A predicted NEDD8 conjugating enzyme gene identified as a Capsicum candidate Rf gene using bulk segregant RNA sequencing

Abstract

Cytoplasmic male sterility (CMS) is an important tool for producing F₁ hybrids, which can exhibit heterosis. The companion system, restorer-of-fertility (<i>Rf</i>), is poorly understood at the molecular level and would be valuable in producing restorer lines for hybrid seed production. The identity of the <i>Rf</i> gene in <i>Capsicum</i> (pepper) is currently unclear. In this study, using bulked segregant RNA sequencing (BSR-seq), a strong candidate <i>Rf</i> 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 <i>Rf</i> gene in pepper and will be useful for other crops utilizing the CMS system.