The Gametophyte factor1 (Ga1) locus in maize confers unilateral cross-incompatibility (UCI), and it is controlled by both pollen and silk-specific determinants. Although the Ga1 locus has been reported for more than a century and is widely utilized in maize breeding programs, only the pollen-specific ZmGa1P has been shown to function as a male determinant; thus, the genomic structure of the Ga1 locus and all the determinants that control UCI at this locus have not yet been fully characterized. Here, we used map-based cloning to confirm the determinants of UCI at the Ga1 locus and maize pan-genome sequence data to characterize the genomic structure of the Ga1 locus. The Ga1 locus comprises one silk-expressed pectin methylesterase gene (PME) (ZmGa1F) and eight pollen-expressed PMEs (ZmGa1P and ZmGa1PL1-7). Knockout of ZmGa1F in Ga1/Ga1 lines leads to the complete loss of the female barrier function. The expression of individual ZmGa1PL genes in a ga1/ga1 background endows ga1 pollen with the ability to overcome the female barrier of the Ga1 locus. These findings, combined with genomic data and genetic analyses, indicate that the Ga1 locus is modulated by a single female determinant and multiple male determinants, which are tightly linked. The results of this study provide valuable insights into the genomic structure of the Ga2 and Tcb1 loci and will aid applications of these loci in maize breeding programs.

Hybrid seed production technology (SPT) using genic recessive male sterility is of great importance in maize breeding. Here, we report a novel SPT based on a maize unilateral cross-incompatibility gene ZmGa1F with an extremely low transgene transmission rate (TTR). Proper pollen-specific ZmGa1F expression severely inhibits pollen tube growth leading to no fertilization. The maintainer line harbors a transgene cassette in an ipe1 male sterile background containing IPE1 to restore ipe1 male fertility, ZmGa1F to prevent transgenic pollen escape, the red fluorescence protein encoding gene DsRed2 for the separation of male sterile and fertile seeds, and the herbicide-resistant gene Bar for transgenic plant selection. When the maintainer line is selfed, gametes of ipe1/transgene and ipe1/- genotypes are produced, and pollen of the ipe1/transgene genotype is not able to fertilize female gametes due to pollen tube growth inhibition by ZmGa1F. Subsequently, seeds of ipe1/ipe1 and ipe1/transgene genotypes are produced at a 1:1 ratio and could be separated easily by fluorescence-based seed sorting. Not a single seed emitting fluorescence is detected in more than 200,000 seeds examined demonstrating that the pollen-tube-inhibition (PTI)-based TTR is lower than what has been reported for similar technologies to date. This PTI-based SPT shows promising potential for future maize hybrid seed production.