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

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    Type
    Article
    Authors
    Hume, Benjamin
    Ziegler, Maren cc
    Poulain, Julie
    Pochon, Xavier
    Romac, Sarah
    Boissin, Emilie
    de Vargas, Colomban
    Planes, Serge
    Wincker, Patrick
    Voolstra, Christian R. cc
    KAUST Department
    Biological and Environmental Sciences and Engineering (BESE) Division
    Marine Science Program
    Red Sea Research Center (RSRC)
    Date
    2018-05-23
    Permanent link to this record
    http://hdl.handle.net/10754/627966
    
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    Abstract
    The 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.
    Citation
    Hume 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.
    Sponsors
    This 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.
    Publisher
    PeerJ
    Journal
    PeerJ
    DOI
    10.7717/peerj.4816
    Additional Links
    https://peerj.com/articles/4816/
    Relations
    Is Supplemented By:
    • [Bioproject]
      Title: Tara Pacific Symbiodinium ITS2 primerPublication Date: 2018-01-15. bioproject: PRJNA430028 Handle: 10754/666397
    ae974a485f413a2113503eed53cd6c53
    10.7717/peerj.4816
    Scopus Count
    Collections
    Articles; Biological and Environmental Science and Engineering (BESE) Division; Red Sea Research Center (RSRC); Marine Science Program

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