Multi-step formation, evolution, and functionalization of new cytoplasmic male sterility genes in the plant mitochondrial genomes

New gene origination is a major source of genomic innovations that confer phenotyp ic changes and biological di- versity. Generation of new mitochondrial genes in plants may cause cytoplasmic male sterility （CMS）, which can pro- mote outcrossing and increase fitness. However, how mitochondrial genes originate and evolve in structure and func- tion remains unclear. The rice Wild Abortive type of CMS is conferred by the mitochondrial gene WA352c （previously named WA352） and has been widely exploited in hybrid rice breeding. Here, we reconstruct the evolutionary tra- jectory of WA352c by the identification and analyses of 11 mitochondrial genomic recombinant structures related to WA352c in wild and cultivated rice. We deduce that these structures arose through multiple rearrangements among conserved mitochondrial sequences in the mitochondrial genome of the wild rice Oryza rufipogon, coupled with sub- stoichiometric shifting and sequence variation. We identify two expressed but nonfunctional protogenes among these structures, and show that they could evolve into functional CMS genes via sequence variations that could relieve the self-inhibitory potential of the proteins. These sequence changes would endow the proteins the ability to interact with the nucleus-encoded mitochondrial protein COXI1, resulting in premature programmed cell death in the anther ta- petum and male sterility. Furthermore, we show that the sequences that encode the COXll-interaction domains in these WA352c-related genes have experienced purifying selection during evolution. We propose a model for the for- mation and evolution of new CMS genes via a ＂multi-recombination/protogene formation/functionalization＂ mecha- nism involving gradual variations in the structure, sequence, copy number, and function.