Re-Sequencing the Mitochondrial Genome Unveils a Novel Isomeric Form of NWB CMS Line in Radish and Functional Verification of Its Candidate Sterile Gene

Radish (Raphanus sativus L.) is a globally significant vegetable and relies on cytoplasmic male sterile (CMS) lines for hybrid seed production. The NWB CMS type is favored over Ogura CMS for its ease in maintainer screening. Despite its varied mitochondrial configurations and unvalidated sterile gene, we re-sequenced the mitochondrial genome of NWB CMS Tibet A and verified the function of the sterility gene via genetic transformation of Arabidopsis thaliana. The mitochondrial genomes of Tibet A could be assembled into circular DNA molecules, with a mitochondrial genome size of 239,184 bp. Our analysis indicated that the specific orf463a was the CMS-associated gene in Tibet A, sharing sequence consistency with the CMS gene in DCGMS and NWB CMS YB-A. Collinearity analysis showed that the mitochondrial genomes of NWB CMS Tibet A, DCGMS, and NWB CMS YB-A share the same mitotype, with structural variations due to recombination via a 9731 bp long repeat sequence and a 508 bp short repeat sequence. Driven by the Ap3 promoter, transgenic Arabidopsis with orf463a exhibited male sterility, confirming the gene’s potential role in CMS. In this study, we assembled a new isomeric form of NWB CMS mitochondrial genome and proved the function of the candidate sterile gene.