Identification of partial male fertility restoration conferred by the Ms2 locus in onion (Allium cepa L.) carrying CMS-S male-sterile cytoplasm

A stable restorer-of-fertility (Rf) gene is essential for efficient F1 hybrid seed production using cytoplasmic male sterility (CMS). Ms and Ms2 are known Rf loci in onion (Allium cepa L.). Although unstable male fertility restoration by Ms2 has been reported in some accessions, its stability in the CMS-S cytoplasm type (cytotype), which is widely used in F1 hybrid breeding, remains uncharacterized. To investigate this, segregating populations for both Rf loci were evaluated over five years for male fertility phenotypes and their corresponding genotypes. In onions with the CMS-S cytotype, the Ms2 locus conferred partial restoration of male fertility. Partially male-fertile (PMF) plants produced fewer pollen grains, many of which were deformed or non-viable. Pollen viability assays consistently demonstrated that plants with the CMS-R cytotype exhibited complete fertility restoration by either Rf locus, whereas the degree of restoration in plants with the CMS-S cytotype varied depending on the Ms2 genotype. PMF plants also produced fewer seeds than fully male-fertile plants when self-pollinated. Transcriptional analysis suggested that partial restoration may result from insufficient suppression of orf725, the CMS-inducing gene, by Ms2. The CMS-S cytotype exhibited higher orf725 expression than CMS-R, indicating that elevated transcription levels in CMS-S may exceed the suppression ability of Ms2. Unexpectedly, the PMF phenotype was also observed in plants with the CMS-S cytotype carrying both dominant Ms and Ms2 alleles. These results suggest a potential interaction between Ms and Ms2 in the restoration of male fertility in onion.