Discovery of Four Novel ORFs Responsible for Cytoplasmic Male Sterility (CMS) in Cotton (Gossypium hirsutum L.) through Comparative Analysis of the Mitochondrial Genomes of Four Isoplasmic Lines

Cytoplasmic male sterility (CMS) is an important feature for achieving heterosis in the development of hybrid crops. Mitochondria contribute to CMS, especially via mitochondrial DNA (mtDNA) rearrangements and chimeric genes. However, the mechanisms of CMS have not been fully elucidated, and the isonuclear alloplasmic lines used in previous studies have limited utility in cotton CMS research. In this study, three CMS lines (J4A-1, J4A-2 and J4A-3) and their isoplasmic maintainer line (J4B) were analyzed for mtDNA structural differences via high-throughput sequencing. The results showed that mtDNA was conserved (with similarities higher than 99%) among the three CMS lines and their isoplasmic maintainer line. All lines harbored 36 known protein-coding genes, 3 rRNAs, and 15 tRNAs. The protein-coding genes with non-synonymous mutations mainly encoded two types of proteins: ATPase and ribosomal proteins. Four new open reading frames (ORFs) (orf116b, orf186a-1, orf186a-2 and orf305a) were identified as candidate ORFs responsible for CMS. Two of the ORFs (orf186a-1 and orf186a-2) were identified as orf4 and orf4-2 of the upland cotton CMS line 2074A (a line with Gossypium harknessii Brandegee CMS-D2-2 cytoplasm), respectively. These findings provide a reference for CMS research in cotton or other crops.