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

Identification of the Genetic Factors that Contribute to Biological Nitrogen Fixation in Sorghum (Sorghum bicolor)


Rafael E. Venado2* (espejelvenad, Emily S. A. Wolf1, Saddie Vela1, Marina Kotvana3, Prakriti Garg3, Saptarshi Pyne3, Sushmita Roy3, Wilfred Vermerris4,5, Jean-Michel Ané2,6


1Plant Molecular and Cellular Biology Graduate Program, University of Florida; 2Department of Bacteriology, University of Wisconsin–Madison; 3Department of Biostatistics and Medical Informatics, University of Wisconsin–Madison; 4Department of Microbiology and Cell Science, University of Florida; 5UF Genetics Institute, University of Florida; 6Department of Plant and Agroecosystem Sciences, University of Wisconsin–Madison



The primary aim of this proposal is to deepen our comprehension of the molecular and cellular mechanisms governing associative nitrogen fixation traits within the mucilage of sorghum plants that form aerial roots. This will be achieved through a multifaceted approach encompassing genetics, synthetic bacterial communities, and systems biology. Our overarching hypotheses posit that both plant and bacterial gene networks play pivotal roles in regulating nitrogen fixation efficiency in sorghum, and unraveling these networks will facilitate the enhancement of nitrogen fixation in sorghum, thus advancing its potential for bioenergy production.


Current agricultural practices rely heavily on applying nitrogen-rich fertilizers, posing significant environmental risks, including pollution of ground and surface water, nitrous oxide emissions, and greenhouse gas emissions during the production of ammonia-based fertilizer. However, adopting biological nitrogen fixation presents a promising avenue for mitigating these risks. Sorghum (Sorghum bicolor L. Moench) is gaining recognition as a sustainable crop due to its resilience to drought and high temperatures, and certain types of sorghum produce high yields of lignocellulosic biomass that can be used to produce renewable fuels and chemicals. Select sorghum accessions exhibit prolific growth of thick aerial roots that secrete a dense, carbohydrate-rich mucilage following rainfall and in humid conditions (Venado et al. 2023). This mucilage is an optimal environment for diazotrophic microorganisms that provide the plant with ammonium. To improve sorghum’s ability to support diazotrophs, breeding programs require a detailed understanding of the genetic factors influencing aerial root development and mucilage production. In pursuit of this objective, we conducted a comprehensive genome-wide association study (GWAS) on the sorghum minicore that consists of 242 landraces and 30 accessions from the sorghum association panel at two locations (Florida and Wisconsin) and with a standard and reduced fertilizer treatment at each location. Through this GWAS, we identified loci associated with the phenotypes of aerial root diameter and the number of nodes with aerial roots. Sequence variations within genes responsible for encoding transcription factors governing phytohormone signaling and root system architecture were associated with these traits (Wolf et al. 2023). In parallel, several breeding populations were developed from crosses between accessions that produce aerial roots and regionally adapted sweet sorghums. Segregating F2 populations were used to validate some of the loci identified in the GWAS, whereas continued inbreeding and selection are expected to result in bioenergy sorghum cultivars capable of obtaining a portion of their nitrogen needs from biological nitrogen fixation. Furthermore, we conducted single-cell RNA sequencing (scRNAseq) on sorghum aerial roots, comparing those with and without mucilage. This analysis uncovered novel gene markers specific to different cell types. Leveraging our scRNAseq data, we constructed gene regulatory networks using various algorithms, including single-cell Multi-Task Network Inference (scMTNI). This approach will enable us to explore genes essential for mucilage production further. Together, these results offer opportunities for enhancing biological nitrogen fixation in cereal crops and reducing our reliance on synthetic fertilizers.


Wolf, E. S. A., et al. 2023. “Identification of Genetic and Environmental Factors Influencing Aerial Root Traits That Support Biological Nitrogen Fixation in Sorghum,” G3: Genes, Genomes, Genetics 14(3). DOI:10.1093/g3journal/jkad285.

Venado, R. E., et al. 2023. “Mucilage Produced by Sorghum (Sorghum Bicolor) Aerial Roots Supports a Nitrogen-Fixing Community,” bioRxiv. DOI:10.1101/2023.08.05.552127.

Funding Information

The authors gratefully acknowledge funding from the U.S. DOE, BER program, grant no. DE-SC0021052.