## 2 Results
### Transcriptome Analysis of Pepper Fruits from Different Samples  
To further explore the expression changes of genes related to VACs in pepper fruit, we sequenced the transcriptome of 36 samples. Sequencing quality and data statistics are shown in Table S4, and pepper reference genome mapping data statistics are shown in Table S5. We then analyzed the gene expression patterns in different samples of pepper fruits based on the FPKM values of genes (Figure S7). Cluster analysis (Figure 4a) showed that the biological repetition of pepper fruit samples from different samples had good classification. According to the PCA analysis results (PC1: 60.86% and PC2: 25.4%), the samples differed significantly in different varieties and stages (Figure 4b). Then, we used \(p<0.05\) and \(\,\mathrm{l}\,\mathrm{log2 FC}\,\mathrm{l}\geq 1\) as the screening conditions to screen the DEGs between different comparison groups. As shown in (Figure 4c,d), the number of DEGs in the four pepper varieties showed an increasing trend first and then decreased (Figure 4c), indicating that the gene expression change is the most active at the breaking stage, which may be the critical period for pepper to synthesize and metabolize VACs. The comparison of different varieties (Figure 4d) showed that there were more DEGs at the breaking stage and at the mature stage, which might be the critical period for the formation of differential VACs in different varieties. Furthermore, DEGs in different comparison groups showed that the number of down-regulated genes was much higher than that of up-regulated genes. Figure 4: Summary analysis of transcriptomes. (**a**) Cluster plot of the transcriptome sequencing samples; (**b**) PCA of 36 pepper fruit samples; (**c**) the number of DEGs during pepper fruit development; (**d**) the number of DEGs between the four pepper varieties. To further study the change of genes at the transcriptional level, we performed Venn analysis on the DEGs (Figures 5a-c and S8A-C). There were 5282, 4790, and 371 common DEGs (Figure 5a-c) at three stage comparison groups of the four varieties (a vs. b, a vs. c, and b vs. c), respectively. While 3273, 4807, and 4548 common DEGs (Figure S8A-C) were in four variety comparison groups at three stages (G) vs. CTJ, HDL1 vs. CTJ, and HDL2 vs. CTJ), respectively. Furthermore, the results of GO enrichment analysis showed that at level 2, a total of 45 functional subclasses were enriched between the comparison groups at stages (Figure 5d-f), including 22 biological processes, 13 cellular components, and 10 molecular functions. A total of 46 functional subclasses, including 21 biological processes, 13 cellular components, and 12 molecular functions, were enriched among different varieties (Figure S8D-F). KEGG enrichment analysis showed that 117, 114, and 57 metabolic pathways were enriched in three comparison groups at three stages. The pathways related to the synthesis and metabolism of VACs mainly included carotenoid biosynthesis (cann00906), fatty acid elongation (cann00062), alpha-Linolenic acid metabolism (cann00592), phenylpropanol biosynthesis (cann00940), and valine, leucine and isoleucine degradation (cann00280), fatty acid degradation (cann00071), and fatty acid biosynthesis (cann00061) (Figure 5g-i). These metabolic pathways were significantly enriched (\(p<0.05\)) in the comparison group of green and maturity stage, green stage and breaking stage, but were generally not significantly enriched in the comparison group of the maturity and breaking stages, indicating that the gene expression related to the synthesis of VACs mainly occurred in the period before the breaking stage, among which the breaking stage is the critical turning point. Common DEGs among different varieties were enriched to 34 metabolic pathways at the green stage, but most pathways were not significant (Figure S8G).