Morphological and Chemical Analysis of 16 Avocado Accessions ( Persea americana ) From China by Principal Component Analysis and Cluster Analysis

The physicochemical composition of avocado fruit has been well reported, but there is little detail on Chinese native avocado varieties. The present study investigated the morphological characteristics, oil contents, and fatty acid compositions of 16 avocado accessions grown in the tropical and subtropical regions of China. Eight fatty acids were identified and quantified by GC-MS. The major fatty acids of avocado pulp were palmitic, oleic, and linoleic acids, accounting for 78-91% of the total fatty acids content. The analysis of one-way variance (ANOVA) of the data revealed morphological and chemical differences between most of avocado accessions. Moreover, 16 avocado accessions were distinguished through a PCA scores scatter plot and cluster analysis based on fatty acid profiles. The results identified some remarkable characteristics of avocado accessions from different places of collection.


Introduction
Avocado (Persea americana Mill.), a member of the family Lauraceae of the order Laurales, is a plant native to Central America, South America, and Mexico (Schaffer et al., 2012).The avocado is among the most economically important subtropical/tropical fruit crops in the world, and production and consumption levels have increased dramatically during the last 150 years (Schaffer et al., 2012).One factor contributing to this marked increase was the constant expansion of avocado products into new markets in parts of the world where avocado was previously unknown or scarcely available (Schaffer et al., 2012).Avocados were first introduced to the Taiwan province of China in 1918 (Papademetriou, 2000), thus allowing the rapid propagation of superior varieties to develop an industry producing avocados with superior plant characteristics and fruit quality.Since the late 1950s, hundreds of avocado varieties have been introduced in China successively from the United States, Mexico, and Central America (Ge et al., 2017a).Breeding and selection programs were undertaken and are ongoing to this day mainly by CATAS, GSSASRI, Guangxi Vocational and Technical College, and other state-owned or private farms, leading to the selection of more than a dozen high-quality avocado varieties (Ge et al., 2017a).Meanwhile, natural hybridization between avocado varieties often occurs, producing many new avocado hybrids in state-owned or private farms.Several local avocado selections have also gradually developed in relatively isolated growing zones because of local unique geographical environments.Nowadays, some Chinese native superior avocado varieties are widely cultivated in the tropical and subtropical regions of China (Ge et al., 2017a).
Particular chemical composition of avocado are associated with nutritive and health effects (Dreher & Davenport, 2013;Galvão et al., 2014;Ge et al., 2017b).In terms of oil content, avocado fruit is exceeded only by the fruits of the palm and olive trees (Knothe, 2013).Remarkably, the lipid content in avocado can reach 5% to 30% of the fruit fresh weight, depending on the seasonality and planting conditions (Ge et al., 2017a).Avocado fruit lipids contains 50% to 60% monounsaturated fatty acids and 10% to 15% polyunsaturated linoleic and linolenic acids (Giraldoa & Moreno-Piraján, 2012;Pedreschi et al., 2016).Furthermore, avocado fruit lipids could be used in non-food industries, for example, as an alternative biodiesel source instead of the conventional petroleum-based diesel fuel (Giraldoa & Moreno-Piraján, 2012;Knothe, 2013).In addition, the high non-saturated content of avocado fruit lipids provided superior skin permeability and sunscreen performance, and could be used in sunscreen cream as emulsifier (Santo et al., 2014).
Globally, the Hass and Fuerte avocado cultivars are the most commercially valuable, accounting for about two-thirds of avocado production (Schaffer et al., 2012).Therefore, these two cultivars have been the subject of most studies on the properties influencing avocado quality (Hurtado-Fernandez et al., 2011, 2014, 2015;Rodríguez-Carpena et al., 2011).However, no similar studies have yet been published on Chinese native avocado accessions.Thus, the objective of the present study was to determine morphological characteristics, oil contents, and fatty acid compositions of avocado fruit from 16 avocado accessions collected from the tropical and subtropical regions of China.The 16 avocados were distinguished using chromatography of its oil combined with principal component analysis and cluster analysis.The resulting information will be used to evaluate the potential avocado germplasms with high-quality properties for use as food or for industrial biodiesel production.

Plant Material, Reagents, and Sample Preparation
The 16 avocado accessions (P.americana var.guatemalensis) used in the present study were obtained as follows: six native avocado accessions (RN-1,   ).18 mature fruits of each accession were randomly collected and immediately transported to the laboratory in standard foam boxes used for export packaging.The fruit samples were maintained at 5 °C to 6 °C.The pulps were separated from the fruits, homogenized using a domestic blender, and then stored at 4 °C for a maximum of one week before analysis.

