Two nuclear loci Rf-m1 and Rf-m2 interplay to determine the fertility restoring efficacy of ZD-type CMS soybean

Two Restorer-of-fertility loci, Rf-m1 and Rf-m2, co-condition fertility restoration in ZD-type CMS soybean though complex genetic interactions in both gametophytic and sporophytic manners. In soybean (Glycine max), the genetic mechanisms underlying fertility restoration of ZD-type cytoplasmic male sterility (CMS-ZD) are not fully understood. By crossing the CMS-ZD line W931A with its strong restorer WR016, we elucidated the inheritance patterns of fertility restoration. We discovered that restoration by WR016 is primarily governed by two Restorer-of-fertility (Rf) loci, Rf-m1 and Rf-m2, located within ~ 220 kb on chromosome 16 and ~ 117 kb on chromosome 9, respectively. They exhibited two distinctive inheritance modes across genotype combinations. In plants with the genotype of -/rf-m1 -/rf-m2, such as F₁ hybrids, gametophytic inheritance mode is predominant. In this case, Rf-m1 in haploid tissue is essential for pollen fertility restoration, and Rf-m2 is a modifier to enhance the effect. Correspondingly, plants displayed low pollen staining rates (sterile to ~ 50% depending on genotype). Conversely, in plants with one locus present as the homozygous Rf/Rf genotype, sporophytic inheritance mode is predominant. Plants exhibited high pollen staining rates (mostly > 80%), and the segregation of heterozygous loci in the next generation followed the Mendelian ratio. Notably, alleles rf-m1 and especially rf-m2 appear to suppress fertility. Further experiments revealed the best candidates of Rf-m1 and Rf-m2 are Glyma.16G139800 and Glyma.09G171200, encoding mitochondrial pentatricopeptide repeat (PPR) proteins GmPPR567 and GmPPR497. Haplotype analysis revealed that lines carrying homozygous Rf /Rf or rf /rf at both loci are rare among domesticated soybean germplasms. Using locus-specific markers and in silico haplotype screening, we identified two effective restorers and one maintainer line in elite germplasms, facilitating hybrid breeding in soybean.