An improved primer set and amplification protocol with increased specificity and sensitivity targeting the Symbiodinium ITS2 region
AuthorsHume, Benjamin C.C.
de Vargas, Colomban
Voolstra, Christian R.
KAUST DepartmentBiological and Environmental Sciences and Engineering (BESE) Division
Marine Science Program
Red Sea Research Center (RSRC)
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AbstractThe Internal Transcribed Spacer 2 (ITS2) rRNA gene is a commonly targeted genetic marker to assess diversity of Symbiodinium, 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 Symbiodinium, 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 Symbiodinium ITS2 PCR protocol developed here delivers improved specificity and sensitivity towards Symbiodinium with apparent minimal sub-genera taxonomic bias across all sample types. In particular, the protocol’s ability to amplify Symbiodinium from a range of environmental sources will facilitate the study of Symbiodinium populations across biomes.
CitationHume BCC, Ziegler M, Poulain J, Pochon X, Romac S, et al. (2018) An improved primer set and amplification protocol with increased specificity and sensitivity targeting the Symbiodinium ITS2 region. PeerJ 6: e4816. Available: http://dx.doi.org/10.7717/peerj.4816.
SponsorsThis project has been funded through the Tara consortium, France Genomique grant number ANR-10-INBS-09, and KAUST baseline and KAUST BESE division funds to Christian R. Voolstra. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.