Construction and Analysis of Pangenome Graphs for Aspergillus flavus and Aspergillus parasiticus to Elucidate Genetic Diversity and Aflatoxin Production

Aspergillus flavus and Aspergillus parasiticus are opportunistic pathogens that can infect various agricultural crops, such as corn and peanuts, and produce carcinogenic secondary metabolites called aflatoxins, threatening safe food production. These species vary in their ability to produce the carcinogenic aflatoxins and other secondary metabolites. To capture the complete genomic diversity within a species, the genomes of multiple strains can be assembled by constructing a pangenome. A linear pangenome for 346 A. flavus isolates called AflaPan was published and was considered a closed pangenome, as the addition of new genomes did not significantly increase the variant pool. However, AflaPan was limited in its ability to depict large structural variations (>50bp). Our goal is to construct a graph based pangenome to provide a more accurate understanding of individual genomes and their relationship with others by capturing larger structure variants. Initially, we assembled and constructed a pangenome graph for the 225 A. flavus isolates we sequenced. This analysis revealed a total of 729,852 variants, including 542,577 single nucleotide polymorphisms (SNPs) and 189,267 (non-SNPs). Notably, the highest variant density was observed on chromosome 3, which contains the aflatoxin biosynthesis pathway gene cluster. Using the genotypes derived from the pangenome graph, we created a phylogenetic tree that revealed a clustering of 46 isolates from Georgia and Mississippi, which exhibited remarkably similar genotypes. These isolates showed a 2 kb insertion on chromosome 1 and an 8.7 kb insertion on chromosome 8. Our next step will be to gradually add publicly available genomes of 332 A. flavus and 51 A. parasiticus isolates to the subset pangenome to test if the pangenome is closed. This will allow us to analyze additional variations and compare them to the previously established AflaPan pangenome. Finally, we will use the pangenome graph as genotyping tool for genome-wide association studies (GWAS) to dissect aflatoxin biosynthesis pathways and other morphological phenotypes of interest.