Evolutionary relationship and a novel method of efficient identification of Lentinula edodes cultivars in China

Lentinula edodes (shiitake in Japanese, xiang-gu in Chinese) is the most productive mushroom in China, and there are more than 500 cultivars names.However, the traditional identification of mushroom cultivars can be challenging due to the problems of the heavy workload, insufficient accuracy and low reproducibility, which greatly affect the industrial development and germplasm innovation process.In this study, we developed and illustrated a methodological and technical system for the identification of L. edodes cultivars and for detecting their evolutionary relationship based on the multiple nucleotide polymorphism marker (MNP) sequences.Specifically, 501 universal MNP markers were screened based on the whole-genome resequencing data of 188 L. edodes strains mainly from China (25 of which were self-tested).The following analysis of detection rate, repeatability rate and accuracy rate showed those MNP markers are satisfactory with 94.4%, 99.92%, and 99.96% respectively.This method of multiplex PCR amplification and high-sequencing resulted in the construction of an MNP sequence library of L. edodes.On this basis, 187 MNP markers were further screened as core MNP markers.An ML phylogenetic tree of 162 wild strains and 78 cultivars was generated by using core MNP sequences, revealing all cultivars can be separated into 24 distinct evolutionary pedigrees.The genetic similarity (GS) value between different pedigrees and all cultivars tested showed that a broad library of pedigrees had GS values less than or equal to 94%, and the different cultivars within the same pedigrees had GS values range of 94-99.5%.The combination of all results indicated that most cultivars of L. edodes were originally from the Northeast Asia except the only cultivar L. edodes from south China, but there has a serious homogenization of L. edodes germplasms in China.In this study, we established a genealogical map of L. edodes cultivars in China, a standard library of MNP markers, and a rapid experimental procedure for genealogical and cultivar identification, which lays out a highly reliable platform for encouraging original innovation and modified cultivar innovation.Finally, a brief prospect for MNP markers identification of mushroom cultivars is addressed.