## 3 Results and Discussion
### Differential Volatile Aroma Screening and OPLS-DA Analysis of VOCs during Ripening of R. roxburghii  
The OPLS-DA analysis of 145 volatile substances in _R. roxburghii_ fruit revealed that the first three stages (I, II, and III) were located in the same quadrant, indicating a similarity in the volatile components of the fruits. In contrast, stages IV and V were distributed across different quadrants, resulting in the formation of three distinct groups (Figure 4B): the first three stages (I, II, and III), stage IV, and stage V. Additionally, Figure 4A illustrates the significant changes in _R. roxburghii_ across five maturation stages through the quantitative visualization of VOCs, thereby confirming the aforementioned classification. For additional information regarding the VOCs depicted in Figure 4A, please refer to Figure S1 in the Supplementary File. The 145 VOCs of _R. roxburghii_ exhibited marked differences at various stages of ripening. Green and purple colors indicate the decrease and increase in substance content, respectively, directly reflecting the changes occurring during the ripening process of _R. roxburghii_. Through the cluster heat map, the 145 VOCs are organized into two distinct clusters, labeled 1 and 2, with cluster 2 further subdivided into two subclusters: 2.1 and 2.2. Cluster 1 was characterized by the changes in VOCs during the initial three stages. In this phase, the concentrations of aldehydes and alcohols gradually decreased, while the levels of esters significantly increased. Research indicates that lipid metabolism serves as a crucial source of aldehydes and alcohols, which not only contribute directly to fruit aroma but also act as precursors for the synthesis of volatile esters [58]. Therefore, compounds with similar biosynthetic pathways are often clustered together. In cluster 1, we identified 12 hexyl esters and their corresponding prohexanols, which are derived from the metabolism of oleic and linoleic acids [59; 60]. Figure 4A illustrates that the content and variety of ester substances continued to rise during the early ripening stage of _R. roxburghii_ fruit. As shown in Table 2, the first three stages predominantly featured flower (1-hexanol, geraniol), fruit (hexyl butyrate, hexyl acetate), green ((E)-2-octenal, (Z)-3-hexenyl acetate, and (Z)-3-hexenyl butanoate), and fat ((E)-2-heptenal, decyl aldehyde) compounds. Cluster 1 was notable for the formation of small clusters at stage III, which comprised 14 distinct volatiles. Among these, seven substances were unique to this stage: cyanamide, N-(3-methyl-2-butten-1-yl)-, hexyl isobutyrate, fema 3498, and so on. In conjunction with the findings presented in Table 2, the predominant aroma characteristic of this stage was a sweet aroma, primarily dominated by (E)-\(\beta\)-ocimene (ROAV \(>\) 100), alongside a notable green aroma, primarily derived from (Z)-3-hexenyl butanoate (ROAV \(>\) 1). Collectively, these components contributed to the distinctive aroma profiles of _R. roxburghii_ fruit at stage III. Figure 4A illustrates that cluster 2.2 was further subdivided into three sub-clusters, which corresponded to the I, II, and IV stages of _R. roxburghii_ ripening, respectively. During stage I, nine volatiles (including 2-Isopropenyl-5-methylhex-4-enal, phenethyl alcohol, and (E,E)-2,4-hexadienal, etc.) were significantly clustered and exhibited higher concentrations than in the other stages. Notably, (E,E)-2,4-hexadienal and phenethyl alcohol were particularly prominent, with phenethyl alcohol having an ROAV greater than 1, contributing to the fruit's sweet and aromatic flavor. As the fruit progresses into stage II, esters became the dominant volatiles, consistent with previous analyses. Seven volatiles (including bis (2-ethylhexyl) adipate, (E)-2-hexenyl acetate, and 2,6-di-tert-butylphenol, etc.) that were unique to this stage could serve as distinguishing markers to differentiate stage II from the  
Figure 4: Clustering heat map and OPLS-DA analysis of VOCs in _R. roxburghii_ fruit at different ripening stages.