## 2 Results
### Analysis of Differential VACs in Pepper Fruits from Different Samples  
The difference in VACs from different pepper fruits was analyzed using the relative content of each of the metabolites. Principal component analysis (PCA) showed that the samples were significantly different (Figure 3a). Hierarchical cluster analysis (HCA) indicated that the experimental data were reliable (Figure 3b). In the cluster heat map (Figure 3c), the VACs of different pepper fruit showed significant differences. Most of them were significantly accumulated in the middle and late stages (breaking and maturation stages), clearly separated from the green stage, while metabolites of specific biochemical pathways were without obvious clustering. Moreover, the accumulation patterns of VACs were different between different varieties at the same stage. The differential accumulation of these volatile aroma substances may be the reason for elucidating the aroma formation mechanism of pepper fruit. Figure 3: Characterization of VACs from different pepper samples; (**a**) PCA of VACs from different pepper samples; (**b**) HCA of VACs in different samples; (**c**) heat map of the species and relative contents (\(\upmu\)g/kg) of volatiles in different samples. (The heatmap was drawn using log2-based relative content values. Red represents high expression, and blue represents low expression). Moreover, we constructed PLS-DA models of VACs to analyze the similarities and differences of VACs in pepper fruits at different development stages, and in different varieties of pepper fruits at the mature stage, based on the fact that fresh peppers are mostly eaten at the mature stage. The quality test results showed that the PLS-DA model was reliable (Figures S3 and S4). Finally, according to the screening conditions VIP > 1, \(p\) < 0.05, and 1 log2 FC \(|\geq\) 1, 70 significantly different metabolites (Table S3 and Figure S5) from all the detected VACs were screened. There were 24 different VACs at three development stages fruits of CTJ (CTJ-a vs. CTJ-b and CTJ-a vs. CTJ-c), among which 10 VACs were significantly up-regulated at the breaking and maturation stages compared with the green stage 30 different VACs in GJ (GJ-a vs. GJ-b and GJ-a vs. GJ-c), 12 VACs were significantly up-regulated and 9 VACs were significantly down-regulated at the breaking and maturation stages compared with the green period, Among them, up-regulated VACs included \(Z\)8 (4-Methyl-pentyl isobutyrate), \(Z\)28, TH4,TH12, T20, and the down-regulated VACs mainly included O4 (2-pentyl-furan), C8 (e-2-hexadecen-1-ol), Q3, Q4, etc. 32 significantly different VACs were found in the HDL1 comparison group at different developmental stages (HDL1-a vs. HDL1-b and HDL1-a-V HDL1-c), 12 VACs such as Z7, Z29, Z31 (valeric acid, benzyl ester), and TH5 (heptane) were significantly upregulated compared to the green stage in the middle and late stages, while Z11, Z12, Z15 (butyric acid, 2-methyl, hexyl ester), C3, C8, and the other 9 VACs were significantly downregulated. In addition, 30 VACs were identified as significant differences in HDL2 (HDL2-a vs. HDL2-b and HDL2-a vs. HDL2-c), including 6 up-regulated and 12 down-regulated VACs in the middle and late stages. Among them, up-regulated VACs included \(Z\)39, Z22 (4-methyl-amyl tglate), C9, TH19 (z-3-heptadecene), etc., and down-regulated VACs mainly included \(Z\)35 (Isopentyl 8-methylnon-6-enoate), Z36 (pentadecanoic acid, 3-methylbutyl ester), Z23 (5-methyl-hexyl 3-methylbutanoate), Z11, Z12, etc. Forty VACs showed significant differences in mature pepper fruits of different varieties (GJ-c vs. CTJ-c, HDL1-c vs. CTJ-c, and HDL2-c vs. CTJ-c), among which volatile esters, aldehydes, alcohols, terpenoids, and hydrocarbons were the main VACs, such as Z12, Z13, Q25 (z-9,17-octadecaniel), C8, T3, Q16, etc. The VACs in pepper fruits of different varieties showed different accumulation patterns compared with CTJ mature fruits, HDL1 and GJ had more up-regulated metabolites, while HDL2 had more down-regulated metabolites.