Fruit morphological diversity
The use of morphological characteristics as phenotypic indicators is important for estimating phenotypic diversity and selecting clonal varieties [38, 39]. The data for fruit characters (fresh weight, FVD, FTD, FSI), kernel characters (grain dry weight, GVD, GTD, ST, GVD/ST), and yield (kernel dry weight, kernel shell dry weight) are summarized in Table 1. The values of morphological characters differed significantly among the 22 C. retusus accessions (P < 0.05). The mean value for fruit fresh biomass was 76.432 ± 20.75 g, ranging from 49.240 ± 0.94 g (B-1) to 121.303 ± 7.51 g (T-8) with a coefficient of variation (CV) of 27.15%. The mean value of grain dry biomass was 24.370 ± 6.52 g, ranging from 10.085 ± 0.94 g (WS-2) to 36.067±0.98 g (T-8) with a CV of 26.75%. The mean value of kernel dry biomass was 12.122 ± 3.21 g, ranging from 7.751 ± 0.54 g (WS-2) to 16.665 ± 1.80 g (T-8) with a CV of 26.52%. The mean values of FVD, FTD, GVD, and GTD were 12.462 ± 1.39 mm, 9.742 ± 1.04 mm, 10.732 ± 1.51 mm, and 6.831 ± 0.65 mm, respectively. C. retusus kernels contain oil and important metabolites, and they are the main organ used by the processing industry. Therefore, Kp is a major economic indicator. The average value for Kp was 50.16%, ranging from 39.90% to 62.59% (Fig. 1). The maximum Kp was in T-2 (62.59%), and this value was significantly higher than those in other accessions (P < 0.05).
Oil contents and fatty acid composition
We analyzed the oil content in the kernels of 22 C. retusus accessions (Table 2). The mean oil content in the kernels of 22 C. retusus accessions was 36.55% ± 5.78%, with a range from 28.3% ± 0.35% (S-3) to 47.5% ± 0.67% (WS-5) and a CV of 15.81% (Table 2). The accession WS-5 had the highest oil content (47.5% ± 0.67%) and S-3 had the lowest (28.3% ± 0.35%), which was not significantly different from those of B-1 (28.9% ± 0.58%), WA-1 (29.0% ± 0.45%), and S-2 (29.2% ± 0.38%) (Table 2). These differences in oil content may be due to variations among accessions and differences in their genetic background .
Fatty acid composition is an important index of oil quality. Analyses of the fatty acid composition of vegetable oil from 22 C. retusus kernels revealed seven main fatty acid constituents (Table 2). The most abundant fatty acid was oleic acid (C18:1), accounting for 49.53% ± 0.06% to 59.67% ± 0.15% of total fatty acids (average, 52.33% ± 3.22%); followed by linoleic acid (C18:2) (range, 27.00% ± 0.80% to 34.80% ± 0.51%; average, 31.31% ± 3.28%); palmitic acid (C16:0) (range, 3.41% ± 0.02% to 5.09% ± 0.02%; average, 4.30% ± 0.49%); stearic acid (C18:0) (range, 1.45% ± 0.01% to 2.88% ± 0.01%; average, 2.12% ± 0.39%); linolenic acid (C18:3) (range, 0.34% ± 0.03% to 0.51% ± 0.05%; average 0.42% ± 0.05%); arachidonic acid (C20:1) (range, 0.26% ± 0.03% to 0.40% ± 0.04%; average, 0.36% ± 0.08%); and arachidic acid (C20:0) (range, 0.12% ± 0.03% to 0.21% ± 0.04%; average, 0.15% ± 0.03%). Notably, arachidonic acid (C20:0) was only detected in the WA, WS, and S accessions. Compared with saturated fatty acids, unsaturated fatty acids are better for human health. Therefore, the content of unsaturated fatty acids is often used as a quality standard for vegetable oil . According to the number of double bonds in the molecule, fatty acids can be categorized into three groups: saturated fatty acids (SFA), monounsaturated fatty acids (MUFA), and polyunsaturated fatty acids (PUFA) The mean value for SFA was 6.49% ± 0.78%, ranging from 5.13% ± 0.06% (S-6) to 7.64% ± 0.20% (B-1) with a CV of 12.02%. The mean value for MUFA was 52.33% ± 3.23%, ranging from 49.87% ± 0.11% (S-3) to 60.00% ± 0.18% (Z-5) with a CV of 6.17%. The mean value for PUFA was 31.88% ± 3.26%, ranging from 27.39% ± 0.83% (S-6) to 35.14±0.53% (WS-3) with a CV of 10.22% (Table 2). Thus, the main component of vegetable oil from C. retusus kernels was unsaturated fatty acids, accounting for 80.55% ± 0.43% to 91.84% ± 0.29%. These findings indicate that C. retusus has potential as a high-quality oil crop.
