Genome-wide association studies and modeling of stomatal gas conductance reveal genetic control of water-use efficiency in sorghum

The increasing frequency and intensity of droughts present significant challenges to global food security. In this study, we examined the genetic and physiological mechanisms underlying drought tolerance and resilience in sorghum (Sorghum bicolor L.) by phenotyping the Sorghum Association Panel (SAP; n = 397) for a broad suite of traits. These included leaf anatomical characteristics (stomatal density [SD], stomatal size, pore area, stomatal pore area per leaf area, and anatomical maximum stomatal gas conductance), physiological traits [net photosynthetic rate (An), stomatal gas conductance (gsw), and intrinsic water-use efficiency (iWUE)], and functional traits (leaf width, leaf thickness, leaf mass area, and chlorophyll content). Substantial natural variation was detected within the SAP, and correlation analyses indicated that leaf anatomical and functional characteristics play key roles in regulating physiological traits, including An, gsw, and iWUE. Genome-wide association studies identified a genomic hotspot on chromosome 1 (77.5-78.6 Mb) region associated with 3 key single-nucleotide polymorphisms (S01_77550396, S01_78561058, and S01_78619413). Haplotype analysis of these loci uncovered 8 distinct allele combinations influencing SD, An, gsw, and iWUE. Application of the Ball-Woodrow-Berry gsw model to these haplotypes demonstrated that accessions from haplotypes 1 to 5 exhibited greater stomatal plasticity, displaying more dynamic responses under well-watered conditions. In contrast, accessions from haplotypes 6 to 8 showed more conservative stomatal behavior under water-limited conditions. These results provide insights into the coordinated genetic control of leaf traits underlying drought resilience in sorghum and offer a predictive framework for breeding cultivars with stable performance across diverse water regimes.