Mitochondrial Graph-Based Pan-Genome Analysis of <em>Hypsizygus Marmoreus</em>: Structural Variation, Adaptive Evolution and Its Implications for Germplasm Resource Improvement

As semi-autonomous organelles, mitochondria function through the coordinated regulation of nuclear genomes and their own genetic material, primarily providing energy for eukaryotic organisms. Currently, high-throughput sequencing technologies have been used to resolve the mitochondrial genomes of various edible fungi. With advances in sequencing technology, species genome characterization has evolved from single genomes to pan-genomes. However, the application of pan-genomes for the analysis of edible mushroom mitochondrial genomes remains unexplored. In this study, we conducted a comparative mitochondrial genome analysis of 31 Hypsizygus marmoreus strains (4 newly sequenced monotypes and 27 public datasets). The results revealed that the mitochondrial genome sizes ranged from 98,284 to 111,087 bp, exhibiting significant structural diversity. This variation is primarily driven by dynamic changes in non-coding regions, particularly intronic polymorphisms in the cox1 gene. This study revealed that tRNA secondary structures exhibit atypical globular and elongated conformations alongside copy number variations. Additionally, codon usage showed a pronounced A/T bias, whereas core respiratory chain genes demonstrated an evolutionary pattern of strong purifying selection. Furthermore, the 31 mitochondrial genomes of H. marmoreus were identified 8 gene rearrangement patterns and 5 genetic clusters, and the pan-genome (220,364 bp, 217 nodes) captured abundant SNPs, InDels and structural variations. This study provides breeding-relevant genetic markers and a genomic framework for germplasm classification, genetic improvement and stress-resilient variety molecular breeding of Hypsizygus marmoreus.