Analysis of partial Glomales SSU rRNA gene sequences: implications for primer design and phylogeny

Abstract

Arbuscular mycorrhizal (AM) fungi, classified in the order Glomales, cannot easily be identified by hyphal morphology. PCR can be used successfully to identify the fungi within the host or soil. Some of the primers that have been used are based on a few sequences of a variable region in the SSU rRNA gene. To verify the specificity of primers of this type which have been used in earlier publications, and to provide data for future primer design, we analysed 51 partial Glomales SSU rRNA gene sequences from 33 glomalean isolates.

To distinguish contaminants from the AM fungi (AMF) all sequences were verified by phylogenetic analyses. Together with sequences from the EMBL database a set of 88 sequences from 58 isolates was analysed, comprising 39 species. The phylogenetic trees reveal at least three distinct well supported Glomus clades and other interesting relationships within the Glomales. Sequence data show that most of the primers published in earlier studies are not useful at the proposed taxonomic level. For example, the VANS1 primer site is not specific for the Glomales, and is homologous to fewer than half of the AMF sequences investigated. Primer regions that have been considered to be family-specific within the Glomales do not show strict specificity. Because clades in the phylogenetic trees fit well to several sequence patterns, priming sites for AMF are discussed and sequence data for future analyses provided.