The graphical barley pangenome reveals micro- and macro-scale genetic variation

Graphical pangenomes and orthologous gene clustering visualisations offer powerful tools to analyse, validate, and extract genomic variations within a species. For crop genomics, integrating these models and algorithms into complex and large genomes is essential to unlock the potential of genomic technology. Barley ( Hordeum vulgare L.) is an important crop for the global malting industry, and faces increasing challenges from climate change and rising production demands. In this study, we integrated pangenome graph and orthologous gene clustering tools and applied them to multiple high-quality barley genome assemblies. This approach revealed a detailed and compact representation of genetic diversity, capturing variation from base-pair level to large structural rearrangements across the entire barley pangenome. Using pangenome graph and gene clustering analyses, we identified substantial structural variation (SV) between barley cultivars and landraces and characterised presence–absence variation (PAV) and copy number variation (CNV) patterns. Through hierarchical edge bundles, network plots, and sequence tube maps of genes controlling row-type, we identified haplotype blocks defined by single-nucleotide polymorphisms (SNPs) and InDels. Linearised genome graph visualisation via the Panache pangenome browser further enabled exploration of PAV regions. Together, these visualisation approaches demonstrate how multiple levels of resolution in a graphical pangenome can provide a unified view of genomic variation. The genomic resources and tools developed here expand the toolkit for barley breeding and genetic improvement.