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

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.

Fungal species concepts in the genomics era

“…At the genetic level, incomplete lineage sorting could also result in false signals in population relationships (Steenkamp et al 2018). With increasing commercial trade and human travel, as well as our increasing influence on the environment, both the genotypic and phenotypic features of fungal populations and species could be severely impacted (e.g., Xu et al 1997;Chen et al 2016;Samarasinghe et al 2020). In the section below, I highlight some of the features of fungi and their implications for fungal species concepts.…”

Fungal DNA barcoding

“…The main problems include (i) the lack of sequence variation between many closely related sister species (i.e., lack of a barcode gap; e.g., Xu et al 2000); (ii) the inability to obtain clear ITS sequences directly using PCR and Sanger sequencing of PCR products due to intrastrain sequence heterogeneity among copies of the ITS within the ribosomal RNA gene cluster (Chen et al 2016); and (iii) the inability of the "universal primers" (ITS1, ITS2, ITS3, and ITS4) to amplify about 10% of the tested fungal specimens (Schoch et al 2012;Stielow et al 2015). As a result, a number of attempts have been made to identify other loci with suitable barcode characteristics.…”

“…With the increasing availability of genome sequences representing all major groups of fungi, better primers targeting the ITS regions could be developed to allow the amplification of (virtually) all fungi to overcome the first problem. The second problem can be overcome with current technologies by either cloning the PCR products first before sequencing using the Sanger approach or directly using nextgeneration DNA sequencing technologies (Buée et al 2009;Chen et al 2016;Mark et al 2016). As discussed in the above section, there is an emerging consensus that TEF1 could be an excellent secondary barcode for the vast majority of examined fungal groups to help solve the third problem.…”

Patterns of PCR Amplification Artifacts of the Fungal Barcode Marker in a Hybrid Mushroom

“…Though intragenomic variability may have limited impacts on species richness estimates (Lindner et al, 2013), such variations can impact PCR and DNA sequence-based identifications of strains, geographic populations, and species (Xu, 2016). For example, in a recent study of a cultivated mushroom – Agaricus subrufescens Peck – collected in Saint-Leon, Chen et al (2016) found that there were three main types of ITS sequences (called types A, B, and C) within a single specimen. Types A and B sequences were similar to those amplified from A. subrufescens specimens collected from the Americas and Europe while type C sequence was close to those found in Oceanian and Asian specimens (Chen et al, 2016).…”

“…For example, in a recent study of a cultivated mushroom – Agaricus subrufescens Peck – collected in Saint-Leon, Chen et al (2016) found that there were three main types of ITS sequences (called types A, B, and C) within a single specimen. Types A and B sequences were similar to those amplified from A. subrufescens specimens collected from the Americas and Europe while type C sequence was close to those found in Oceanian and Asian specimens (Chen et al, 2016). These three types of ITS sequences differ from each other at 7–9 nucleotide positions, and the similarities among the three types are about 98.8%.…”