Fine Mapping and Candidate Gene Analysis of <i>Male Sterile 4</i> Promotes Complete Male Sterility in Common Wild Rice ( <i>Oryza rufipogon</i> )

The transverse filament (TF) protein of the synaptonemal complex (SC) is crucial for rice meiosis, but its role in male sterility remains unexplored. We used Yuanjiang common wild rice (YJCWR) as a donor parent and developed a series of introgression lines (ILs, BC₄) in the genetic background of YUNDAO 1. <i>Male Sterile 4</i> (<i>MS4</i>) was mapped to a 49.8 kb region containing <i>OsZEP1</i> (<i>Os04g37960</i>), which encodes a transverse filament (TF) protein. Homozygous sterile lines exhibited shorter, weaker, pale yellow anthers and sterile pollens compared to fertile lines. SEM analysis reveals that <i>OsZEP1-CWR</i> impairs anther cuticle and pollen wall formation. Transverse semithin sections of sterile anthers show abnormal cuticle, pollen exine formation, delayed tapetum degradation, and defective microspores. CRISPR/Cas9 <i>OsZEP1</i> knockouts validated <i>OsZEP1</i> as the <i>MS4</i> candidate gene, causing complete male sterility. This study highlights the role of <i>MS4</i> in male fertility, and the newly developed KASP markers will be useful for marker-assisted breeding.