Cytoplasmic male sterility (CMS) is pivotal in plant breeding and widely employed in various crop hybrids, including pepper. However, the functional validation of the restorer of fertility (Rf) gene in pepper has been lacking until now. This study identifies and characterizes CaRf, a single dominant locus crucial for restoring CMS in the pepper strong recovery inbred line Zhangshugang. The CaRf gene encodes a mitochondria-targeted pentatricopeptide repeat protein, validated through the induction of male sterility upon its silencing in hybrid F₁ plants. To enhance pepper breeding efficiency, 176 important pepper breeding parent materials were resequenced, and a PepperSNP50K liquid-phase breeding chip was developed, comprising 51 172 markers. Integration of CaRf functional characterization and PepperSNP50K facilitated the development of a high-quality red pepper hybrid. These findings provide significant insights and practical strategies for advancing molecular-designed breeding in peppers.

Pepper (Capsicum annuum L.) is an important horticultural crop whose growth, development, and yield formation are severely constrained by heat stress. The Mediator complex is a key transcriptional co-regulator in plants and plays important roles in developmental processes and stress responses. However, the MED gene family and its functions in heat stress responses remain largely unexplored in pepper. Using the chromosome-level reference genome of the cultivated pepper (Capsicum annuum var. annuum) cultivar Zhangshugang, a total of 49 CaMED genes were identified and classified into four conserved Mediator modules, namely the head, middle, tail, and kinase modules. Comprehensive bioinformatic analyses showed that CaMED genes are evolutionarily conserved across species, whereas differences in gene structure and sequence characteristics among family members may contribute to their functional diversification. Promoter analysis further showed that these genes contain abundant cis-acting elements related to light, phytohormone, and stress responses. Transcriptome analysis of the 49 identified CaMED genes showed distinct tissue-specific expression patterns, with many members showing preferential expression during early flower development and late placenta development. Furthermore, expression profiling of all CaMED genes using publicly available transcriptome datasets under 42 °C heat-stress conditions, followed by RT-qPCR validation of selected candidates, showed that CaMED25a displayed a relatively stable heat-responsive expression pattern. Virus-induced gene silencing of CaMED25a compromised heat tolerance in pepper plants under heat stress, as evidenced by increased H₂O₂ accumulation and significantly reduced expression of heat defense-related genes, including CaHSP18, CaHSP25.9, and CaHSP70.1. Taken together, this study provides an integrated analysis of the pepper CaMED gene family and reveals the positive contribution of CaMED25a to heat stress tolerance. These findings lay the groundwork for subsequent studies on CaMED gene function and the molecular regulation of high-temperature responses in pepper.

Research on the CBP gene family in plants is scarce, with only sporadic reports on its association with immune responses. No systematic study has explored how CBP family genes regulate pepper resistance against Phytophthora capsici. Here, we focused on pepper CaCBP2, an RNA-binding protein, whose expression was significantly induced by P. capsici. Functional validation via VIGS and heterologous overexpression confirmed CaCBP2 as a negative regulator of pepper resistance to P. capsici. Based on physiological assays, transcriptome sequencing and WGCNA, we speculate it may mediate immune responses by regulating antioxidant systems, defense hormone metabolism, and disease resistance-related genes. Our findings fill the relevant research gap, enrich the role of RNA-binding proteins in plant anti-phytophthora defense, and provide a novel target for crop disease-resistant breeding.