The automated eukaryotic pangenome pipeline EukPan reveals accessory genome differentiation beyond core-gene phylogeny in Aspergillus oryzae

The automated eukaryotic pangenome pipeline EukPan has unveiled previously undetected accessory genome differentiation in the filamentous fungus Aspergillus oryzae. Traditional pangenome analysis has shown that genes can vary beyond the confines of a single reference genome, but applying this technique to eukaryotes has been difficult due to inconsistent gene annotation.

ANNEVO, a gene model predictor, can generate gene models from genome FASTA assemblies without requiring RNA-seq data. To address these challenges, a team has created EukPan, an automated post-annotation pipeline that standardizes gene annotation files, selects representative gene variants, constructs protein databases, identifies gene orthogroups, and builds a concatenated alignment of single-copy core proteins.

This pipeline then highlights shared accessory proteins while excluding those found only in a single genome.

When applied to 123 strains of Aspergillus oryzae, the EukPan pipeline identified 11,245 core orthogroups and 4,407 shared accessory orthogroups. By comparing the core-protein phylogeny, the study found that it aligns well with the previously established A-H classification for this species. However, when examining the accessory genomes, the EukPan analysis clearly separated the 33 group-A strains from the remaining 90 strains.

By analyzing the distribution of accessory genes, the researchers found that 62 group-A-associated and 158 group-A-depleted orthogroups existed. Among these, genes from the major facilitator superfamily (MFS) transporter family and fungal Zn2Cys6 transcription-factor domains were particularly prominent in the depleted set.

Moreover, among the 93 orthogroups that were present in all non-A strains but absent from group-A strains, 59 were mapped to 10 segments of the RIB40 genome—the standard reference genome for A. oryzae. Interestingly, this reference genome is from a non-A (group-F) strain. These findings demonstrate that EukPan can provide reproducible, coordinated core- and accessory-pangenome analysis from eukaryotic genome assemblies, opening up new avenues for understanding the diversity and evolution of complex eukaryotic genomes.