Multi-locus genetic basis of stomatal density and size in maize revealed by integrated QTL and GWAS

Stomatal patterning is a fundamental aspect of plant photosynthetic performance and water-use efficiency, yet its genetic basis in C₄ grasses such as maize remains poorly understood. Here, we integrated linkage analysis across three recombinant inbred line populations and a genome-wide association study in a diverse maize panel to dissect the genetic architecture of six stomatal traits. This approach identified 33 quantitative trait loci and 51 association loci for 6 stomatal traits and revealed a significant negative genetic correlation between stomatal density (SD) and size, with multiple genomic intervals showing co-localization of effects on both trait categories. By integrating reported cell-type-specific transcriptomic data, we prioritized high-confidence candidate genes, including conserved stomatal regulators as well as previously uncharacterized candidates involved in processes such as cell wall biosynthesis. Functional analyses of EMS-induced mutants for candidates ZmbHLH140 and ZmCESA6 demonstrated that loss of ZmbHLH140 reduces SD, whereas mutation of ZmCESA6 has the opposite effect. In addition, the signature of selection at the ZmbHLH140 locus is consistent with a role for stomatal regulation during maize domestication. Together, our results establish a comprehensive genetic framework for stomatal development in maize, highlighting both conserved and grass-specific mechanisms that shape stomatal patterning and offering a foundation for future functional studies of stomatal development in cereals.