Pan-genome Analysis Reveals Hidden Diversity and Selection Signatures of Auxin Response Factors (ARFs) Associated with Breeding in Barley

Auxin Response Factors (ARFs) play a pivotal role in regulating plant growth and development, yet their evolutionary dynamics and functional divergence remain poorly understood in barley ( Hordeum vulgare L.) In this study, we conducted a comprehensive genome-wide analysis of the ARF gene family across a 76-accession barley pan-genome. By integrating gene presence/absence variation (PAV) and copy number variation (CNV), phylogeny, expression profiling, transposable element (TE)-mediated regulation, and selection signatures, we characterized 1,911 ARF-coding genes and their structural, transcriptional, and functional variation at the population level. Phylogenetic analysis revealed lineage-specific expansion and dynamic duplications, particularly within the HvARF13 clade. Co-expression networks and tissue-resolved transcriptomes showed that many HvARFs are preferentially expressed in inflorescence and meristematic tissues. Selective sweep and haplotype analyses identified HvARF3 as a candidate gene under selection during European barley breeding. A favorable haplotype of HvARF3 , enriched in European cultivars, was significantly associated with increased grain size and weight, demonstrating the utility of pan-genome-enabled frameworks for accelerating gene-trait association and candidate gene discovery. This study highlights the power of multi-omics integration in decoding gene family complexity and provides valuable insights for functional genomics and trait improvement in cereal crops.