Insights into intraspecific variation and genotyping of Ganoderma lingzhi through pan-mitogenome analysis

Ganoderma lingzhi is a medicinal fungus characterized by its large fruiting bodies. In this study, we collected 151 cultivated Ganoderma strains from across China and performed whole-genome resequencing. By integrating 30 publicly available G. lingzhi datasets, we successfully assembled a total of 181 complete Ganoderma mitochondrial genomes. We conducted a systematic analysis of their genomic features, intron distribution, gene order, non-synonymous/synonymous substitution rates (Ka/Ks), and phylogenetic relationships. Our results revealed that among the 151 strains we collected, 19 exhibited discordances between genetic identity and labeled names, highlighting the prevalent issue of strain misidentification in the current commercial market of G. lingzhi. The size of G. lingzhi mitogenomes ranged from 49,233 to 70,498 bp. We identified 20 distinct introns whose presence/absence was highly dynamic across the G. lingzhi samples. Based on intron distribution patterns, the samples were classified into two major groups. Comparative genomics revealed a conserved gene order across the genus, and Ka/Ks analysis indicated that the 15 core protein-coding genes were under purifying selection compared to neutral expectations. Phylogenetic analysis based on the mitogenome confirmed the monophyly of G. lingzhi. This study presents the first large-scale pan-mitogenomic analysis of G. lingzhi, revealing that intron dynamics are the primary driver of intraspecific genomic variation and differentiation. These results can be used for the precise identification, traceability, and breeding of G. lingzhi strains.