Complete mitochondrial genome map and transcriptome reveal the mechanisms of a novel neau type CMS in Brassica rapa

Cytoplasmic male sterility (CMS) is a widespread phenomenon in crops and is beneficial for commercial hybrid breeding. Although CMS systems in Brassica species are extensively utilized, they are derived from relatively limited sources, and the underlying molecular mechanisms are not well understood. In this study, a novel CMS line, named Neau, was identified through distant hybridization between Matthiola incana and Brassica rapa. Neau CMS exhibits agronomic traits similar to its maintainer line of Chinese cabbage and displays sterility at the tetrad stage. The mitochondrial genomes of Neau CMS and its maintainer line were assembled to 359596 bp and 219771 bp, respectively, encoding 99 and 66 unknown open reading frames (orfs). Six orfs were identified as specific to Neau CMS with sequence alignments, with orf154 and two copies of orf138 identified as candidate genes based on their transmembrane structures and their ability to encode toxic proteins that inhibited Escherichia coli cell growth. The expression of orf154 was induced during the tetrad stage by quantitative real-time polymerase chain reaction(qRT-PCR), whereas orf138a showed higher expression at the microspore mononuclear stage in Neau CMS. Transcriptome analysis revealed reduced expressions of most pollen development-related genes, as well as genes involved in peroxidase/oxidative stress responses and the electron transfer chain in Neau CMS. Additionally, Neau CMS exhibited increased levels of O₂^.-, H₂O₂, and malondialdehyde (MDA), alongside reduced activities of SOD, CAT, and POD. Both ATP content and ATPase activity were down-regulated in Neau CMS. Overexpression of orf138 and orf154 in Arabidopsis thaliana resulted in reductions in pollen quantity, viability, and seed production. These findings suggest that orf138 and orf154 are key candidate genes responsible for the sterility observed in Neau CMS in Chinese cabbage.