	63.088	49.85 **	94.978	2.070	1.636	3.116	1.266	1.906	96.823
Oleic acid (C18:1n-9)	853.657	106.001 **	98.906	1.386	0.172	0.161	8.053	0.933	99.838
Linoleic acid (C18:2n-6)	27.434	4.628 *	76.644	2.432	0.410	6.794	5.928	16.561	91.857
Linolenic acid (C18:3n-3)	0.336	16.782 **	66.142	0.152	7.597 **	29.921	0.020	3.937	68.852
Unsaturated fatty acid (UFA)	185.992	111.317 **	99.110	0.000	0.000	0.000	1.671	0.890	100.000
Mean	3972.620	81.806	83.504	210.381	4.091	11.524	22.580	4.972	87.627

⁽¹⁾ “*” indicted p < 0.05, “**”indicted p < 0.01.

). Therefore, fresh fruit weight, oleic acid content, and other indicators can be used as important indicators to distinguish wild C. oleifera from the cultivars.

3.5. Comparative Analysis of Fruit Yield Traits and Seed Chemical Composition between Wild C. oleifera and Cultivars

The comparative analysis of fruit yield traits and seed chemical composition between wild C. oleifera and the cultivars in this study showed that wild C. oleifera had significantly lower fresh fruit weight, fresh seed yield, oil rate of kernel, oleic acid content, and unsaturated fatty acid content traits than the cultivars (p < 0.001) (Figure 4; Supplemental Table S11). The stearic acid content, palmitic acid content, linoleic acid content, linolenic acid content, and saturated fatty acid content of wild C. oleifera were significantly higher than those of the cultivars (p < 0.001) (Figure 4; Supplementary Table S11).

3.6. Cluster Analysis

In this study, the relationships between 13 wild C. oleifera populations and 434 cultivated C. oleifera varieties were analyzed by hierarchical cluster analysis. The results showed that all the germplasm resources could be classified into six different groups. The 13 populations of wild C. oleifera showed a clustering trend, of which 11 populations were clustered in Group I, and the other 2 populations were clustered in Group III (Figure 5; Supplementary Table S12).

Group I contained 124 germplasm resources, accounting for 27.740% of the total accessions. This group was characterized by the smallest fresh fruit weight (Group I: 15.175 g; Wild: 5.952 g), the lowest oil rate of kernel (Group I: 38.777%; Wild: 41.443%) and the highest linolenic acid content (Group I: 0.331%; Wild: 0.430%) (Figure 5;

Table 2. Phenotypic characteristics of the cluster groups (X ± SE) ⁽¹⁾.

Groups	Fresh Fruit Weight (g)	Fresh Seed Yield (%)	Oil Rate of Kernel (%)	Fatty Acid Composition of C. oleifera Oils(%)
				Stearic Acid (C18:0)	Palmitic Acid (C16:0)	Saturated Fatty Acid (SFA)	Oleic Acid (C18:1n-9)	Linoleic Acid (C18:2n-6)	Linolenic Acid (C18:3n-3)	Unsaturated Fatty Acid (UFA)
I	15.175 ± 0.535 d	43.781 ± 0.357 b	38.777 ± 0.469 c	1.892 ± 0.044 bc	8.568 ± 0.085 b	10.483 ± 0.085 b	79.710 ± 0.266 c	8.516 ± 0.209 ab	0.331 ± 0.016	88.714 ± 0.114 a
II	15.979 ± 0.604 d	55.272 ± 0.947 a	42.816 ± 0.719 b	1.811 ± 0.093 c	8.710 ± 0.163 b	10.521 ± 0.172 b	80.341 ± 0.450 bc	7.939 ± 0.459 bc	0.309 ± 0.021	88.589 ± 0.249 ab
III	15.708 ± 0.532 d	35.030 ± 0.680 d	43.556 ± 0.628 b	2.058 ± 0.060 ab	8.460 ± 0.106 bc	10.523 ± 0.112 b	81.162 ± 0.243 ab	7.300 ± 0.205 cd	0.282 ± 0.011	88.782 ± 0.136 bc
IV	50.387 ± 3.117 a	28.946 ± 3.372 e	38.781 ± 1.853 c	2.158 ± 0.263 a	9.143 ± 0.291 a	11.301 ± 0.379 a	78.487 ± 1.038 d	9.338 ± 0.975 a	0.328 ± 0.029	88.153 ± 0.374 a
V	25.896 ± 0.460 c	44.541 ± 0.544 b	44.385 ± 0.513 b	1.943 ± 0.048 abc	8.059 ± 0.130 cd	10.002 ± 0.117 c	81.897 ± 0.262 a	7.062 ± 0.212 cd	0.281 ± 0.015	89.240 ± 0.094 c
VI	34.531 ± 0.747 b	41.179 ± 0.641 c	49.165 ± 0.623 a	2.173 ± 0.079 a	7.893 ± 0.114 d	10.065 ± 0.131 c	82.161 ± 0.325 a	6.809 ± 0.257 d	0.285 ± 0.016	89.255 ± 0.131 c

⁽¹⁾ Different lowercases in the same column indicate the significant difference at 0.05 level.