Morphological Characteristics and Chemical Assays
The length, breadth, and weight of each fruit were measured.Nine replicates (fruits and seeds) were randomly selected to measure per accession.
Oil content was evaluated using the description of Ge et al. (2017b).Nine mature fruits were randomly collected from 18 fruits of each accession, and the pulps of theirs were mixed respectively.The avocado pulps were dried and ground to a powder, and the dry powders (5 g) were transferred to a cylindrical filter paper, to which absolute ether (50 °C) was added at a material-to-solution ratio of 1:20.The mixture solutions were stood over night.The pulp oils were sequentially extracted using a Soxhlet extractor for 6 h.Finally, the extracted solutions were evaporated using a rotary evaporator, and the residue was weighed.The total lipid content was expressed as g/100 g on a fresh weight (FW) basis.The experiments were performed in triplicate for each accession.
The fatty acid profiles were determined as described by Ge et al. (2017b).Nine mature fruits were randomly collected from 18 fruits of each accession, and the pulps of theirs were mixed respectively.The oils extracted from avocado pulps (40 μL) were saponified at 80 °C for 30 min after adding 5 mL NaOH-MeOH (0.2 mol/L).After cooling, 2.5 mL BF 3 -MeOH (14%) was added to the mixtures to allow methyl esterification at 80 °C for 30 min.After the addition of 2 mL saturated NaCl and 4 mL n-hexane, the solutions were heated under reflux for 15 min.The upper layers were collected, filtered through a 0.22-μm filter membrane, and then analyzed for fatty acids by GC-MS.
GC-MS conditions were evaluated using the method of Ge et al. (2017b).The sample volume injected in the gas-chromatograph was 1 μL.The mass spectrometer was operated in the electron impact mode at 70 eV in the scan range of 35 to 400 m/z.The fatty acid methyl esters (FAMEs) were identified by comparing the retention times of the peaks with those of commercial standards, and by computer matching of their corresponding mass spectra with those reported in the NIST 2011 library.The FAMEs were quantified against methyl nonadecanoate, which was added as an internal standard and then quantified using the calibration curves of the respective FAMEs (R 2 ≥ 0.995).The contents of the FAMEs were expressed as mg/100 g FW.The experiments were carried out using three replicates.

Statistical Analyses
Significant differences and principal components analysis (PCA) were analyzed using SPSS version 20.0 (SPSS Inc., Chicago, IL, USA).Significant differences among the fruit characteristics, oil contents, and fatty acid compositions of the 16 avocado accessions were verified by one-way analysis of variance, and Duncan's multiple comparison test was used to determine the statistical significance of differences between means at a 95% confidence level.The results were presented as the mean±standard deviation of three measurements.The distance matrix was subjected to cluster analysis by the unweighted pair-group method (UPGMA, Sneath & Sokal, 1973), a SAHN clustering technique (Sneath & Sokal, 1973), which compresses the patterns of variation into two-dimension branch diagrams (dendrograms).The dendrogram was constructed using a Jaccard's formula with NTSYS pc 2.1 statistical package (Rohlf, 2000).

