Genetic Analyses of the Internal Transcribed Spacer Sequences Suggest Introgression and Duplication in the Medicinal Mushroom Agaricus subrufescens.

Magalie Moinard,Jianping Xu,Marie Foulongne-Oriol,Shouxian Wang,Jie Chen,Kevin D. Hyde,Ruilin Zhao,Philippe Callac

doi:10.1371/journal.pone.0156250

Abstract

The internal transcribed spacer (ITS) region of the nuclear ribosomal RNA gene cluster is widely used in fungal taxonomy and phylogeographic studies. The medicinal and edible mushroom Agaricus subrufescens has a worldwide distribution with a high level of polymorphism in the ITS region. A previous analysis suggested notable ITS sequence heterogeneity within the wild French isolate CA487. The objective of this study was to investigate the pattern and potential mechanism of ITS sequence heterogeneity within this strain. Using PCR, cloning, and sequencing, we identified three types of ITS sequences, A, B, and C with a balanced distribution, which differed from each other at 13 polymorphic positions. The phylogenetic comparisons with samples from different continents revealed that the type C sequence was similar to those found in Oceanian and Asian specimens of A. subrufescens while types A and B sequences were close to those found in the Americas or in Europe. We further investigated the inheritance of these three ITS sequence types by analyzing their distribution among single-spore isolates from CA487. In this analysis, three co-dominant markers were used firstly to distinguish the homokaryotic offspring from the heterokaryotic offspring. The homokaryotic offspring were then analyzed for their ITS types. Our genetic analyses revealed that types A and B were two alleles segregating at one locus ITSI, while type C was not allelic with types A and B but was located at another unlinked locus ITSII. Furthermore, type C was present in only one of the two constitutive haploid nuclei (n) of the heterokaryotic (n+n) parent CA487. These data suggest that there was a relatively recent introduction of the type C sequence and a duplication of the ITS locus in this strain. Whether other genes were also transferred and duplicated and their impacts on genome structure and stability remain to be investigated.

Highlights

The internal transcribed spacer (ITS) region of nuclear ribosomal DNA has been proposed as a universal DNA barcode marker for fungi [1]
Intra-strain or intra-species variation in ITS sequences have been reported in several fungal genera and species such as Fusarium [9], Scutellospora [10], Ganoderma [11], Xanthophyllomyces [12], Laetiporus [4] and more recently in Ophiocordyceps sinensis [5]
Since double or triple peaks were observed in chromatograms by direct sequencing, PCR products of the heterokaryotic parental strain CA487 were cloned to reveal the ITS sequence diversity within the strain

Summary

Introduction

The internal transcribed spacer (ITS) region of nuclear ribosomal DNA (rDNA) has been proposed as a universal DNA barcode marker for fungi [1]. It is widely used in fungal taxonomy and phylogeny due to a high success rate in PCR amplification and a high degree of interspecific variation that can distinguish between most of the closely related fungal species. Numerous studies revealed that the multicopy genes, such as ITS, do not perfectly follow the concerted evolution, and exhibit intra-strain and intra-species variation in a wide variety of organisms [3, 5, 8]. Intra-strain or intra-species variation in ITS sequences have been reported in several fungal genera and species such as Fusarium [9], Scutellospora [10], Ganoderma [11], Xanthophyllomyces [12], Laetiporus [4] and more recently in Ophiocordyceps sinensis [5]

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Conclusion