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Cytoplasmic male‐sterile (CMS) lines are being used to produce hybrid seeds. Thus far, four CMS sources in soybean [Glycine max (L.) Merr.] have been reported in China. However, they are not sufficient or efficient in meeting the requirements of commercial soybean hybrid seed production. In this study, 33 varieties were tested for CMS using 45 crosses among 37 landraces and 17 annual wild soybean accessions (Glycine soja Sieb. et Zucc.). The cross of N23661 × N23658 showed partial to complete male sterility in backcross generations, while the corresponding reciprocal cross showed normal male fertility. Thus, the cytoplasm of N23661 is male‐sterile, the continuously backcrossed line is a male‐sterile line (designated NJCMS4A), and N23658 is its maintainer (designated NJCM4B). The male fertility of NJCMS4A was restored by another accession, Nansheng9403. Accordingly, NJCMS4A along with its maintainer and restorer composes a complete set of three lines for producing hybrid soybean. Using mitochondrial markers and sequence analyses, NJCMS4A is a CMS line with its cytoplasm not identical to the four previously reported CMS sources in soybean.

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In soybean, heterosis achieved through the three-line system has been gradually applied in breeding to increase yield, but the underlying molecular mechanism remains unknown. We conducted a genetic analysis using the pollen fertility of offspring of the cross NJCMS1A×NJCMS1C. All F1 plants’ pollen was semi-sterile; in F2, the ratio of pollen-fertile plants to pollen semi-sterile plants was 208:189. This result indicates NJCMS1A is gametophyte sterile, and the fertility restoration of NJCMS1C to NJCMS1A is a quality trait controlled by a single gene locus. Using bulked segregant analysis, the fertility restorer gene Rf in NJCMS1C was located on chromosome 16 between the markers BARCSOYSSR_16_1067 and BARCSOYSSR_16_1078. Sequence analysis of genes in that region showed that GmPPR576 was non-functional in rf cultivars. GmPPR576 has one functional allele in Rf cultivars but three non-functional alleles in rf cultivars. Phylogenetic analysis showed that the GmPPR576 locus evolved rapidly with the presence of male-sterile cytoplasm. GmPPR576 belongs to the RFL fertility restorer gene family and is mitochondrial-targeted. GmPPR576 was knocked out in soybean N8855 using CRISPR/Cas9. The T1 plants showed sterile pollen, and T2 plants produced few pods at maturity. The results indicate that GmPPR576 is the fertility restorer gene of NJCMS1A.

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