Analysis of Rfo-Mediated Network in Regulating Fertility Restoration in Brassica oleracea

Ogura cytoplasmic male sterility (CMS) lines play a crucial role in the utilization of heterosis. However, valuable traits, such as disease resistance genes from Ogura CMS hybrids, are challenging to incorporate for germplasm innovation, particularly in cabbage and broccoli. To date, the <i>Rfo</i>-mediated network regulating fertility restoration remains largely unexplored. In this study, we conducted a transcriptomic analysis of broccoli flower buds from Ogura CMS SFB45 and its <i>Rfo</i>-transgenic fertility restoration line, pRfo, at different stages of pollen development. Gene Ontology (GO) terms such as "pollen exine formation", "flavonoid metabolic and biosynthetic processes", and "pollen wall assembly", along with Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways including "flavonoid biosynthesis", "MAPK signaling pathway-plant", and "ABC transporters", were significantly enriched. We identified five differentially expressed genes (DEGs) involved in tapetum-mediated callose metabolism, thirty-four DEGs related to tapetum-mediated pollen wall formation, three DEGs regulating tapetum programmed cell death (PCD), five MPKs encoding DEGs, and twelve DEGs associated with oxidative phosphorylation. Additionally, yeast two-hybrid and bimolecular fluorescence complementation (BiFC) assays demonstrated that RFO directly interacts with ORF138 at the protein level. These findings provide valuable insights into the fertility recovery mechanisms regulated by <i>Rfo</i> in broccoli and offer important clues for breeders aiming to enhance Ogura CMS hybrids in <i>Brassica oleracea</i>.