European ash pangenome reveals widespread structural variation and a diverse genetic basis for low ash dieback susceptibility

European Ash (Fraxinus excelsior) is a keystone forest tree species, whose populations are being decimated by Ash Dieback disease (ADB). Uncovering the genetic basis of low susceptibility to this devastating disease relies on a comprehensive understanding of genetic variation present in F. excelsior. A linear reference genome from a single individual cannot contain the total sequence variability within a species, including its genic regions; a pangenome more fully captures total sequence content. In this study, we developed a F. excelsior pangenome reference using a new chromosomal-level phased linear reference genome, and de novo assemblies and long-read data from a geographically diverse set of fifty F. excelsior samples. We identified 362,965 structural variants (SVs), including 174Mb of sequence absent from the linear reference genome (22% of the linear reference size). We demonstrated that failing to explicitly link SVs with gene sequences can lead to substantial overestimation of dispensable genes (those that vary in their presence/absence between individuals) due to variability in the annotation process. Controlling for this, we identified 3,065 dispensable genes including 133 with gene ontologies associated with low disease susceptibility. We used the pangenome to analyse existing genomic data from over 1,200 individuals to identify loci associated with reduced susceptibility to ADB. This analysis revealed that different genetic backgrounds harbour distinct sets of loci associated with low susceptibility, highlighting the complexity of this trait but also the potential benefits of combining variants from different genetic backgrounds to boost efforts to ensure the future survival of F. excelsior.