A simple and reliable method for SSU rRNA gene DNA extraction, amplification, and cloning from single AM fungal spores

Abstract

We describe a method that allows quick and easy PCR amplification and cloning of nearly complete SSU rRNA genes from arbuscular mycorrhizal fungi. The procedure tested on spores from 37 different glomalean isolates was based on magnetic separation with Dynabeads, followed by nested PCR with two primer pairs. All trials led to visible amplification products of the expected size. Thereafter, the PCR fragments could be quickly and efficiently cloned by means of a topoisomerase-activated vector (pCR2.1-TOPO). The technique is rapid, uncomplicated and comparatively inexpensive. The use of single spores for DNA extraction has some advantages over multispore-preparations, e.g. it is less susceptible to contamination with other organisms present in the cultures. The method can be used for the quick and reliable preparation of a large number of samples and is highly reproducible. It could also be used for genes other than the SSU rRNA gene.

Former Land Use and Host Genotype Influence the Mycorrhizal Colonization of Poplar Roots

“…The ITS regions were amplified by a NESTED PCR using the universal primer pair GEOA1 and ART4 [26] for a first PCR reaction and the four pairs of taxon-specific primers targeting the 18S rDNA genes (GmF-GmR Glomus mosseae group, GiF-GiR Glomus intraradices group, GhF-GhR Glomus hoi, SsF-SsR Scutellospora spp. [19]) for the second reaction.…”

Molecular diversity of arbuscular mycorrhizal fungi colonising arable crops

“…The DNA primer AM1 was designed by Helgason et al [19] to match all small subunit ribosomal RNA (SSU) sequences known at that time from the Glomales, but to di¡er from corresponding plant SSU sequences. It has become apparent that the AM1 primer excludes a number of fungal types from previously unrecognised groups, the Archaeosporaceae and Paraglomaceae identi¢ed by Morton and Redecker [2,22,23]. Thus, AM1 can be used in combination with the general eukaryotic primer NS31 [17] in a polymerase chain reaction (PCR) to amplify sequences from the three traditional families of Glomales from colonised roots.…”

Advances in Molecular Diversity of Arbuscular Mycorrhizal Fungi (Phylum Glomeromycota) in Forest Ecosystems

“…In the past decade, considerable effort has been expended to understand the keystone ecological position of AM symbioses, most studies have been limited in scope to recording organism occurrences and identities, as determined from morphological characters and ribosomal sequence markers for characterization of AMF, leading to important advances in our understanding of the phylogeny Schwarzott et al, 2001), ecology (Helgason et al, 1998;Helgason et al, 2002;Husband et al, 2002a, b;Kowalchuk et al, 2002), genetics (GianinazziPearson et al, 2001;Harrison, 1999), and evolution (Gandolfi et al, 2003;Sanders, 2002) of this group of obligatory symbiotic fungi. rRNA genes have become the most widely use targets for detection of AMF in environmental samples (Clapp et al, 2002).…”