A New Method for Constructing High‐Resolution Phylogenomic Topologies Using Core Gene‐Associated MNP Markers: A Case Study From Agaricus bisporus

ABSTRACT Accurate strain identification is essential for economically significant fungi, as it aids in understanding their diverse agronomic traits, pathogenicity, and other important characteristics. However, traditional methods often face challenges related to limited accuracy, high workloads, and reproducibility issues. Recently, multiple nucleotide polymorphism (MNP) markers have been employed in mushroom strain identification, demonstrating significantly improved accuracy and reproducibility. Nevertheless, the identification of strains across different species still heavily depends on specific and often overly complex MNP markers. In this study, we address these challenges by developing a novel method for constructing high‐resolution phylogenomic topologies using core gene‐associated multiple nucleotide polymorphism (cgMNP) markers, focusing on Agaricus bisporus (button mushroom). Utilising resequencing data from 213 cultivated and wild strains of A. bisporus , we identified 84 cgMNP markers within 83 core genes from 1011 MNP markers. Phylogenetic analysis based on cgMNP sequences and the genetic distance between strain pairs allowed for precise identification of all strains. Moreover, the successful transferability of these cgMNP markers to an additional 385 A. bisporus strains and other fungal species, including Flammulina filiformis (enoki mushroom) and Saccharomyces cerevisiae (yeast), highlights their cross‐species applicability. The high resolution and strong congruence of cgMNP markers with whole‐genome data provide a robust and reliable method for strain‐level discrimination in fungi. The success of this approach in A. bisporus sets a promising precedent for its application to a broader range of fungal taxa.