; Supplementary Table S11). Group II contained 46 germplasm resources, representing 10.291% of the total accessions, with the highest fresh seed yield (55.272%) and the lowest stearic acid content (1.811%). Group III contained 97 resources, accounting for 21.700% of the total accessions, with the lowest linolenic acid content (0.282%). Group IV contained 18 germplasm resources, representing 4.027% of the total accessions, and this group was characterized by the highest fresh fruit weight (50.387 g), palmitic acid (9.143%), linoleic acid (9.338%), and saturated fatty acid content (11.301%) and the lowest fresh seed yield (28.946%), oleic acid (78.487%), and unsaturated fatty acid content (88.153%). Group V contained 102 germplasm resources, representing 22.819% of the total accessions. This group was characterized by the lowest content of saturated fatty acids (10.002%). Group VI contained 60 germplasm resources, representing 13.423% of the total accessions, and was characterized by the highest oil rate of kernel (49.165%), stearic acid (2.173%), oleic acid (82.161%), and unsaturated fatty acid content (89.255%) and the lowest palmitic acid (7.893%) and linoleic acid content (6.809%) (Figure 5; Table 2).

3.7. TOPSIS Comprehensive Evaluation

By using the TOPSIS method, we conducted a comprehensive score rank of wild and cultivated C. oleifera (Supplementary Table S13). LS, LFS, and JGS were the top three wild C. oleifera. Yunyoucha 9, Xianning 15youzhu, yunyoucha 14, Minzayou 22, Yangxintongcha 208, Shihe Youzhu No.2, Yongkangyouzhu 7, Wanning 2, Minzayou 25, Nanzheng 1 were the top 10 C. oleifera varieties (

Table 3. Composite scores and groupings of the top 13 ranked wild C. oleifera and top 20 ranked cultivated C. oleifera.

Cultivated				Wild
Variety	Cluster Group	Comprehensive Score	Rank	Population	Cluster Group	Comprehensive Score	Rank
Yunyoucha 9	VI	0.690	1	LS	III	0.440	260
Xianning 15youzhu	I	0.681	2	LFS	I	0.428	285
Yunyoucha 14	VI	0.644	3	JGS	I	0.398	335
Minzayou 22	VI	0.636	4	NL	I	0.354	387
Yangxintongcha 208	III	0.628	5	DLC	I	0.335	401
Shihe Youzhu No.2	I	0.623	6	TK	I	0.328	407
Yongkangyouzhu 7	VI	0.616	7	HK	I	0.327	409
Wanning 2	V	0.607	8	HLT	I	0.322	415
Minzayou 25	VI	0.606	9	DCP	I	0.321	416
Nanzheng 1	VI	0.604	10	EM	III	0.317	421
Yangxinyanggang 48youzhu	I	0.604	11	BR	I	0.312	428
Minlong No.31	II	0.602	12	QL	I	0.309	430
Xianning 11danzhu	III	0.601	13	YBS	I	0.290	438
Wanhui 2	I	0.600	14
Minzayou 24	VI	0.599	15
Minzayou 1	V	0.598	16
Yuyoucha 7	VI	0.596	17
Wanqi 2	V	0.595	18
Minzayou 9	V	0.591	19
Wanqi 3	V	0.589	20

).

In order to better illustrate the changes in the traits of cultivated C. oleifera, we showed the trends of the five traits with the largest differentiation coefficients mentioned above. From the results, we can see that the fresh fruit weight, oleic acid content, and unsaturated fatty acid content of the top 20 germplasm resources of cultivated C. oleifera were significantly higher than those of wild C. oleifera, and stearic acid and saturated fatty acid content were significantly lower than those of wild C. oleifera (p < 0.001) (Figure 6).

