Location: National Peanut Research Laboratory
Project Number: 6044-21600-001-006-S
Project Type: Non-Assistance Cooperative Agreement
Start Date: Sep 1, 2026
End Date: Jul 28, 2030
Objective:
1. To assemble a representative collection of A. flavus isolates from harvested peanuts from every producing state in the United States.
2. To sequence the genomes of all isolates of A. flavus obtained in the collection.
3. To phenotype the obtained isolates for aflatoxin production, pathogenicity-related traits, and fungicide resistances for genome-wide association analyses (GWAS).
4. To functionally characterize putative aflatoxin regulatory genes identified in the GWAS analysis.
Approach:
As with Phase 1, Phase 2 will heavily rely on partnerships and participation with national and regional shellers and buying point operators to obtain peanut samples. Samples remaining after grading will be requested for all Seg3-graded lots obtained from participating buying points with a maximum of 25 samples per buying point. In the event there are insufficient Seg3-graded lots, Seg1 lots can be sampled to provide geographical coverage of the buying point service areas. This process will need to be performed in every peanut producing state in order to provide a complete survey of US peanut producing regions. Once collected, the peanut samples will be shelled, and a portion of the seeds will be plated on Aspergillus-selective medium (modified rose bengal agar) for A. flavus isolation. Purified isolates will then be cultured on liquid medium and then used for DNA isolation with a QIAcube Connect automated process. Whole genome sequencing will then be performed on each isolate on an Illumina NovaSeq platform at HudsonAlpha. Obtained sequencing data will be filtered and used for whole genome assembly for each isolate. Population structure will then be evaluated using STRUCTURE and other bioinformatic pipelines to examine for regional and varietal/market type variation in A. flavus populations. Unique gene content will be examined between regions with an emphasis on pangenomic assembly generation representing A. flavus in the US. In total, up to 3,000 isolates are expected to be sequenced, equating to 250 isolates from each peanut producing state (Alabama, Arkansas, Florida, Georgia, Louisiana, Mississippi, New Mexico, North Carolina, Oklahoma, South Carolina, Texas, and Virginia). The collection is expected to take place over two years. Collected isolates will also be phenotyped for aflatoxin production for association analyses to identify novel aflatoxin regulatory genes. Sensitivities will also be examined for some select fungicides to evaluate the prevalence of fungicide resistances across peanut producing regions. If novel aflatoxin regulatory genes are identified, functional validation testing will be performed through direct mutagenesis using CRISPR-Cas9 or similar editing approach to generate knock out mutants for evaluation. Gene expression and pathway testing appropriate to the suspected biochemical pathways affected will then be performed to determine the specific role the gene(s) may play in aflatoxin biosynthesis.