Development of male-sterile lines of Setaria viridis to accelerate C ₄ model plant genetics

Setaria viridis is a diploid C ₄ grass in the Poaceae family, notable for its rapid life cycle of 6–8 weeks from sowing to seed—much shorter than the 4–5 months required by crops such as Zea mays and Sorghum bicolor . This fast growth makes S. viridis a valuable model for C ₄ crop research. Genetic crosses are essential for studying gene function, but manual crossing is labor-intensive and time-consuming. To address this, we developed a male-sterile line by targeting the S. viridis ortholog of Setaria italica NO POLLEN 1 ( SiNP1 ), which encodes a glucose–methanol–choline oxidoreductase required for pollen exine formation. Using Cas9 and TREX2-mediated genome editing, we generated SiNP1 knockouts in both the S. viridis ME034V and A10.1 backgrounds that were fully male-sterile. Backcrossing T ₀ male-sterile plants to ME034V yielded a stable line homozygous for a 59 bp deletion, easily genotyped by PCR. Using this line, we developed a simple and efficient crossing protocol that eliminates the need for emasculation. This method enables a single person to perform up to 100 crosses per day—compared to 15 using traditional methods—and yields 20–32 F ₁ hybrid seeds per panicle with 100% genetic purity. We also quantified pollen flow and outcrossing frequencies under greenhouse conditions to develop optimal bagging strategies and prevent unintended pollination.This resource accelerates genetic research in S. viridis , enhancing its utility as a premier C ₄ model for mapping and functional genomics.