4. Discussion

Genetic diversity is a fundamental component of biological diversity. Phenotypic diversity is an important area of genetic diversity research [19]. The coefficient of variation and the coefficient of phenotypic differentiation can reflect the degree of difference between different phenotypic traits [20]. By analyzing 25 phenotypic traits of 13 wild C. oleifera populations, it was found that wild C. oleifera varied significantly among different populations and that inter-population variation was greater than intra-population variation. Similar results were found in the genetic structure analysis of wild C. oleifera by Cui et al. [10]. This is mainly due to the fact that wild C. oleifera is widely distributed in the Wuyi Mountain Range, Luoxiao Mountain Range, Nanling Mountain Range of Guangdong Province, Huangshan Mountain Range, and other low mountainous areas, where the habitat differences between individual populations are large and gene flow is somewhat hindered [10]. Bioactive compounds such as tocopherols and squalene found in C. oleifera have antioxidant, anti-inflammatory, and other health benefits. It has been shown that tocopherols and squalene can be used to identify potential markers for C. oleifera oils [21]. In this study, we found that wild C. oleifera with high Shannon–Wiener index scores for oil content (1.681), tocopherols (1.548), and squalene (1.201) contained rich variants, and these rich variants may be important fingerprints for the identification and evaluation of C. oleifera [5,21].

In this study, 434 C. oleifera cultivars were selected, and there was a wide variation in traits among the cultivars (H’: 0.267 to 1.626; CV: 1.307% to 49.423%). The study suggests that plant domestication by artificial selection is diverse and driven by cultural traits, crop characteristics, and geo-environmental factors [22]. For example, there are more than 2,000 cultivated olive varieties in the Mediterranean basin, with a wide variety of fruit morphology, stone size, and shape [23]. The samples selected for this study were collected, as far as possible, from selected varieties within the main habitats of C. oleifera, geographically covering a wide area of the middle and lower reaches of the Yangtze River, with obvious differences in habitat and culture between regions. Therefore, the current major C. oleifera varieties in China have a high diversity of traits rather than a single variety. Through cluster analysis, we found that Group I was characterized by smaller fruits (15.175 g) and a higher linolenic acid content (0.331%), Group II varieties had a higher fresh seed yield (55.272%), Group IV varieties had a larger fresh fruit weight (50.387 g), and Group VI varieties had a higher oil content (49.165%) and oleic acid content (82.161%). Further correlation analysis with latitude revealed that fresh the fruit weight, oil content, and saturated fatty acid content of the cultivars were significantly negatively correlated with latitude, and unsaturated fatty acid content was significantly positively correlated with latitude (Figure 2). The ability to regulate membrane lipid fluidity by altering unsaturated fatty acid levels is an important characteristic of plants domesticated by environmental stress [24]. In plants, an increase in fatty acid unsaturation helps to increase the fluidity of cell membranes, prevent stress-induced membrane hardening and membrane damage, and maintain the structural and functional integrity of cell membranes, thus improving the plant’s resistance to environmental stress [25,26,27]. Xie et al. [28] found significant gene enrichment in the fatty acid elongation pathway in C. oleifera during cold domestication. Thus, the increase in unsaturated fatty acids with latitude in the cultivars may be related to the evolution of their adaptations to cold stress. In summary, the domestication of C. oleifera is driven by a combination of artificial directional selection and environmental factors [12,23].

The domestication of wild plants to produce high-yielding and high-quality crops is an important event in the advancement of human civilization [16,29]. Strong human selection pressure on crop plants can rapidly alter phenotypic traits in crops [30]. Cultivated plants typically show changes in traits adapted to the cultivated environment, such as the greater morphological integrity of individual plants, increased yields, altered nutrient content, and reduced defenses, compared to wild species [31,32]. For example, cultivated olives have heavier fruits, larger leaves, and a significantly higher oil content than wild olives [33,34]. The results of the present study also showed that among the 10 selected traits, fresh fruit weight, fresh seed yield, seed kernel oil content, oleic acid, and unsaturated fatty acid content were significantly higher, while stearic acid, palmitic acid, linoleic acid, linolenic acid, and saturated fatty acid content were significantly lower in the cultivars compared to wild C. oleifera (Figure 4; Supplementary Table S11). This indicates that after nearly a thousand years of artificial selection and cultivation, the cultivars are clearly distinguishable from wild C. oleifera.

