Optical topometry and machine learning to rapidly phenotype stomatal patterning traits for maize QTL mapping

Stomata are adjustable pores on leaf surfaces that regulate the tradeoff of CO2 uptake with water vapor loss, thus having critical roles in controlling photosynthetic carbon gain and plant water use. The lack of easy, rapid methods for phenotyping epidermal cell traits have limited discoveries about the genetic basis of stomatal patterning. A high-throughput epidermal cell phenotyping pipeline is presented here and used for quantitative trait loci (QTL) mapping in field-grown maize (Zea mays). The locations and sizes of stomatal complexes and pavement cells on images acquired by an optical topometer from mature leaves were automatically determined. Computer estimated stomatal complex density (SCD; R2 = 0.97) and stomatal complex area (SCA; R2 = 0.71) were strongly correlated with human measurements. Leaf gas exchange traits were genetically correlated with the dimensions and proportions of stomatal complexes (rg = 0.39-0.71) but did not correlate with SCD. Heritability of epidermal traits was moderate to high (h2 = 0.42-0.82) across two field seasons. Thirty-six QTL were consistently identified for a given trait in both years. Twenty-four clusters of overlapping QTL for multiple traits were identified, with univariate versus multivariate single marker analysis providing evidence consistent with pleiotropy in multiple cases. Putative orthologs of genes known to regulate stomatal patterning in Arabidopsis (Arabidopsis thaliana) were located within some, but not all, of these regions. This study demonstrates how discovery of the genetic basis for stomatal patterning can be accelerated in maize, a C4 model species where these processes are poorly understood.