## 2 Results
### The Expression Changes of Key Genes in the Biosynthesis Pathway of VACs  
Volatile esters (especially branched-chain esters) and aldehydes, which are the most abundant and changing substances in pepper fruits, are mainly produced by the fatty acid pathway (FAP) and branched-chain amino acid pathway (BCAAP). According to transcriptome annotation and homologous sequence alignment, 90 genes involved in FAP and 35 genes involved in BCAAP were identified with differential expression (Figure 6 and Table S6). In the FAP (Figure 6 (left)), we identified 13 genes encoding LOX, 1 HPL, 11 ADH, and 3 AAT, among which most of the DEGs were significantly downregulated in pepper fruit development (\(p<0.05\)), which is consistent with the change of VACs of straight-chain fatty acids such as hexanal and (e)-2-hexanal, indicating that they may play an important role in this process. Similarly, the genes encoding ACC, MAT, FAD, AOS, and ACX showed similar expression patterns. In addition, we found the _LOC107850471_ gene encoding SAD, the _LOC107839570_ gene encoding 9S-LOX, and the _LOC107867711_ gene encoding ADH had significantly high expression at the breaking and maturation stages, indicating that these genes may be associated with the significant accumulation of VACs of straight-chain fatty acids such as hexyl hexanoate in middle and late pepper fruits. Compared with different varieties, most of the DEGs showed lower expression levels compared with CTJ in GJ, HDL1, and HDL2 pepper fruits of the same period. Branched-chain aminotransferase (BCATs) perform the last step of synthesis of L-leucine, L-isoleucine, and L-valine and the first step of catabolism to produce volatile branched-chain esters in the BCAAP (Figure 6 (right) and Table S6). Three BCAT genes showed significant differences in different samples, among which _LOC107851037_ was significantly downregulated during pepper fruit development, while _LOC107867296_, showed higher expression at the breaking and maturation stages, indicating that the gene of this family may have some control in the generation of volatile esters in BCAAP comprehensively and coordinately. Interestingly, the expression level of _LOC107867296_ was significantly higher in HDL2, which may be related to the significant accumulation of 4-methylpentyl 8-methylmonanoate (_Z39_) in pepper fruit at the breaking stage of HDL2, but was almost undetectable in the other three pepper varieties. As the key enzyme that catalyzes the straight-chain aldehydes to produce acids in BCAAP, seven genes encoding Aldehyde  
Figure 5: Functional enrichment analysis of DEGs in different developmental stages. (**a**,**b**) Venn diagram of the DEGs four in pepper varieties at different growth periods; (**d**–**f**) gene ontology (GO) classifications of overlapping DEGs in (**a**–**c**); (**g**–**i**) Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment of DEGs in (**a**–**c**). dehydrogenase (ALDH) were identified, and most of these genes' expression are significantly up-regulated in the fruit development, among which LOC107862407 has more significant expression, indicating that it may have a more critical role in the synthesis of branched ester. In our study, 10 Carboxylesterase (CXE) genes were identified, among which _LOC107872419_ and _LOC107859022_ gene expression were significantly reduced in the middle and late stages of pepper fruit development, which may be related to the significant increase of most branched-chain esters in pepper fruit at breaking and maturation stages. In addition, we found that the _LOC107867571_ and _LOC107859022_ genes encoding CXE were more highly expressed in HDL2 than the other three varieties, which may associate with 4-methylpentyl 8-methylnonanoate (Z39) significant accumulation at the mature stage. Figure 6: Expression profiles of volatile-related genes involved in the fatty acid (**left**) and branched-chain amino acid (**right**) metabolism pathway in different pepper fruit samples (red represents high expression, and blue represents low expression).