Cytoplasmic male sterility (CMS) is an important tool for producing F₁ hybrids, which can exhibit heterosis. The companion system, restorer-of-fertility (Rf), is poorly understood at the molecular level and would be valuable in producing restorer lines for hybrid seed production. The identity of the Rf gene in Capsicum (pepper) is currently unclear. In this study, using bulked segregant RNA sequencing (BSR-seq), a strong candidate Rf gene, Capana06g002866, which is annotated as a NEDD8 conjugating enzyme E2, was identified. Capana06g002866 has an ORF of 555 bp in length encoding 184 amino acids; it can be cloned from F₁ plants from the hybridization of the CMS line 8A and restorer line R1 but is not found in CMS line 8A. With qRT-PCR validation, Capana06g002866 was found to be upregulated in restorer accessions compared to sterile accessions. The relative expression in flower buds increased with the developmental stage in F₁ plants, while the expression was very low in all flower bud stages of the CMS lines. These results provide new insights into the Rf gene in pepper and will be useful for other crops utilizing the CMS system.

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