Individual fatty acids have their own specific properties, and they can alter metabolism, gene expression, responsiveness to hormones, and patterns of secondary metabolite production . Unsaturated fatty acids play an important role in human body. They can reduce cholesterol, regulate blood lipids, and enhance immunity . The unsaturated fatty acids content is one of the important indexes for woody oil crops. In fact, the main goal for peanut breeding is to produce varieties with high levels of oleic acid . In this study, the average proportion of unsaturated fatty acids in oil from kernels of C. retusus was 88.51%, which is equivalent to the content of unsaturated fatty acids in oils from common woody oil crops such as olive (89.72%), Camellia oleifera (83.48%), and Acer truncatum (91.71%) [42-44]. Our analyses indicate that the main unsaturated fatty acid in C. retusus kernels is oleic acid (55.17% of total fatty acids). Thus, C. retusus is a woody oil crop with a high oleic acid content. Oleic acid is the most important unsaturated fatty acid. It is a monounsaturated fatty acid that can reduce cholesterol levels in serum, thereby ameliorating or preventing cardiovascular and cerebrovascular diseases [45, 46]. Oleic acid can reduce the concentrations of total cholesterol and low-density cholesterol (harmful cholesterol) in human blood to a certain extent, while maintaining the concentration of high-density cholesterol (beneficial cholesterol) [10, 11]. In 2018, the Food and Drug Administration (FDA) issued food health guidelines allowing for edible oil with an oleic acid content of > 70% to state, “20 g per day can reduce the risk of cardiovascular disease” on the product label . In this study, the oleic acid contents in kernels were significantly higher (P < 0.05) in WS-2 (59.58% ± 0.08%), WS-3 (59.40% ± 0.10%), and WS-5 (59.73% ± 0.18%) than in the other tested accessions (Table 2). Thus, our results identify WS-2, WS-3, and WS-5 as excellent germplasm resources with a high oleic acid content.
Linoleic acid is a biosynthetic precursor of γ-linolenic acid and arachidonic acid, particularly in the skin. The deficiency of this essential fatty acid results in the breakdown of skin integrity and an inability to prevent transdermal water loss . Linoleic acid lowers blood cholesterol and low-density lipoprotein cholesterol concentrations, particularly when it replaces common saturated fatty acids [47, 48]. Other fatty acids detected in C. retusus kernels were linolenic acid (0.42% ± 0.05%), arachidonic acid (0.15% ± 0.03%), and arachidonic acid (0.36% ± 0.08%), all of which can enhance human immune function, reduce pro-inflammatory factors, prevent infarction, and inhibit allergic reactions [14-21]. The ratio of saturated fatty acids, monounsaturated fatty acids, and polyunsaturated fatty acids in oil from C. retusus kernels was 6:55:33, similar to the composition of rapeseed oil and olive oil. Thus, the oil from C. retusus kernels is a potential edible vegetable oil.
