The specific goals for this project are envisioned as below.Goal 1: Testing NUE-related phenotypic effects of the beneficial microbes. Potentially beneficial microbes will be selected from our culture collection for sequence validation. Phenotypic testing will be conducted in sterile gemination bags and completely enclosed pots to test whether the selected GEM lines can be stably colonized and whether any of the candidate beneficial microbes will have positive effects on NUE-related traits. The confirmed beneficial microbes will be further tested in the LemnaTec greenhouse to maturity.Goal 2: Validating microbiome-associated GWAS signals using an unrelated population. Field trials will be conducted using the GEM-DH population (N=250 lines) in two consecutive years with standard N and low N conditions. The 16S sequencing data will be collected in the 2nd year to profile the microbiome composition of the population. And ASVs will be called from the 16S sequencing data to conduct GWAS. In parallel, the UAV imagery data will be collected from these field trials to calculate NUE-related traits.Goal 3: Developing genomic selection models to enhance NUE-related traits. Mediation analysis will be conducted to understand the portion of phenotypic variance explained by the microbiome as compared to the host genome. Then, by combining SNP data from the host genome and ASV data from the root-associated microbiome, the microbiome-enabled genomic selection models will be developed to facilitate NUE-related traits prediction.