*3.1. ORF178 and ORF103c Identified as CMS Candidate Mitochondrial Genes of SXCMS5A*

In plants, the production of most CMS was closely related to the variation, recombination and rearrangement of mitochondrial genome, resulting in a large number of new chimeric ORFs, which changed the transcription and translation products of genes, affected the expression and loss of function of genes, and led to male sterility in plants [12,26,27]. In rice, the CMS of the wild abortion type and Honglian type had been intensively investigated. Among the genes involved in these CMS variants, *WA352*, a CMS sterility gene, interacted with mitochondrial protein COX11 to stimulate the degradation of tapetum in anther, which in turn led to pollen abortion [28]. ORFH79, a protein expressed by a CMS sterility gene of the rice Honglian type, interacted with p61, a small subunit of mitochondrial respiratory electron transport chain complex III, resulting in ATP concentration decreased and ROS amount increased, which eventually led to cytoplasmic male sterility [29]. In this study, 13 DEGs included 12 ORFs and 1 *COX2* were mitochondrial genome genes between SXCMS5A and SXCMS5B of which three (*ORF103c*, *ORF151* and *ORF178*) were expressed almost exclusively in differential soybean CMS lines. Furthermore, both *ORF103c* and *ORF178* encode transmembrane proteins, which is one of the main characteristics of CMS genes [14,22]. Interestingly, *ORF178* was formed during the process of genome recombination in a soybean CMS line NJCMS1A, and it was also found that *ORF178* was expressed in NJCMS1A, NJCMS4A and NJCMS5A [22]. However, *ORF103c* was found in SXCMS5A, SXCMS6A and SXCMS7A, but not in NJCMS1A. In addition, *ORF261*, another CMS candidate gene in NJCMS1A, was downregulated in SXCMS5A compared to SXCMS5B, which might be caused by cytoplasmic differences. Thus, the upregulated expression of *ORF178* and *ORF103c* might change the transcription and translation products of genes and affect gene expression and loss of function, which was related to pollen abortion and male sterility in SXCMS5A. Since the role of these DEGs in pollen development had not been previously reported, these findings offered a new direction for investigations of the molecular mechanisms underlying soybean CMS.

### *3.2. Down-Regulation of DEGs Associated with Pollen Wall Development Is One of the Key Factors of Pollen Development Defect in SXCMS5A*

The development of the pollen wall in pollen grains was a requirement for plant sexual reproduction, and most of the characters associated with male sterility were related to abnormal development of the pollen wall [30]. Pectin metabolism played an important role in pollen development; thus, inhibition of pectin metabolism during pollen development would lead to delayed pollen development, male sterility, and a lower seed setting rate [31,32]. In this study, we identified 11 PMEs, all of which were downregulated in the CMS line SXCMS5A compared to SXCMS5B. These genes were predicted to be correlated with pectinesterase activity. In addition, three PLs were also downregulated in the CMS line SXCMS5A compared to SXCMS5B. PL and PME were two important enzymes involved in the degradation of plant pectin and the formation of pollen walls in plants [23,24]. PL, Exo-PG, and PME were associated with male fertility restoration of the CMS line in pepper, and PL and PME played an important role in pollen development [33]. Thus, the downregulated expression of PL and PME genes might result in abnormal pollen wall development, which was related to pollen abortion and male sterility in SXCMS5A.