Table 3 shows the total phytosterols content and phytosterols composition of oil from the kernels of 22 C. retusus accessions. The total phytosterol content in oil from tested accessions varied significantly at P < 0.05, ranging from 225.71 ± 1.21 mg/100 g (S-5) to 360.59 ± 1.54 mg/100 g (WA-2) with a mean value of 280.33 ± 43.96 mg/100 g and a CV of 15.68%. Among more than 250 different phytosterols found in plants, the most common ones are β-sitosterol, campesterol, and stigmasterol [22, 24]. Capillary GC analyses of C. retusus phytosterols revealed seven phytosterols, with β-sitosterol as the dominant compound (average, 191.14 ± 28.51 mg/100 g; range, 155.93 ± 6.67 mg/100 g (T-10) to 240.67 ± 1.07 mg/100 g (WA-2)), followed by β-sitostanol (average, 36.37 ± 4.56 mg/100 g; range, 31.66 ± 0.02 mg/100 g (B-2) to 49.65 ± 0.64 mg/100 g (T-8). The other detected phytosterols were campestanol (range, 12.09–42.90 mg/100 g), campesteranol (range, 10.07–19.30 mg/100 g), campesterol (10.99–22.95 mg/100 g), and Δ5, 24-stigmasterol (range, 2.26–9.87 mg/100 g) (Table 3). This is the first report of the phytosterols content and profile in C. retusus oil. Although some studies have shown that dietary phytosterols can lower low-density lipoprotein cholesterol, others have shown that the consumption of phytosterols reduces the absorption and plasma concentrations of some fat-soluble vitamins and antioxidants . The FDA tentatively issued a health claim related to phytosterol consumption and coronary heart disease risk; that is, the daily dietary intake to achieve the claimed effect of phytosterols is 2 g per day . The phytosterol content was much higher in C. retusus oil than in oils of olive (Olea europaea L. subsp. Oleaster) (206.82 mg/100 g), yellow horn (Xanthoceras sorbifolium Bunge) (185.3 mg/100 g), and peanut (Arachis hypogaea) (135 mg/100 g) [51-53]. Thus, C. retusus oil has potential as a functional ingredient to produce foods enriched with phytosterols.
Tocopherols content and composition
This is the first report on the content and composition of tocopherols in C. retusus oil. The total tocopherols content and composition in oil varied significantly (P < 0.05) among the 22 C. retusus accessions (Table 4). The average tocopherols content in oil across the 22 C. retusus samples was 578.31 ± 58.66 µg/g, ranging from 480.94 ± 2.11 µg/g (Z-1) to 654.22 ± 1.94 µg/g (T-10), with a CV of 10.14%. Tocopherols is a viscous oily light yellow substance that is soluble in organic solvents. Tocopherols can be classified into eight types according to the saturation of the chain and the number and position of methyl groups. In vegetable oil, tocopherols mainly exists in the form of tocopherols . Among several natural tocopherols, α-tocopherol has the highest absorption and metabolic efficiency in the human body, while γ-tocopherol has the strongest antioxidant capacity . In this study, the types of tocopherols in the 22 C. retusus samples were determined by liquid chromatography. Four tocopherols were detected in C. retusus oil, with γ-tocopherol being the most abundant (526.90 ± 51.36 µg/g) followed by α-tocopherol (34.1 ± 7.56 µg/g). The concentrations of δ-tocopherol and β-tocopherol were 9.60 ± 3.05 and 7.50 ± 1. µg/g, respectively. Tocopherols helps to prevent or ameliorate a variety of major human diseases, such as cancers, cardiovascular diseases, eye disease, and nervous system diseases [32-34, 55, 56]. The main source of tocopherols in the human body is vegetable oils in the diet . The FDA recommends that adults should consume 30 mg tocopherols per day . Some studies have shown that the absorption of 7–9 mg α-tocopherol per day can ensure the normal function of the human central nervous system and vascular system, and the absorption of 100–1000 mg α-tocopherol per day can delay human aging and protect against ultraviolet radiation . Our analyses show that the tocopherols content is higher in C. retusus oil than in oils from soybean (Glycine max (Linn.) Merr.) (285 µg/g), olive (O. europaea L.) (347.5 µg/g), and C. oleifera (280 µg/g) [37, 58, 59]. Thus, C. retusus oil has potential applications as an ingredient to produce fortified foods enriched with tocopherols.
