Analysis of the Beneficial Associations of Sorghum with Arbuscular Mycorrhizal Fungi Studied with Genetics, Genomics, and Microbiomics
Jeff Bennetzen1*, Phil Brailey-Jones1, Tom Pendergast1, Shufan Zhang1, Beatrice Bock2, Amanda Bouffier-Landrum1, Jia Hwei Cheong1, Xiomy Pincha Davila1, Josue Fernandez1, Ching-Ting Huang1, Ben Long1, Sarah Mondibrown1, Rachel Rackers1, Sedona Spann2, Isaac Torres1, Richard Trippe2, Jonathan Arnold1, Anny Chung1, Katrien Devos1, and Nancy Collins Johnson2
1University of Georgia; and 2Northern Arizona University
This project is designed to investigate the interactions between sorghum genes, arbuscular mycorrhizal fungi (AMF), the sorghum root-associated microbiome, and numerous environmental factors that contribute to sorghum biomass production. A key focus is on host genetics, so the team is investigating 337 Bioenergy Association Panel accessions to perform a full genome-wide association study (GWAS) analysis.
In first-year pilot studies, researchers determined that very different AMF populations were associated with sorghum roots at two field locations in Georgia and Arizona. Root types and developmental stages were also identified that were consistent for AMF abundances, infection structures, and percent colonization, thereby decreasing the number of sample extractions that will be needed in all future experiments. These first-year studies also indicated that a good deal of microbiome analysis could be performed most effectively by shotgun sequence analysis, rather than amplicon analysis.
In second-year studies of input effects, performed at a Georgia field location, the team determined efficient/reproducible methods for sample production/collection, and generated the full set of 4044 samples (337 genotypes * three replications * four treatments) to determine the effects of phosphate and nitrogen levels on all of the investigated field/sample properties in a randomized complete block design. These measured properties included quantification and type characterization of AMF and other microbes on or within the sorghum roots, expression of eukaryotic genes on or within the sorghum roots, microscopic analysis to classify and quantitate AMF infection structures, and agronomic sorghum traits, such as plant height, plant biomass, plant tillering, plant flowering time, and plant mineral content. Most of these analyses are still in the data generation stage, but researchers did observe a trend of high nitrogen increasing tiller number, but high phosphate decreasing plant height, potentially indicating disruption of mycorrhizal associations at the Georgia field site.
The team has also made major technical advances, including creating and training an automated imaging platform to allow high throughput classification and segmentation of AMF structures, such as arbuscules and vesicles, which in turn provided the size, density, and percent colonization of AMF structures in field-grown sorghum roots for GWAS analysis. Over 100 fungal morphotypes have been cultured from sorghum roots in Georgia, including three AMF species that are being propagated on sorghum as pure cultures. In addition, 100+ endophytic bacteria and fungi have been isolated and cultured from first-year studies in Arizona, and about half have been shown to exhibit drought tolerance by a polyethylene glycol assay. These cultured microbes will be used for planned greenhouse tests of hypotheses generated from descriptive field data from the major GWAS fields in Arizona and Georgia.