FEMS Microbiology Ecology, 96, 2020, faa110 doi: 10.1093/femsec/faa110 Advance Access Publication Date: 3 June 2020 Research Article RESEARCH ARTICLE A novel PCR-clamping assay reducing plant host DNA amplifcation signifcantly improves prokaryotic endo-microbiome community characterization Emilie Lef ` evre 1 , Courtney M. Gardner 2 and Claudia K. Gunsch 1, * ,† 1 Department of Civil and Environmental Engineering, Duke University, 127 Hudson Hall, Box 90287, Durham, NC 27708, USA and 2 Department of Civil and Environmental Engineering, Washington State University, 405 Spokane street, Sloan 101, Box 642910, Pullman, WA 99164, USA ∗ Corresponding author: Department of Civil and Environmental Engineering, Duke University, 127 Hudson Hall, Box 90287, Durham, NC 27708, USA. Tel: +1 (919) 660-5208; E-mail: ckgunsch@duke.edu One sentence summary: In this study, we are presenting a novel PCR-clamping assay reducing the co-amplifcation of plant chloroplast and mitochondrial DNA and signifcantly improving the characterization of endo-microbiome prokaryotic communities. Editor: Angela Sessitsch † Claudia K. Gunsch, http://orcid.org/0000-0002-8555-0313 ABSTRACT Due to the sequence homology between the bacterial 16S rRNA gene and plant chloroplast and mitochondrial DNA, the taxonomic characterization of plant microbiome using amplicon-based high throughput sequencing often results in the overwhelming presence of plant-affliated reads, preventing the thorough description of plant-associated microbial communities. In this work we developed a PCR blocking primer assay targeting the taxonomically informative V5-V6 region of the 16S rRNA gene in order to reduce plant DNA co-amplifcation, and increase diversity coverage of associated prokaryotic communities. Evaluation of our assay on the characterization of the prokaryotic endophytic communities of Zea mays, Pinus taeda and Spartina alternifora leaves led to signifcantly reducing the proportion of plant reads, yielded 20 times more prokaryotic reads and tripled the number of detected OTUs compared to a commonly used V5-V6 PCR protocol. To expand the application of our PCR-clamping assay across a wider taxonomic spectrum of plant hosts, we additionally provide an alignment of chloroplast and mitochondrial DNA sequences encompassing more than 200 terrestrial plant families as a supporting tool for customizing our blocking primers. Keywords: blocking primers; chloroplast DNA; mitochondrial DNA; PCR clamping; plant microbiome; prokaryotic endophytes INTRODUCTION Over the last decade, the increasing amount of surveys applying next-generation sequencing technologies to the study of micro- bial communities has unquestionably improved our apprecia- tion of the extent of the microbial taxonomic and functional diversity, as well as the importance of the microbial world for ecosystem functioning (Delgado-Baquerizo et al. 2016). Plant- associated microbial communities are no exception to this trend. Surveys describing the structure and diversity of plant- associated fungal and prokaryotic communities across various host species and habitats have led to the realization that they are a source of incredible biological diversity and are essen- tial to key ecosystem processes (Moyes et al. 2016; Laforest- Lapointe et al. 2017; Fitzpatrick et al. 2018a; Christian, Herre and Clay 2019). However, the taxonomic characterization of plant microbiome using direct amplicon sequencing can pose impor- tant methodological challenges. Due to the sequence homology Received: 10 April 2020; Accepted: 1 June 2020 C  FEMS 2020. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com 1 Downloaded from https://academic.oup.com/femsec/article/96/7/fiaa110/5850752 by guest on 14 February 2023