By comparing the differentiation coefficients of wild and cultivated C. oleifera traits, we found that the differentiation coefficients of five traits, including fresh fruit weight, oleic acid, unsaturated fatty acid, stearic acid, and saturated fatty acid, were greater than 95% (Table 1), which is a very high degree of differentiation and may be a potential domestication trait for C. oleifera. Camellia oleifera varieties are rich in trait variation, and the direction of selection and breeding of cultivated C. oleifera traits, as well as the degree of domestication, varies in different regions and at different times, resulting in the formation of a rich diversity of cultivated C. oleifera varieties, which can be classified into six major groups according to the degree of trait domestication (Figure 5). In order to further verify that the above five traits are the main indicators of domestication, our study used the TOPSIS method to rank the C. oleifera cultivars in terms of their comprehensive scores and selected the top 20 cultivars (mainly clustered in the V and VI branches) for a comparison with wild C. oleifera, which showed a more significant trend in the differentiation of fresh fruit weight, oleic acid, unsaturated fatty acid, stearic acid, and saturated fatty acid (Figure 6). Studies have shown that heterogamous pollinated perennials are susceptible to domestication bottlenecks due to factors such as generation overlap, generation reduction, and hybridization. These bottlenecks manifest themselves in the form of domestication that is not readily achieved or reduced trait differentiation, especially for some composite traits such as seed yield and oil content [35]. As a self-incompatible perennial flowering plant, the cultivars of pear (Pyrus) are mainly propagated by grafting. This has resulted in a low number of sexual generations in the history of pear domestication, which may also have led to insufficient selection pressure and phenotypic differentiation during the pear domestication process [36]. Similarly, C. oleifera is also a self-incompatible perennial woody oilseed plant, and most cultivars are selected from wild C. oleifera plants and then propagated by grafting. Such asexual lines may not undergo further selection and breeding in the later stages of propagation, which may result in the under-domestication of some complex traits of C. oleifera [1]. Therefore, composite traits such as fresh seed yield and oil content may still require longer-term continuous selection over multiple generations. Furthermore, through results such as PCA analysis, we found that cultivated C. oleifera differed significantly from wild C. oleifera only in terms of fresh fruit weight and oleic acid content (Figure 3). In crops where the fruit is an economically important organ, fruit size is a key component of crop yield and has been a typical trait for crop domestication [34,37]. Th enhancement of fruit size significantly increases C. oleifera oil production [20,38,39]. Therefore, according to the results of this study, fresh fruit weight is an important trait in the domestication process of C. oleifera.

Another important domestication trait of C. oleifera is the increase in oleic acid content in the seed. First, according to previous studies, crop seeds with high oleic acid content can effectively improve the oxidative stability and significantly increase the shelf life of seeds. Soybean seeds with high oleic acid content can be better preserved and germinated and can be easily selected by directional selection [40,41]. Similarly, wild C. oleifera seeds with high oleic acid content also have better antioxidant activity, which not only improves seed germination but also improves the shelf life of the pressed tea oil [42,43]. In addition, the modern breeding standard for C. oleifera (GBT28991-2020) also specifies the oleic acid content (≥78%) of cultivars. This has led to a further increase in the oleic acid content of cultivated C. oleifera. In the fatty acid synthesis pathway, since the domestication of C. oleifera is directed to increase oleic acid synthesis, this results in a corresponding decrease in the synthesis of stearic, linoleic, and linolenic acids. Studies have shown that in the fatty acid synthesis pathway of C. oleifera, the SAD gene (stearoyl-ACP desaturase) primarily catalyzes the desaturation of stearic acid to produce oleic acid [44]. The gene FAD2 (fatty acid desaturase 2) mainly regulates the desaturation of oleic acid to linoleic acid [45]. The high expression of the SAD gene and low expression of the FAD2 gene are regulatory mechanisms for oil accumulation in C. oleifera seeds [46]. The genes FAD3, FAD7, and FAD8 are key regulators of the conversion of linoleic acid to linolenic acid, and the reduced expression of these genes at later stages of seed development also contributes to the accumulation of oleic acid [38,45,46]. With the increasing demand for high-quality edible oil and the rapid development of molecular breeding, the results of our study can provide better theoretical support for the selection and breeding of C. oleifera varieties in the future.

5. Conclusions

In this study, 25 quantitative traits of wild C. oleifera fruit yield characteristics and seed chemical composition were determined and comprehensively evaluated, and 10 major traits were selected for comparative analyses with cultivated varieties. The results of this study showed that wild C. oleifera phenotypic traits contain rich variation, and fresh fruit weight and oleic acid content can be potential domestication traits for cultivated C. oleifera. The results of this study can help the effective excavation and utilization of wild C. oleifera genetic resources and enable positive explorations into C. oleifera breeding.