A novel strategy for developing CMS and restorer lines in pepper using intermediate isocytoplasmic materials

Abstract Cytoplasmic male sterility (CMS) is a crucial system for hybrid seed production in pepper ( Capsicum annuum L.), as it eliminates the need for manual emasculation and improves seed purity and production efficiency. However, the instability of CMS and corresponding restorer lines has constrained its large-scale utilization. In this study, a novel breeding strategy was established by combining cytoplasmic sterility genes from existing CMS lines with fertility-restoring genes from restorer lines through the creation of an intermediate restorer line, S( RfRf ). This intermediate, carrying sterile cytoplasm and fertile nuclei, served as a genetic bridge for the introgression of desirable agronomic traits from elite germplasm into new CMS and restorer lines. Both cytoplasmic and nuclear genotypes were accurately identified using CMS-SCAR 130 and CRF-SCAR molecular markers, confirming segregation patterns consistent with Mendel’s first law. Through systematic selection and marker-assisted breeding, isocytoplasmic CMS and restorer lines were developed, exhibiting uniform fertility and favorable agronomic performance. This approach reduced the land area and time required for breeding while enhancing the efficiency of line conversion. Overall, the method provides a reliable and scalable framework for the development of CMS-based hybrid systems in pepper, demonstrating its genetic stability and practical applicability for precision breeding.