Genomic Science Program
U.S. Department of Energy | Office of Science | Biological and Environmental Research Program

Accelerating Discovery of Genes Regulating Stomatal Patterning and Water Use Efficiency in C4 Crops with Novel High-Throughput Methods for Mutagenesis and Phenotyping


Grace Tan1* (, Redeat Tibebu2, Erik Alan Myers2, Degao Liu2, Hui Jiang3, Collin Luebbert3, Daniel Tejeda-Lunn1, Colby G. Starker2, Andrew D. B. Leakey1, Daniel F. Voytas2, and Ivan Baxter3


1University of Illinois–Urbana-Champaign; 2University of Minnesota; and 3Donald Danforth Plant Science Center


Bioenergy feedstocks need to be deployed on marginal soils with minimal inputs to be economically viable and have a low environmental impact. Currently, crop water supply is a key limitation to production. The yields of C4 bioenergy crops such as Sorghum bicolor have increased through breeding and improved agronomy. Still, the amount of biomass produced for a given amount of water use (water-use efficiency, or WUE) remains unchanged. Therefore, this project aims to develop novel technologies and methodologies to redesign the bioenergy feedstock sorghum for optimal WUE. Within this broader context, this subproject is using Setaria viridis as a rapid cycling model for gene discovery. The project aims to develop and demonstrate novel methods and resources to accelerate both the production of genetic variants and phenotyping of WUE traits as part of reverse and forward genetics approaches to discover genes regulating stomatal patterning and WUE.


Stomata regulate the exchange of CO2 and water vapor between the leaf and atmosphere, and therefore play a key role in determining WUE. However, relatively little is known about the genes that regulate stomatal patterning and WUE in C4 grasses. Previous work has identified several hundred candidate genes through a combination of genome-wide association study and transcriptome-wide association study. To validate these discoveries and advance efforts to engineer improved WUE of bioenergy crops, the team is developing novel methods to accelerate the use of forward and reverse genetics for gene discovery. Researchers conducted a forward genetic screen of 155 families of an N-nitroso-N-methylurea–mutagenized Setaria population, of which 100 lines show small stature and/or altered leaf color phenotypes. Whole-plant WUE is being assessed by imaging and automated lysimeters. These families are part of a larger population which is being fully sequenced by DOE Joint Genome Institute to create a sequence indexed mutant population. This data is being paired with screening for abnormalities in stomatal patterning. To accelerate a reverse genetic screen, researchers developed new methods for viral delivery of mutagenesis reagents in both Setaria and tobacco and are using these methods for studying stomatal developmental genes through loss- and gain-of-function mutations. Both the forward and reverse genetic approaches utilize high throughput optical tomography imaging to generate high-resolution images of the leaf surface.

A robust machine learning model was developed for identifying the size, shape, and number of epidermal cells in maize, and the team is adapting the model for Setaria. This work demonstrates a positive feedback loop of high-throughput phenotyping to genotyping in which genes of interest can be quickly identified and tested for a role in water-use efficiency. Success in this effort could be leveraged to accelerate research on a wide range of other traits and species.

Funding Information

This research was supported by the DOE Office of Science, Biological and Environmental Research (BER) Program, grant no. DE-SC0023160 and DE-SC0018277.