Abstract
Cis-regulatory elements are specific DNA sequences that control gene expression in a spatiotemporal manner, and variation within these elements represents a major source of phenotypic diversity and evolutionary innovation. Nevertheless, how regulatory elements evolve and shape gene expression remains poorly understood, particularly in plants. The well-resolved phylogeny of allopolyploid peanut (Arachis hypogaea) and its diploid progenitors, A. duranensis and A. ipaensis, provides an ideal system to investigate the regulatory evolution at a lineage-specific level. By integrating comparative analyses of sequence similarity, chromatin accessibility, histone modifications, conserved noncoding sequences (CNSs), and gene expression, we reconstructed the evolutionary trajectories of Accessible Chromatin Regions (ACRs), where regulatory elements typically reside, and revealed their distinct contributions to homeolog expression bias, unequal expressions between homeologs. Most ACRs exhibited high sequence similarity, comparable chromatin accessibility, and conserved states for H3K4me3, H3K56ac, and H3K36me3, indicating regulatory stability after hybridization and polyploidization. However, a subset of novel ACRs emerged de novo from previously non-regulatory regions or through sequence mutations in preexisting ACRs, arising at different rates and evolutionary stages. Notably, even highly sequence-conserved ACRs exhibited substantial variation in chromatin accessibility, consistent with CNS composition differences and minor sequence variation, although causal relationships remain to be demonstrated. Our analyses further revealed a complex spectrum of CNS dynamics across the diploid-polyploid framework. Overall, our study provides empirical insights into the fine-scale evolution of plant regulatory landscapes and complements previous large-scale comparisons across distant lineages.