An improved primer set and amplification protocol with increased specificity and sensitivity targeting the Symbiodinium ITS2 region

The Internal Transcribed Spacer 2 (ITS2) rRNA gene is a commonly targeted genetic marker to assess diversity of , a dinoflagellate genus of algal endosymbionts that is pervasively associated with marine invertebrates, and notably reef-building corals. Here we tested three commonly used ITS2 primer p...

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Bibliographic Details
Published in:PeerJ (San Francisco, CA) CA), 2018-05, Vol.6, p.e4816-e4816, Article e4816
Main Authors: Hume, Benjamin C C, Ziegler, Maren, Poulain, Julie, Pochon, Xavier, Romac, Sarah, Boissin, Emilie, de Vargas, Colomban, Planes, Serge, Wincker, Patrick, Voolstra, Christian R
Format: Article
Language:English
Subjects:
Analysis
Biodiversity
Climate Change Biology
Coral Reef
Coral reefs
Dinoflagellates
Ecology
Genetic aspects
Genetic markers
Identification and classification
Life Sciences
Marine Biology
Metabarcoding
Metagenomics
Microbial Biology
Microbiology and Parasitology
Molecular Biology
Next-generation sequencing
Protistology
Symbiodinium
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Summary:The Internal Transcribed Spacer 2 (ITS2) rRNA gene is a commonly targeted genetic marker to assess diversity of , a dinoflagellate genus of algal endosymbionts that is pervasively associated with marine invertebrates, and notably reef-building corals. Here we tested three commonly used ITS2 primer pairs (SYM_VAR_5.8S2/SYM_VAR_REV, ITSintfor2/ITSReverse, and ITS-DINO/ITS2Rev2) with regard to amplification specificity and sensitivity towards , as well as sub-genera taxonomic bias. We tested these primers over a range of sample types including three coral species, coral surrounding water, reef surface water, and open ocean water to assess their suitability for use in large-scale next generation sequencing projects and to develop a standardised PCR protocol. We found the SYM_VAR_5.8S2/SYM_VAR_REV primers to perform superior to the other tested ITS2 primers. We therefore used this primer pair to develop a standardised PCR protocol. To do this, we tested the effect of PCR-to-PCR variation, annealing temperature, cycle number, and different polymerase systems on the PCR efficacy. The ITS2 PCR protocol developed here delivers improved specificity and sensitivity towards with apparent minimal sub-genera taxonomic bias across all sample types. In particular, the protocol's ability to amplify from a range of environmental sources will facilitate the study of populations across biomes.
ISSN:2167-8359
2167-8359
DOI:10.7717/peerj.4816