Morphological Characteristics Analyses
Fruit weight is one of the most important indices in avocado production (Schaffer et al., 2012).As shown in Table 1, large differences in fruit weight were observed between most of the avocado accessions (p < 0.05).The fruit of RN-15 was the heaviest (0.51±0.04 kg), whereas the fruit of RN-12 was the lightest (0.09±0.01 kg).With the exception of RN-12, the weights of the fruits of the 14 native Chinese avocado accessions all exceeded that of the Hass cultivar.The fruit lengths of the 16 avocado accessions ranged from 5.47±0.71cm (RN-12) to 19.95±1.87cm (RN-21), with significant differences between most of the accessions (p < 0.05; Table 1).The fruit breadths of the 16 avocado accessions were also presented in Table 1, showing small differences between most accessions (p < 0.05).The highest fruit breadth was found in RN-15 (8.92±0.38 cm), while the lowest was found in RN-12 (5.42±0.31cm).Schaffer et al. (2012) suggested that the fruit shapes of the avocado cultivars was exceedingly rich around the world, such as Bacon and Hass for ovate, Ettinger for pyriform, and Fuerte for pyriform with a distinct neck, etc.Based on the fruit lengths, breadths, and appearances (Table 1 and Figure 1), the fruit shapes of the avocado accessions were mainly pyriform (RN-1, RN-11, RN-21, RN-23, and RN-27) and ovate (RN-5, RN-15, Hass, RN-22, RN-24, RN-26, RN-28, and RN-29).The exceptions were RN-12, which was round, and RN-16 and RN-25, which were pyriform with long, narrow necks.1999, 2002;Villa-Rodríguez et al., 2011;Vinha et al., 2013); these previously reported values were mostly higher than those found in the present study.We inferred that the different cultivation conditions of the avocado accessions could have the main effect on the variation of the total lipid levels of the pulps.The fatty acid compositions of the pulps of the 16 avocado accessions were presented in Table 2.The same eight fatty acids were found in the pulps of all 16 avocado accessions, although the compositions were significantly different among most of the accessions (p < 0.05).The major fatty acids (≥ 15%, the percentage of the individual fatty acid out of the total fatty acid content) in the avocado pulp oil quantified in the present study were palmitic acid (C16:0), linoleic acid (C18:2), and oleic acid (C18:1), and intermediate amounts (1% to 15%, the percentage of the individual fatty acid out of the total fatty acid content) of palmitoleic acid (16:1), linolenic acid (C18:3), and stearic acid (C18:0) were detected.Small amounts (≤ 1%, the percentage of the individual fatty acid out of the total fatty acid content) of arachic acid (C20:0) and myristic acid (C14:0) were found.These findings agreed with previous studies reporting that palmitic, oleic, and linoleic acids were the dominant fatty acids in avocado pulp oil (Ozdemir & Topuz, 2004;Pedreschi et al., 2016;Rohman et al., 2016).Previous studies indicated that the content of oleic acid had a considerably higher than those of other fatty acids (Dreher & Davenport, 2013;Galvão et al., 2014;Ferreyra et al., 2016), while the content of oleic acid had almost the same as that of linoleic acid and slightly lower than that of palmitic acid in this study.In the present study, more than 67% of the total fatty acids (TFA) in the avocado pulp oil were unsaturated, with the remaining 33% being saturated (Table 3).The total content of unsaturated fatty acids (ΣUFA) ranged from 1090.75±23.78mg/100 g FW in RN-28 to 5689.39±140.63 mg/100 g FW in RN-5.The total content of saturated fatty acids (ΣSFA) varied between 492.26±24.16mg/100 g FW in RN-28 and 2930.02±56.58mg/100 g FW in RN-5, while TFA varied from 1583.01±47.94mg/100 g FW in RN-28 to 8619.41±197.21mg/100 g FW in RN-5.Palmitic acid was the most abundant SFA, with noticeable differences in palmitic acid content observed between avocado samples (p < 0.05); RN-5 had the greatest content of palmitic acid (2813.95±54.15mg/100 g FW), while RN-28 had the lowest content (435.65±23.47mg/100 g FW).The UFAs linoleic and oleic acids were the second most abundant fatty acids and were found in similar amounts.The ΣUFA/ΣSFA ratios in the 16 avocado accessions ranged from 1.49 to 2.41, which were in accordance with those of the Collinson and Barker cultivars but below that of the Fortuna cultivar (3.49) (Galvão et al., 2014).Note.Different letters within the same row are significantly different (p < 0.05); ΣSFA = total saturated fatty acids; ΣUFA = total unsaturated fatty acids; TFA = total fatty acid

Principal Component Analysis
The PCA results of eight fatty acids from 16 avocado accessions were obtained using NTSYS pc 2.1 software and were shown in Figure 3. PCA generalized eight fatty acids to two principal components which accounted for 55.44% of the total variation.The first component F1 explained 33.97% of the total variation and was mainly associated with linoleic acid, palmitic acid, oleic acid, stearic acid, arachic acid, and myristic acid.The second component F2 accounted for 21.47% of the total variation and was fundamentally defined by linolenic acid and palmitoleic acid.The 16 avocado accessions were classified into two groups through PCA, approximately separated the avocado accessions from Hainan province from the avocado accessions from Yunnan province (Figure 4). jas.ccsenet.
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Table 1 .
Fruit characteristics (mean value±standard deviation, n = 9) of 16 avocado accessions grown in southern China

Table 2 .
Fatty acid compositions (mean value±standard deviation, mg/100 g FW, n = 3) in the pulps of 16 avocado accessions grown in southern China