High-Resolution Core Gene-Associated Multiple Nucleotide Polymorphism (cgMNP) Markers for Strain Identification in the Wine Cap Mushroom Stropharia rugosoannulata

Abstract

Stropharia rugosoannulata, an ecologically valuable and economically important edible mushroom, faces challenges in strain-level identification and breeding due to limited genomic resources and the lack of high-resolution molecular markers. In this study, we generated high-quality genomic data for 105 S. rugosoannulata strains and identified over 2.7 million SNPs, unveiling substantial genetic diversity within the species. Using core gene-associated multiple nucleotide polymorphism (cgMNP) markers, we developed an efficient and transferable framework for strain discrimination. The analysis revealed pronounced genetic differentiation among cultivars, clustering them into two distinct phylogenetic groups. Nucleotide diversity (π) across 83 core genes varied significantly, highlighting both highly conserved loci under purifying selection and highly variable loci potentially associated with adaptive evolution. Phylogenetic analysis of the most variable gene, Phosphatidate cytidylyltransferase mitochondrial, identified 865 SNPs, enabling precise differentiation of all 85 cultivars. Our findings underscore the utility of cgMNP markers in addressing challenges posed by horizontal gene transfer and phylogenetic noise, demonstrating their robustness in cross-species applications. By providing insights into genetic diversity, evolutionary dynamics, and marker utility, this study establishes a foundation for advancing breeding programs, conservation strategies, and functional genomics in S. rugosoannulata. Furthermore, the adaptability of cgMNP markers offers a universal tool for high-resolution strain identification across diverse fungal taxa, contributing to broader fungal phylogenomics and applied mycology.

Keywords: core gene-associated MNP markers; phylogenetic tree; phylogenomics; strain identification.

Figure 1

Circos plot of the Stropharia rugosoannulata genome, illustrating features from the outermost to innermost circles as follows: (i) the 16 longest scaffolds of the genome, (ii) GC content, (iii) SNP density, and (iv) positions of core gene-associated MNPs.

Figure 2

Phylogenetic tree of Stropharia rugosoannulata based on core gene-associated MNPs from 105 strains. This neighbor-joining tree, constructed from MNPs within core genes, depicts genetic relationships among wild and cultivated strains of S. rugosoannulata, with branch colors representing pileus color (brown/yellow). The innermost ring indicates geographic origin, and the outermost ring distinguishes between wild and cultivated strains, illustrating genetic diversity, geographic distribution, and domestication effects.

Figure 3

Heatmap illustrating pairwise genetic similarity (GS) values among 85 Stropharia rugosoannulata cultivars. Each cell represents the GS value between two strains, with color intensity indicating the degree of genetic similarity. Cultivars are grouped into two distinct pedigrees (G1 and G2) based on a GS threshold of 69.5%, enabling clear separation of genetic lineages. This analysis reveals both intra- and inter-pedigree variation, providing insights into the genetic structure of domesticated S. rugosoannulata.

Figure 4

Genetic and functional characterization of the 12 highest diverse genes in Stropharia rugosoannulata. (A) Nucleotide diversity (π) profiles of the 12 genes with the highest variability, calculated using a sliding window approach (window size: 300 bp; step size: 50 bp). The Y-axis represents nucleotide diversity (π), and the X-axis denotes relative position along each gene. (B) Gene Ontology (GO) functional classification of these 12 genes based on Level 2 annotations. GO terms are grouped into three major categories: biological process, cellular component, and molecular function. The left Y-axis indicates the number of genes assigned to each GO category, while the right Y-axis shows the proportion of genes within each functional group.

Figure 5

Phylogenetic tree of Stropharia rugosoannulata based on the phosphatidate cytidylyltransferase mitochondrial gene from 85 cultivars. The phylogenetic tree was constructed using 865 SNPs identified within the Phosphatidate cytidylyltransferase mitochondrial gene, demonstrating the genetic relationships among 85 cultivated strains of S. rugosoannulata. The outer ring highlights the classification of these strains into two distinct genetic pedigrees (G1–G2), providing clear visualization of their evolutionary divergence. This analysis underscores the utility of this gene as a high-resolution marker for precise strain differentiation and classification.