## 4 Discussion
Taiwanese \"Taoyuan\" and Okinawan accessions which were plotted along the PC1 axis on the PC2 positive direction were rich in hexanal, indicating that its specific green aroma may enrich odor properties of the leaves [21]. On the other hand, Kagoshima \"Tokunoshima 1\", Taiwanese \"Nantou\", and Okinawan accessions which were located on the PC2 negative direction were abundant in monoterpenes (limonene, p-cymene, \(\gamma\)-terpinene, and linalool) and sesquiterpenes (\(\beta\)-caryophyllene, germacrene B, and elemol). These volatile constituents are known to have typical citrus and woody-spicy odors, respectively [19; 20]. These leaves can be used to enhance the aroma of steamed food or brewed tea because they are rich in monoterpenes, which are abundant in _C. depressa_ fruits. The genetic and VOC data of the leaves were partially associated with the Kagoshima accessions; however, the outcome did not fully agree with the differentiation of Okinawan and Taiwanese accessions (Figure 2). Nevertheless, the metabolic and physicochemical traits of citrus differ despite the commonality of accessions and cultivation environments [11; 17]. Although _C. depressa_ usually inherits the parents' genetic information as a polyembryonic plant, there are occasions where mutations occur in nuclear embryos [9; 10; 18; 22]. Therefore, genetic and VOC differences in _C. depressa_ accessions may be caused by genetic changes and environmental factors [9; 10]. The OPLS-DA visualization plot allowed the grouping of the three regions based on their VOCs, and further indicated that monoterpenes were useful for the identification of leaves of Kagoshima origin, sesquiterpenes from Okinawa, and hexanal from Taiwan (Figure 3e,f). Moreover, volcano plots allowed for further distinct volatile components when the two cultivation regions were compared (Figure 4, Supplementary Table S3). In detail, the VOC markers which can identify _C. depressa_ accessions according to their cultivated regions are as follows: 5,9,9-trimethyl-spiro[3.5]non-5-en-1-one for Okinawa; piperitone, citronellal, (Z)-\(\beta\)-ocimene, 1,8-cineo, \(\alpha\)-phellandrene, and nerol for Kagoshima; and hexanal for Taiwan. Compared to Okinawan accessions, leaves of Kagoshima origin have a citrus-minty odor, while Taiwanese leaves have a green-floral aroma [19; 21]. Piperitone and citronellal were particularly promising as volatile markers that can distinguish between cultivation origins, given the large range in absolute values of log2 Fc in Kagoshima compared to the other two regions (6.38-7.48 and 5.17-6.57, respectively). In Southeast Asia, the leaves of citrus fruits are often used for seasoning purposes that contribute to the flavor quality of cooking; thus, _C. depressa_ leaves are potential sources for such food applications [23]. The leaves from accessions such as \"Tokunoshima 1\" (Kagoshima) and \"Nantou\" (Taiwan), as well as Okinawan \"Iriomote\", \"Kaachi\", \"Nakamoto seedless\", and \"Ogimi 1\", have typical aroma qualities from monoterpenes similar to its fruit, therefore they can be useful for flavoring dishes. Because leaves contain more biomass than fruits, they can be acquired any time of the year, and citrus leaves can be a great source of VOCs with antioxidant and potent antibacterial activities [24; 25]. Taken together, the VOC profile data for each accession of _C. depressa_ could be used more easily than those for fruits to classify the distinctiveness of each cultivation region, and the leaves are potential aroma resources for cooking and other food applications. The edible fruit parts of Taiwanese accessions containing more VOCs than those from Okinawa and Kagoshima were elicited using heat maps and PCA plots (Figure 5a,b). Taiwanese \"Nantou\" could be characterized by its high monoterpene levels; this line was found to have the genetic ability to produce greater amounts of terpen such as \(\alpha\)-terpinol and linalool [11].