## 4 Discussion  
Physicochemical traits provided basic information for differentiating the primary qualities of the edible fruit parts of three major cultivated shikuuwasha lines and calamansi (Table 1). The ratio of TSS/TA in the \"Izumi kugani-like\" line (25.15) is comparable with that in mandarin and pummelo (26.30 and 27.78, respectively), while the ratios in \"Izumi kugani\" and \"Ogimi kugani\" are within the range recorded for grapefruit (6.32-12.00) [15]. On the other hand, the TSS/TA ratio of calamansi (3.18) is between those for lemon and grapefruit (1.79-6.32) [13]. These results indicate that shikuuwasha and calamansi can be primarily distinguished by the soluble solids and acidity in their edible parts; thus, these simple traits can be used to differentiate the basic fruit qualities of the two citrus cultivars when they are used for raw fruit or flesh juice consumption. The red indices (a* values) of the shikuuwasha and calamansi fruits were within the range of other citrus varieties such grapefruit and lemon, while their yellow index (b* value) was similar to that  
Figure 4: PCA plots of metabolites and VOCs in shikuuwasha and calamansi juices: **(a)** component scores of whole juices; **(b)** Factor loadings of whole juices; **(c)** component scores of flesh juices; **(d)** factor loadings of flesh juices. of mandarin [20]. These color indices are important factors that determine the impression of consumers when evaluating the quality of fruits and fruit juices [9] and can be used to differentiate the color of shikuwasha from that of calamansi. The edible fruit parts of calamansi were characterized by a higher content of carotenoids and pigments for red, yellow, and orange than those in the shikuwasha lines, and thus the two cultivars could be visually differentiated (Figure 1). Compared to the other citrus varieties, shikuwasha features similar levels of carotenoids to those of lemon and pomelo (0.03-0.65 and 0.04-1.32 mg/100 g FW, respectively), while the carotenoid concentration of calamansi is slightly lower than that in mandarin oranges (1.85-2.49) [10]. The pigments found in most citrus varieties are \(\beta\)-cryptoxanthin and violaxanthin; mandarin tends to be rich in \(\beta\)-cryptoxanthin and orange in violaxanthin [10; 21]. These carotenoids are beneficial to health because they are precursors to vitamin A, which is essential for humans, while \(\beta\)-cryptoxanthin may prevent osteoporosis, boost immunity, and act as an antioxidant [22]. A positive correlation between carotenoid content and a* values has also been reported for other citrus varieties, as well as vegetables such as tomatoes and pumpkins [12; 23; 24]. The red or orange color of the shikuwasha and calamansi may intensify as carotenoid content increases (Figure 1b,d); thus, these pigments are responsible for distinguishing the color quality of the fruits. Simple sugars such as monosaccharides and disaccharides are the main sources of sweetness in citrus fruits, while organic acids such as citric, malic, and malonic acids provide a sour taste [14; 25]. The shikuwasha lines were primarily associated with higher concentrations of sucrose and choline (Figure 2a-e). \"Izumi kugani\" and \"Izumi kugani-like\", which were positioned close to one another on the PCA component scores plot, had overall similar profiles regarding metabolite composition, while the lower sucrose and higher malonate contents in \"Ogimi kugani\" were the main reasons for its separation from the other two lines. Accordingly, \"Izumi kugani\" may be perceived sweeter and less sour than \"Ogimi kugani\", as reported previously [14]. In contrast, calamansi, which had higher citric and malic acid contents, could be distinguished by its high sourness intensity. The two citrus cultivars likely provide different levels of beneficial nutrients, that is, choline in shikuwasha and GABA in calamansi (Figure 2e).