title: Performance of 16s rDNA Primer Pairs in the Study of Rhizosphere and Endosphere Bacterial Microbiomes in Metabarcoding Studies.
abstract: Next-generation sequencing technologies have revolutionized the methods for studying microbial ecology by enabling high-resolution community profiling. However, the use of these technologies in unraveling the plant microbiome remains challenging. Many bacterial 16S rDNA primer pairs also exhibit high affinity for non-target DNA such as plastid (mostly chloroplast) DNA and mitochondrial DNA. Therefore, we experimentally tested a series of commonly used primers for the analysis of plant-associated bacterial communities using 454 pyrosequencing. We evaluated the performance of all selected primer pairs in the study of the bacterial microbiomes present in the rhizosphere soil, root, stem and leaf endosphere of field-grown poplar trees (Populus tremula × Populus alba) based on (a) co-amplification of non-target DNA, (b) low amplification efficiency for pure chloroplast DNA (real-time PCR), (c) high retrieval of bacterial 16S rDNA, (d) high operational taxonomic unit (OTU) richness and Inverse Simpson diversity and (e) taxonomic assignment of reads. Results indicate that experimental evaluation of primers provide valuable information that could contribute in the selection of suitable primer pairs for 16S rDNA metabarcoding studies in plant-microbiota research. Furthermore, we show that primer pair 799F-1391R outperforms all other primer pairs in our study in the elimination of non-target DNA and retrieval of bacterial OTUs.
authors.affiliation: Centre for Environmental Sciences, Hasselt University Diepenbeek, Belgium.
authors.forename: Bram
authors.initials: B
authors.lastname: Beckers
authors.affiliation: Centre for Environmental Sciences, Hasselt University Diepenbeek, Belgium.
authors.forename: Michiel
authors.initials: M
authors.lastname: Op De Beeck
authors.affiliation: Centre for Environmental Sciences, Hasselt University Diepenbeek, Belgium.
authors.forename: Sofie
authors.initials: S
authors.lastname: Thijs
authors.affiliation: Centre for Environmental Sciences, Hasselt University Diepenbeek, Belgium.
authors.forename: Sascha
authors.initials: S
authors.lastname: Truyens
authors.affiliation: Centre for Environmental Sciences, Hasselt University Diepenbeek, Belgium.
authors.forename: Nele
authors.initials: N
authors.lastname: Weyens
authors.affiliation: Department of Plant Systems Biology, Flanders Institute for Biotechnology (VIB)Gent, Belgium; Department of Plant Biotechnology and Bioinformatics, Ghent UniversityGent, Belgium.
authors.forename: Wout
authors.initials: W
authors.lastname: Boerjan
authors.affiliation: Centre for Environmental Sciences, Hasselt University Diepenbeek, Belgium.
authors.forename: Jaco
authors.initials: J
authors.lastname: Vangronsveld
keywords: 16S rDNA metabarcoding; 454 pyrosequencing; chloroplast DNA; endophytes; plant microbiome