Phenotyping stomatal closure by thermal imaging for GWAS and TWAS of water use efficiency-related genes

Abstract

Stomata allow CO₂ uptake by leaves for photosynthetic assimilation at the cost of water vapor loss to the atmosphere. The opening and closing of stomata in response to fluctuations in light intensity regulate CO₂ and water fluxes and are essential to maintenance of water-use efficiency (WUE). However, little is known about the genetic basis for natural variation in stomatal movement, especially in C₄ crops. This is partly because the stomatal response to a change in light intensity is difficult to measure at the scale required for association studies. High-throughput thermal imaging was used to bypass the phenotyping bottleneck and assess 10 traits describing stomatal conductance (g_s) before, during and after a stepwise decrease in light intensity for a diversity panel of 659 sorghum accessions. Results from thermal imaging significantly correlated with photosynthetic gas-exchange measurements. g_s traits varied substantially across the population and were moderately heritable (h² up to 0.72). An integrated genome-wide and transcriptome-wide association study (GWAS/TWAS) identified candidate genes putatively driving variation in stomatal conductance traits. Of the 239 unique candidate genes identified with greatest confidence, 77 were orthologs of Arabidopsis genes related to functions implicated in WUE, including stomatal opening/closing (24 genes), stomatal/epidermal cell development (35 genes), leaf/vasculature development (12 genes), or chlorophyll metabolism/photosynthesis (8 genes). These findings demonstrate an approach to finding genotype-to-phenotype relationships for a challenging trait as well as candidate genes for further investigation of the genetic basis of WUE in a model C₄ grass for bioenergy, food, and forage production.