Mitochondrial Graph-Based Pan-Genome Analysis of Hypsizygus marmoreus: Structural Variation, Adaptive Evolution, and Its Implications for Germplasm Resource Improvement

Mitochondria regulate nuclear genomes and their own genetic material, primarily to provide energy in eukaryotes. Currently, high-throughput sequencing technologies are being used to resolve the mitochondrial genomes of various edible fungi. 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 (four newly sequenced monotypes and 27 public datasets), ranging from 98,284 to 111,087 bp. This variation was determined to be primarily driven by dynamic changes in non-coding regions, particularly intronic polymorphisms in the cox1 gene. Further, transfer RNA (tRNA) secondary structures exhibited 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 found to harbor eight gene rearrangement patterns and five genetic clusters, and the pan-genome analysis (220,364 bp, 217 nodes) captured abundant single-nucleotide polymorphisms (SNPs), insertions/deletions (InDels), and structural variations. This study provides breeding-relevant genetic markers and a genomic framework for H. marmoreus germplasm classification, genetic improvements, and the molecular breeding of stress-resilient varieties.