Development of new molecular markers for phylogeny and molecular identification of arbuscular mycorrhizal fungi (Glomeromycota)

Because of the high genetic variability of the nuclear-encoded ribosomal RNA genes within single spores of arbuscular mycorrhizal fungi (AMF, Glomeromycota), new alternative marker genes are needed to better resolve closely related taxa. Based on published nucleotide sequences of the mitochondrial large ribosomal subunit (mt-LSU), new fungal-specific primers were constructed and successfully used for the amplification and sequencing of a partial sequence of the mt-LSU from arbuscular mycorrhizal fungi. A specific primer for Glomus intraradices and Glomus proliferum was constructed and successfully tested on field samples. In contrast to nuclear encoded ribosomal genes, there was no evidence for genetic variability of the mt-LSU sequences of G. intraradices and G. proliferum within fungal isolates. Furthermore, different isolates of G. intraradices could be clearly distinguished from each other by their mt-LSU sequences. Phylum-level phylogenetic analyses of the mt-LSU in the true fungi did not provide compelling support for the relationships between Glomeromycota and other fungal lineages, because the sequences are too variable among phyla. Nevertheless, G. intraradices and G. proliferum were clearly distinguished. Different numbers of introns in the partial sequences of the mt-LSU were identified in several G. intraradices isolates. The varying number of introns among the different isolates of G. intraradices and the absence of introns in G. proliferum, suggest that the intron gain or removal events occurred relatively recently in evolutionary history. Further studies of the distribution of the introns will provide evidence of how frequently they are transferred. An open reading frame (ORF) of a putative homing endonuclease from the LAGLIDADG2 family was identified in mt-LSU introns of several G. intraradices isolates. The phylogeny of the protein sequences showed that the closest known relatives of the LAGLIDADG endonucleases were found in mitochondrial introns of Ascomycetes. As occasional horizontal transfer of these introns is likely, it is not unexpected that the phylogeny obtained is in conflict with established fungal phylogenies based on other marker genes. However, peculiar properties of the homing endonucleases offer promising potential for future studies. Overall, the mt-LSU and its introns are promising high-resolution molecular markers to analyze communities and populations of AMF. The RPB1 gene was analyzed as a possible new nuclear-encoded molecular marker for the Glomeromycota. New Glomus group A-specific primers were designed and successfully tested on several members of this group. No evidence for genetic variability was found within the isolates. Based on the new RPB1 sequences, phylogenetic analyses were performed. The phylum-level …