Aroma is a crucial attribute affecting the quality of pepper and its processed products, which has significant commercial value. However, little is known about the composition of volatile aroma compounds (VACs) in pepper fruits and their potential molecular regulatory mechanisms. In this study, HS-SPME-GC-MS combined with transcriptome sequencing is used to analyze the composition and formation mechanism of VACs in different kinds and development stages of pepper fruits. The results showed that 149 VACs, such as esters, alcohols, aldehydes, and terpenoids, were identified from 4 varieties and 3 development stages, and there were significant quantitative differences among different samples. Volatile esters were the most important aroma components in pepper fruits. PCA analysis showed that pepper fruits of different developmental stages had significantly different marker aroma compounds, which may be an important provider of pepper’s characteristic aroma. Transcriptome analysis showed that many differential genes (DEGs) were enriched in the metabolic pathways related to the synthesis of VACs, such as fatty acids, amino acids, MVA, and MEP in pepper fruits. In addition, we identified a large number of differential transcription factors (TFs) that may regulate the synthesis of VACs. Combined analysis of differential aroma metabolites and DEGs identified two co-expression network modules highly correlated with the relative content of VACs in pepper fruit. This study confirmed the basic information on the changes of VACs in the fruits of several Chinese spicy peppers at different stages of development, screened out the characteristic aroma components of different varieties, and revealed the molecular mechanism of aroma formation, providing a valuable reference for the quality breeding of pepper.

The wide application of pepper is mostly related to the content of capsaicin, and phenylpropanoid metabolism and its branch pathways may play an important role in the biosynthesis of capsaicin. The expression level of MYB24, a transcription factor screened from the transcriptome data of the pepper fruit development stage, was closely related to the spicy taste. In this experiment, CcMYB24 was cloned from Hainan Huangdenglong pepper, a hot aromatic pepper variety popular in the world for processing, and its function was confirmed by tissue expression characteristics, heterologous transformation in Arabidopsis thaliana, and VIGS technology. The results showed that the relative expression level of CcMYB24 was stable in the early stage of pepper fruit development, and increased significantly from 30 to 50 days after flowering. Heterologous expression led to a significant increase in the expression of CcMYB24 and decrease in lignin content in transgenic Arabidopsis thaliana plants. CcMYB24 silencing led to a significant increase in the expression of phenylpropanoid metabolism pathway genes PAL, 4CL, and pAMT; lignin branch CCR1 and CAD; and capsaicin pathway CS, AT3, and COMT genes in the placenta of pepper, with capsaicin content increased by more than 31.72% and lignin content increased by 20.78%. However, the expression of PAL, pAMT, AT3, COMT, etc., in the corresponding pericarps did not change significantly. Although CS, CCR1, and CAD increased significantly, the relative expression amount was smaller than that in placental tissue, and the lignin content did not change significantly. As indicated above, CcMYB24 may negatively regulate the formation of capsaicin and lignin by regulating the expression of genes from phenylpropanoid metabolism and its branch pathways.

To streamline germplasm preservation, enhance resource utilization, and improve breeding efficiency, a core germplasm bank was established using 24 phenotypic traits and DNP markers from 155 pepper resources across various regions. Selection of the optimal core germplasm was based on intra-group retention ratio, overall retention scale, and intra-group stepwise clustering retention. Evaluation of phenotypic trait data for the core germplasm utilized mean, variance, range, and coefficient of variation, with principal component analysis confirming the selection. For molecular evaluation, the core germplasm pre-selection plan was based on SSR clustering, allele retention ratio, and Shannon-Weaver diversity index. This approach resulted in a core germplasm of 41 resources, including 6 var. fasciculatum, 7 var. grossum, 3 var. cerasiorme, 6 var. conoides, and 19 var. longum, representing the maximum phenotypic retention and genetic diversity of the 155 pepper resources. Additionally, a core germplasm of 32 resources was generated based on SSR markers, retaining all 54 polymorphic loci. By integrating phenotypic and molecular core collections, a combined core collection of 57 varieties was developed. This collection achieved a 92.55% phenotype retention ratio and a 100% polymorphism site retention ratio. With a 90% compression ratio, it encapsulates the broad genetic diversity of the original germplasm, serving as a comprehensive resource for further research and breeding applications.

Aroma is an important processing and consumption quality trait of fruits and vegetables, and terpenes produced from the terpenoid metabolic pathway are a critical component of chili fruit flavor. This pathway involves the participation of at least eighteen enzymes, such as AACT, HMGS, HMGR, MVK, PMK, MVD, FPPS, GGPPS, DXS, DXR, MCT, CMK, MECPS, HDS, HDR, GPPS, IDI, and TPS. In this study, the genome wide information, expression characteristics, and relationship with terpenoids of terpenoid pathway genes are analyzed in C. annuum. The results showed that C. annuum has sixty-seven genes related to terpene metabolic pathways. Non-targeted metabolomics studies found that the content of aromatic terpenoids α-calacorene, α-cubene, and cis-β-farnesene increased with fruit development in HDL fruits, while linalool and nerolidol were much higher in GLD608. Correlation analyses between qRT-PCR and metabolome data showed that the expression levels of CaHMGS-3, CaMVD-1, CaCMK-1, and CaGGPPS-2 were positively correlated with the content of linalool, a flavor monoterpene alcohol. CaMECPS-1 was positively correlated with cis-β-farnesene, and there was also a significant positive regulatory relationship between CaTPS-5 and nerolidol relationship. In conclusion, the present study provides genetic resources for further studies on the gene regulatory mechanisms of flavor synthesis and terpenoid metabolic pathways in chili.