Open AccessNew primers to amplify the fungal ITS 2 region – evaluation by 454‐sequencing of artificial and natural communities
Author(s) -
Ihrmark Katarina,
Bödeker Inga T.M.,
CruzMartinez Karelyn,
Friberg Hanna,
Kubartova Ariana,
Schenck Jessica,
Strid Ylva,
Stenlid Jan,
BrandströmDurling Mikael,
Clemmensen Karina E.,
Lindahl Björn D.
Publication year - 2012
Publication title -
fems microbiology ecology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.377
H-Index - 155
eISSN - 1574-6941
pISSN - 0168-6496
DOI - 10.1111/j.1574-6941.2012.01437.x
Subject(s) - biology , amplicon , primer (cosmetics) , internal transcribed spacer , genetics , computational biology , amplicon sequencing , gene , polymerase chain reaction , ribosomal rna , 16s ribosomal rna , chemistry , organic chemistry
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 internal transcribed spacer ( ITS ) region of the ribosome encoding genes is a commonly used marker for many fungal groups. Here, we describe three new primers – f ITS 7, g ITS 7 and f ITS 9, which may be used to amplify the fungal ITS 2 region by targeting sites in the 5.8 S encoding gene. We evaluated the primers and compared their performance with the commonly used ITS 1f primer by 454‐sequencing of both artificially assembled templates and field samples. When the entire ITS region was amplified using the ITS 1f/ ITS 4 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 preserve the quantitative composition of the template. In addition, the new primers yielded more diverse amplicon communities than the ITS 1f primer.