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

Abstract

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.

Keywords: Brassica napus; CMS; Multi-omics analysis; Polima; Rfp; orf224.

Fig. 1

Stereomicroscopy and scanning electron microscopy analyses of differences in anther development and pollen development in sterile and reintroduction materials. (A, C, E, G, I) pol CMS sterile plants anthers, flower buds, and pollen grains in the corresponding period. (B, D, F, H, J) pol CMS recultivated plant stamens mature anthers, flower buds, pollen grains during differentiation

Fig. 2

Overview of metabolomics data. a The metabolome type and the number of species in each group. b The principal component analysis showed significant differences between the two traits

Fig. 3

Differences in metabolites between sterile and restorer lines in the metabolome. a Volcano plot of differential metabolites of the metabolome. b Histogram of the top 20 substances in the differential metabolites of the metabolome

Fig. 4

Differentially abundant proteins (DAPs) between the sterile line and its iso-nuclear maintainer line. Volcano plot of DAPs (fold > 1.5)

Fig. 5

Gene Ontology (GO) classifications of differentially abundant proteins (DAPs) between the sterile line and its reproductive line. The x-axis represents the number of DAP proteins in each category, and the y-axis represents each GO term

Fig. 6

Transcriptome analysis results. a Volcano plot of the results of transcriptome analysis of significantly differentially expressed gene clusters. b KEGG pathway functional annotation gene distribution map of significantly differentially expressed genes

Fig. 7

A Possible Mechanism Contributing to the Occurrence of Sterility and Recovery of Fertility in Pol CMS Lines of B. napus. a Multi-omics association analysis of candidate genes using yeast to screen for related genes. b A proposed a pol CMS working model showing the pol CMS mechanism and infertility mechanism of Rfp and orf224