Intra- and inter-isolate variation of ribosomal and protein-coding genes in Pleurotus: implications for molecular identification and phylogeny on fungal groups.

BACKGROUND: The internal transcribed spacer (ITS), RNA polymerase II second largest subunit (RPB2), and elongation factor 1-alpha (EF1α) are often used in fungal taxonomy and phylogenetic analysis. As we know, an ideal molecular marker used in molecular identification and phylogenetic studies is homogeneous within species, and interspecific variation exceeds intraspecific variation. However, during our process of performing ITS, RPB2, and EF1α sequencing on the Pleurotus spp., we found that intra-isolate sequence polymorphism might be present in these genes because direct sequencing of PCR products failed in some isolates. Therefore, we detected intra- and inter-isolate variation of the three genes in Pleurotus by polymerase chain reaction amplification and cloning in this study. RESULTS: Results showed that intra-isolate variation of ITS was not uncommon but the polymorphic level in each isolate was relatively low in Pleurotus; intra-isolate variations of EF1α and RPB2 sequences were present in an unexpectedly high amount. The polymorphism level differed significantly between ITS, RPB2, and EF1α in the same individual, and the intra-isolate heterogeneity level of each gene varied between isolates within the same species. Intra-isolate and intraspecific variation of ITS in the tested isolates was less than interspecific variation, and intra-isolate and intraspecific variation of RPB2 was probably equal with interspecific divergence. Meanwhile, intra-isolate and intraspecific variation of EF1α could exceed interspecific divergence. These findings suggested that RPB2 and EF1α are not desirable barcoding candidates for Pleurotus. We also discussed the reason why rDNA and protein-coding genes showed variants within a single isolate in Pleurotus, but must be addressed in further research. CONCLUSIONS: Our study demonstrated that intra-isolate variation of ribosomal and protein-coding genes are likely widespread in fungi. This has implications for studies on fungal evolution, taxonomy, phylogenetics, and population genetics. More extensive sampling of these genes and other candidates will be required to ensure reliability as phylogenetic markers and DNA barcodes.

Phylogenetic relationship of two popular edible Pleurotus in China, Bailinggu (P. eryngii var. tuoliensis) and Xingbaogu (P. eryngii), determined by ITS, RPB2 and EF1α sequences.

The aims of this study are to assess the utility of the internal transcribed spacer (ITS) region, and partial translation elongation factor (EF1α) and RNA polymerase II (RPB2) genes, for differentiation of Bailinggu, P. eryngii, and P. nebrodensis; to reconstruct phylogenetic relationships between the three species; and to confirm the taxonomic status of Bailinggu based on ribosomal and protein-coding genes. Pairwise genetic distances between Bailinggu, P. eryngii, and related Pleurotus strains were calculated by using the p-distance model, and molecular phylogeny of these isolates was estimated based on ITS, RPB2, and EF1α using maximum parsimony and Bayesian methods. Differences in ITS, RPB2, and EF1α sequences show that Bailinggu, P. eryngii, and P. nebrodensis are distinct at the species level. Phylogenetic analyses reveal that P. eryngii is closer to P. nebrodensis than to Bailinggu. Sequence analyses of ribosomal and protein-coding genes confirm that P. eryngii var. tuoliensis is identical to Bailinggu. P. eryngii var. tuoliensis should be raised to species level or a new name should be introduced for Bailinggu after a thorough investigation into Pleurotus isolates from Ferula in Xinjiang Province. This study helps to resolve uncertainty regarding Bailinggu, P. eryngii and P. nebrodensis, improving the resource management of these strains. ITS, EF1α, and RPB2 sequences can be used to distinguish Bailinggu, P. eryngii and P. nebrodensis as three different species, and P. eryngii var. tuoliensis should be the scientific name for Bailinggu at present.