Intraspecific Variation and Phylogenetic Relationships Are Revealed by ITS1 Secondary Structure Analysis and Single-Nucleotide Polymorphism in Ganoderma lucidum.

Fengfeng Zhou,Junshe Sun,Zhangyang Xu,Xiaoyan Tan,Zhou Chen,Xiuqing Zhang,Bin Yang,Jing Hu,Haisheng Pei

doi:10.1371/journal.pone.0169042

Abstract

Ganoderma lucidum is a typical polypore fungus used for traditional Chinese medical purposes. The taxonomic delimitation of Ganoderma lucidum is still debated. In this study, we sequenced seven internal transcribed spacer (ITS) sequences of Ganoderma lucidum strains and annotated the ITS1 and ITS2 regions. Phylogenetic analysis of ITS1 differentiated the strains into three geographic groups. Groups 1–3 were originated from Europe, tropical Asia, and eastern Asia, respectively. While ITS2 could only differentiate the strains into two groups in which Group 2 originated from tropical Asia gathered with Groups 1 and 3 originated from Europe and eastern Asia. By determining the secondary structures of the ITS1 sequences, these three groups exhibited similar structures with a conserved central core and differed helices. While compared to Group 2, Groups 1 and 3 of ITS2 sequences shared similar structures with the difference in helix 4. Large-scale evaluation of ITS1 and ITS2 both exhibited that the majority of subgroups in the same group shared the similar structures. Further Weblogo analysis of ITS1 sequences revealed two main variable regions located in helix 2 in which C/T or A/G substitutions frequently occurred and ITS1 exhibited more nucleotide variances compared to ITS2. ITS1 multi-alignment of seven spawn strains and culture tests indicated that a single-nucleotide polymorphism (SNP) site at position 180 correlated with strain antagonism. The HZ, TK and 203 fusion strains of Ganoderma lucidum had a T at position 180, whereas other strains exhibiting antagonism, including DB, RB, JQ, and YS, had a C. Taken together, compared to ITS2 region, ITS1 region could differentiated Ganoderma lucidum into three geographic originations based on phylogenetic analysis and secondary structure prediction. Besides, a SNP in ITS 1 could delineate Ganoderma lucidum strains at the intraspecific level. These findings will be implemented to improve species quality control in the Ganoderma industry.

Highlights

Ganoderma lucidum (Chizhi) is a wood-rotting fungus that is a prestigious medicine [1] and has been continuously used in Asia for approximately two thousand years [2]
In a specific analysis with Chinese spawn strains of Ganoderma lucidum, we found that strain compatibility is correlated with the single-nucleotide polymorphism (SNP) site in ITS1
The species of Ganoderma lucidum could be separated into three groups by phylogenetic analysis of ITS1 sequences (Fig 1)

Summary

Introduction

Ganoderma lucidum (Chizhi) is a wood-rotting fungus that is a prestigious medicine [1] and has been continuously used in Asia for approximately two thousand years [2]. The active ingredients of Ganoderma lucidum, extracted using hot water or ethanol, include polysaccharides [3,4,5], proteins [6,7] and ganoderic acid [8,9] These ingredients can be effective in the treatment of cancer, hypertension and viral infections, since numerous reports have indicated that Ganoderma lucidum has anti-tumor [10,11,12], anti-aging [13,14], immune system-enhancing, [15,16] and anti-hypertension [16,17,18] activities. Due to the high demand of medical use and the potential profits, the identification and quality control of Ganoderma lucidum is a critical point for further industry development

Methods

Results

Conclusion