Primary metabolites in OCC and its donors
As shown in Table 2, twenty-one primary metabolites were identified in peels. Based on the significant analysis of statistics, the peels of OCC (OCP) shared more similarities with that of C (CP) in these profiles. Among them, the 4-aminobutanoic acid, shikimic acid and palmitic acid were exclusively detected in OCP and CP, suggesting that these 4 compounds of OCP were only produced by CP. In the contrary, sorbose was specific to the peels of O (OP), with no detectable evidence in OCP and CP. There were higher concentration of the acids (except 2-ketoglutaric acid) and alcohols detected in OCP and CP, whereas there were lower content of sugars (excpet fucose) detected in OCP and OP.
Notably, some particular chemical characteristics were observed in OCP. Among those identical profiles detected in three samples, carbamic acid and fucose exhibited the highest level in OCP; but in total metabolites, OCP showed a significantly lower value than that in any of the donors.
For the juice sacs, there were 18 primary substances listed in Table 3 among the juice sacs of O (OJ), OC (OCJ) and C (CJ). In the present study, no-significant differences were found according to the total primary metabolites between OCJ and the two donors. Interestingly, there were 5 profiles (quininic acid, xylose, arabinose, turanose and scyllo-Inositol) in OCJ were significantly different from two donors. Among them, arabinose and quininic acid in OCJ were the highest and the lowest of three cultivars, respectively. The remaining 3 profiles in OCJ were significantly different between two donors. In addition, there were 10 profiles in OCJ consistent with one or two donors. However, these profiles actually showed more similarities with O, for example, 4-aminobutanoic acid, palmitic acid and allose were common to OCJ and OJ, but were not presented in CJ. Conversely, the sorbose was only specifically existed in CJ, but undetectable in OCJ and OJ.
Interestingly, there were 3 compounds showed some hereditary differences in OCJ (oxalic acid, sorbose and rhamnose). Among them, either oxalic acid or rhamnose were undetectable only in OCJ, which caused the obvious discrepancies between OCC and its donors. However, the sorbose was missing in both OCJ and its layer source donor O.
Volatile compositions between OCC and its donors
With regards to the volatiles in the peels of three cultivars, there were 36 substances listed in Table 4, including monoterpenes, sesquiterpenes, alcohols, aldehydes, phenol and others. The monoterpenes were the most abundant of profiles quantified, with d-limonene as the dominant compound, accounting for 88.65%, 81.23% and 80.77% of the total volatiles in OP, OCP and CP, respectively. After d-limonene, followed by γ-Terpinene, β-Myrcene and α-Pinene, they were the main compounds for three samples had in common.
The result showed that OCP had a stronger correlation with CP than with OP. Firstly, according to the significance analysis, 14 volatiles had no significant difference between OCP and CP, but only 3 volatiles between that of OCP and OP. This indicated, CP had the dominant position in the regulation of chemical profiles in OCP and more chemical traits in OCP were inherited from CP. Secondly, the main volatiles of OCP were completely consistent with that of CP, including d-limonene, γ-terpinene, Germacrene D, β-myrcene and α-pinene (sort from high to low concentration), but the main volatiles order in OP were divergent (d-limonene, γ-terpinene, β-myrcene, α-pinene, β-elemene). This was mainly because the Germacrene D was significantly higher in OCP and CP than OP, and so strongly suggest that Germacrene D was mainly originated from CP and OP had less impact on the development of OCP. Thirdly, it is worth noting that 2,4-di-t-butylphenol was really unique, which was only detected in OP, and this is the only one volatile that OCC and C exclusively possessed in common.
In addition, most of volatiles in OCP were either inclined to one donor, or maintained some degree between two donors. However, only (E)-3-Hexen-1-ol and 3-Hexenal in OCP exceeded significantly that in two donors.
For the edible juice sacs, the volatiles contained up to 19 constituents (Table 5). OCJ was highly correlated with OJ in total volatiles and monoterpenes (the leading volatiles). Especially in dominant substances, d-limonene, its concentrations of OJ and OCJ were significantly higher than CJ, occupied 78.07%, 72.64% of the total volatiles in OJ and OCJ, respectively, but only 60.03% in that of CJ. Meanwhile, besides d-limonene, there were also significant similarities between OJ and OCJ in methyl nonanoate, copaene and octanal, and we considered that all these compounds in OCJ were originated from O to a great extent.
Moreover, typical volatiles quantitative inheritance traits were observed in OCJ. For example, nootkatone and pentadecanal were presented the largest amounts in OCJ. Instead, γ-terpinene in OCJ was significantly lower than that in any of the donors. Furthermore, what we were particularly interested in was α-ylangene, which was only detected in OCJ but not in two donors, and this volatile was hardly ever been reported in any citrus species.
Carotenoid constituents between OCC and its donors
As it shown in Table 6, a total of 9 carotenoids were detected among OCC and two donors. Generally, the contents and types of carotenoids in OCC were very close to C in peels, and inclined to O in juice sacs.
Obviously, the donor O had the highest contents of all carotenoid components in both peels and juice sacs among three genotypes. In the peels, the carotenoids other than violaxanthin and Lutein in OCP were all significantly consistent with the donor CP. According to the juice sacs, the carotenoids in OCJ were intermediate between two donors. Actually, all of the carotenoids detected in OJ and OCJ were particularly higher than those in CJ. It is remarkable that α-carotene accumulated much less than other carotenoids both in the peels and juice sacs.
The dominant components were different between peels and juice sacs in OCC and two donors. Violaxanthin was primary in peels, and β-cryptoxanthin was dominant in juice sacs. The main carotenoids in OCJ, such as β-cryptoxanthin, phytoene and phytofluene, changed much more than those in OCP, which maintained the color of flesh in OCC compared with layer donor O.
Correlation of total carotenoids between OCC and two donors was analyzed to make out the source donor in tissue coloration. It was suggested that the carotenoids accumulation of OCC had obvious donor bias and was different between peels and juice sacs (Table 7). In peels, total of carotenoids of OCP were significantly correlated with CP and OP, respectively. In the juice sacs, only the correlation coefficient between OCJ and donor OJ was statistically significant (0.957). This donor bias of carotenoids in the peel and juice sac of OCC maturation can partly explain why the peel of OCC is light yellow, similar to donor C, whereas the juice sac is dark orange, similar to donor O.
PCA analysis of metabolites in peels and juice sacs of OCC and two donors
In terms of three categories of metabolites, principal component analysis (PCA) was performed according to different tissues between OCC and two donors.
In the PC1 direction of the score map, there was a clear distinction between the donor O and the other genotypes (OCC and donor C) in primary metabolites (Figure 2A-1), volatiles (Figure 2A-2) and carotenoids (Figure 2A-3) according to the peels.
In the juice sacs, the donor C was clearly distinguished in the primary metabolites (Figure 2B-1), volatiles (Figure 2B-2) and carotenoids (Figure 2B-3) from OCC and donor O according to the PC1 direction of the score map. However, OCJ was separated from OJ in the PC1 direction (Figure 2B-3), indicating a novel profiling of accumulation pattern in carotenoids in the chimera.