Evaluation of six primer pairs targeting the nuclear rRNA operon for characterization of arbuscular mycorrhizal fungal (AMF) communities using 454 pyrosequencing

• We compared six AMF primer pairs specific for use in high throughput sequencing.
• AMV4.5NF–AMDGR, AML1–AML2 and NS31–AML2 were the most AMF specific primer pairs.
• AMV4.5NF–AMDGR favored amplification of Glomeraceae sequences.
• AMV4.5NF–AMDGR together with NS31–AML2 or AML1–AML2 is suitable for tandem use.

In the last few years, 454 pyrosequencing-based analysis of arbuscular mycorrhizal fungal (AMF; Glomeromycota) communities has tremendously increased our knowledge of the distribution and diversity of AMF. Nonetheless, comparing results between different studies is difficult, as different target genes (or regions thereof) and primer combinations, with potentially dissimilar specificities and efficacies, are being utilized. In this study we evaluated six primer pairs that have previously been used in AMF studies (NS31–AM1, AMV4.5NF–AMDGR, AML1–AML2, NS31–AML2, FLR3–LSUmBr and Glo454–NDL22) for their use in 454 pyrosequencing based on both an in silico approach and 454 pyrosequencing of AMF communities from apple tree roots. Primers were evaluated in terms of (i) in silico coverage of Glomeromycota fungi, (ii) the number of high-quality sequences obtained, (iii) selectivity for AMF species, (iv) reproducibility and (v) ability to accurately describe AMF communities. We show that primer pairs AMV4.5NF–AMDGR, AML1–AML2 and NS31–AML2 outperformed the other tested primer pairs in terms of number of Glomeromycota reads (AMF specificity and coverage). Additionally, these primer pairs were found to have no or only few mismatches to AMF sequences and were able to consistently describe AMF communities from apple roots. However, whereas most high-quality AMF sequences were obtained for AMV4.5NF–AMDGR, our results also suggest that this primer pair favored amplification of Glomeraceae sequences at the expense of Ambisporaceae, Claroideoglomeraceae and Paraglomeraceae sequences. Furthermore, we demonstrate the complementary specificity of AMV4.5NF–AMDGR with AML1–AML2, and of AMV4.5NF–AMDGR with NS31–AML2, making these primer combinations highly suitable for tandem use in covering the diversity of AMF communities.