A Molecular Marker System Based on Whole-Genome Sequencing for Mating Type Identification in Major Chinese Cultivars of Lentinula edodes

Lentinula edodes (shiitake) is a globally significant edible mushroom, with China being its largest producer. Efficient breeding is fundamental to sustaining its industry, yet it is often hindered by the labor-intensive and time-consuming nature of traditional mating type identification methods. To develop a rapid genotyping tool, we elucidated the polymorphism of key mating-type genes and established a practical molecular marker system. In this study, we focused on four major cultivated L. edodes varieties in China (0912, 9608, L808, and W1). Whole-genome sequencing, assembly, and annotation revealed the allelic diversity of HD1 of the A mating type and rcb1 and rcb5 of the B mating type in monokaryotic strains of these varieties. Sequence alignment indicated that HD1 could be classified into five types, rcb1 into four types, and rcb5 into four types. Based on SNPs and InDels, co-dominant primers capable of distinguishing all allelic types of HD1, rcb1, and rcb5 were designed, generating clear molecular fingerprints for the 0912, 9608, L808, and W1 varieties. Notably, this system also demonstrated robust discriminatory power when applied to different Chinese varieties and international L. edodes varieties from Japan, Korea, Thailand, and Canada, confirming its reliability across diverse genetic backgrounds. This highly accurate and efficient marker system offers robust theoretical and technical support for parental selection, germplasm identification, and new variety protection for L. edodes, presenting significant potential to improve horticultural mushroom production efficiency.