Correlations among fruit morphology, fatty acid composition, and oil, phytosterol, and tocopherols contents
Spearman’s correlation coefficients between fruit morphology, oil content, SFA, MUFA, PUFA, and total phytosterols content in 22 C. retusus accessions are shown in Table 5. The fruit fresh weight was significantly positively correlated with FTD (r=0.589, P < 0.01), FSI (r = 0.902, P < 0.01), GVD (r = 0.907, P < 0.01), GVD/ST (r = 0.810, P < 0.01), kernel weight (r=0.794, P < 0.01), and kernel rate (r = 0.790, P < 0.01). The FTD was significantly positively correlated with FSI (r =0.448, P < 0.05), grain weight (r=0.566, P < 0.01), GVD (r=0.663, P < 0.01), GTD (r=0.953, P < 0.01), and GVD/ST (r=0.556, P < 0.01). The FSI was positively correlated with GVD (r=0.787, P < 0.01), GVD/ST (r=0.844, P < 0.01), and kernel rate (r=758, P < 0.01); and negatively correlated with grain weight (r=0.478, P < 0.05). A very significant positive correlation between GVD/ST and kernel rate (r= 0.763, P < 0.01) was detected. These results indicated that fruit fresh weight would be a useful selection trait for high kernel dry biomass and high kernel rate in C. retusus breeding. Regarding oil, phytosterols, and tocopherols contents, the total oil content was negatively correlated with the total phytosterols content (r= −0.653, P < 0.01) and positively correlated with kernel weight (r=0.762, P < 0.01). We detected very significant negative correlations between SFA and PUFA (r=−0.561, P < 0.01), between MUFA and PUFA (r= −0.693, P < 0.01), between MUFA and total phytosterols content (r= −0.548, P < 0.01), and between PUFA and total phytosterols content (r= −0.811, P < 0.01). We detected a very significant negative correlation between total phytosterols content and tocopherols content (r= −0.744, P < 0.01).
Principal component analyses of fatty acids, phytosterols, and tocopherols composition
Principal component analyses provide data that are useful for breeding improved varieties. For example, Liang et al. (2019) studied the composition of fatty acids and sterols in 22 Acer truncatum plants, and used a principal component analysis to separate the plants into groups on the basis of their components . Two-dimensional biplots for the first two principal components (PCs) obtained from PCAs based on fatty acids, phytosterols, and tocopherols composition and contents are shown in Fig. 2. For fatty acids composition and content, the first two PCs explained 71.10% of the total variance of fatty acids composition, and the tested C. retusus accessions were separated into four groups (Fig. 2A). Group I included WS-2, WS-3, WS-4, and WS-5, which were characterized by a high oil content and large proportions of PUFA and linoleic acid. Group II consisted of T-2, T-4, T-5, T-9, T-10, T-11, Z-1, Z-4 and Z-5, which were characterized by high proportions of MUFA, SFA, palmitic acid, stearic acid, and oleic acid. Group III included B-1, B-2, WA-1, S-2, S-3, and S-5 which were characterized by high levels of arachidic acid and arachidonic acid. Group IV included WA-2 and S-6, accessions with a medium level of the studied parameters (Fig. 2A). On the basis of these results, WS-2, WS-3, WS-4, and WS-5 were identified as ideal C. retusus germplasm resources with high oil content and high linoleic acid content, and T-2, T-4, T-5, T-9, T-10, T-11, Z-1, Z-4, and Z-5 were identified as C. retusus germplasm resources with a high oleic acid content.
For phytosterols composition and content, the first two PCs explained 72.2% of the total variance of phytosterols composition, and the tested accessions were separated into three groups (Fig. 2B). Group I included WA-1, WA-2, WS-2, WS-3, WS-4, and WS-5, which were characterized by a high level of total phytosterols, β-sitosterol, campestanol, Δ5-avenosterol, and Δ5, 24- stigmasterol. Group II included T-2, T-4, T-5, T-8, T-9, T-10, T-11, B-1, B-2, Z-1, Z-4, and Z-5, which were characterized by high levels of campesterol, campesteranol, and β-sitostanol. Group III included S-2, S-3, S-5 and S-6, which showed no significant difference in sterol composition and content compared with other groups. The results of the PCA identified WA-1, WA-2, WS-2, WS-3, WS-4, and WS-5 as C. retusus germplasm resources with a high phytosterols content.
For tocopherols composition and content, the first two PCs explained 91.2% of the total variance of tocopherols composition and the tested samples were separated into five groups (Fig. 2C). Group I included T-4, T-5, T-8, T-10, WS-4, and WS-5, which were characterized by high contents of total tocopherols, γ-tocopherol, and δ-tocopherol. Group II included T-2, T-9, WS-2, WS-3, and WA-2, which were characterized by a high α-tocopherol content. Group III included B-1, B-2, and T-11, which were characterized by a high β-tocopherol content. Group IV (S-2, S-6, and WA-1) and group V (S-3, S-5, Z-1, Z-4, and Z-5) showed no significant difference in phytosterols composition and content compared with other groups. These results identified T-4, T-5, T-8, T-10, WS-4, and WS-5 as C. retusus germplasm resources with a high tocopherols content.