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

Benjamin C.C Hume,Xavier Pochon,Emilie Boissin,Sarah Romac,Julie Poulain,Serge Planes,Patrick Wincker,Colomban De Vargas,Maren Ziegler,Christian R Voolstra

doi:10.7717/peerj.4816

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.

Highlights

Coral reefs sustain some of the highest levels of biodiversity on Earth and provide a range of services to communities totalling millions of people (Moberg & Folke, 1999; Plaisance et al, 2011)
Of the microbial components that make up the coral holobiont—the consideration of the animal host and its associating microbes as a functional ecological unit—the algal symbionts of the genus Symbiodinium have received the most attention for their role in affording resilience to the coral host (Hume et al, 2016; Thornhill et al, 2017)
The coral-Symbiodinium association is generally obligate with the algal symbiont providing towards the nutritional needs of the animal host, in exchange for a stable, beneficial environment (Muscatine, 1990)

Summary

Introduction

Coral reefs sustain some of the highest levels of biodiversity on Earth and provide a range of services to communities totalling millions of people (Moberg & Folke, 1999; Plaisance et al, 2011). These ecosystems are being lost at an alarming rate (Hughes et al, 2017; Norstrom et al, 2016). This loss is primarily due to anthropogenic stressors degrading the scleractinian corals that build and support these reefs. The ability to resolve taxa within the genus Symbiodinium is essential to better understanding resilience of the coral holobiont and reef ecosystems as a whole

Methods

Discussion

Conclusion