Primer test
Design and test new primers to be used to amplify the fungal ITS2 region by targeting sites in the 5.8S encoding gene.
With recent methodological advances, molecular markers are increasingly used for semi-quantitative analyses of fungal communities. The aim to preserve quantitative relationships between genotypes through PCR places new demands on primers to accurately match target sites and provide short amplicons. The ITS region of the ribosome encoding genes is a commonly used marker for many fungal groups. Here we describe three new primers, which may be used to amplify the fungal ITS2 region by targeting sites in the 5.8S encoding gene. We evaluated the primers and compared their performance with the commonly used ITS1f primer by 454-sequencing of both artificially assembled templates and natural samples. When the entire ITS region was amplified, using the ITS1f - ITS4 primer combination, we found strong bias against species with longer amplicons. This problem could be overcome by using the new primers, which produce shorter amplicons and better preserved the quantitative composition of the template. In addition, the new primers yielded more diverse amplicon communities than the ITS1f primer. Furthermore our results corroborate that the error frequency associated with 454-sequencing is particularly high for fungal ITS sequences, calling for caution when estimating diversity.