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Background  Cytoplasmic male sterility (CMS), which naturally exists in higher plants, is a useful mechanism for analyzing nuclear and mitochondrial genome functions and identifying the role of mitochondrial genes in the plant growth and development. Polima ( pol ) CMS is the most universally valued male sterility type in oil-seed rape. Previous studies have described the  pol  CMS restorer gene  Rfp  and the sterility-inducing gene  orf224  in oil-seed rape, located in mitochondria. However, the mechanism of fertility restoration and infertility remains unknown. Moreover, it is still unknown how the fecundity restorer gene interferes with the sterility gene, provokes the sterility gene to lose its function, and leads to fertility restoration.    Result  In this study, we used multi-omics joint analysis to discover candidate genes that interact with the sterility gene  orf224  and the restorer gene  Rfp  of  pol  CMS to provide theoretical support for the occurrence and restoration mechanisms of sterility. Via multi-omics analysis, we screened 24 differential genes encoding proteins related to RNA editing, respiratory electron transport chain, anther development, energy transport, tapetum development, and oxidative phosphorylation. Using a yeast two-hybrid assay, we obtained a total of seven  Rfp  interaction proteins, with  orf224  protein covering five interaction proteins.    Conclusions  We propose that  Rfp  and its interacting protein cleave the transcript of  atp6/orf224 , causing the infertility gene to lose its function and restore fertility. When  Rfp  is not cleaved,  orf224  poisons the tapetum cells and anther development-related proteins, resulting in  pol  CMS mitochondrial dysfunction and male infertility. The data from the joint analysis of multiple omics provided information on pol CMS’s potential molecular mechanism and will help breed  B. napus  hybrids.    Supplementary Information  The online version contains supplementary material available at 10.1186/s12870-021-02852-7.

High-generation near-isogenic lines combined with multi-omics to study the mechanism of polima cytoplasmic male sterility