PCR Primers for Metazoan Nuclear 18S and 28S Ribosomal DNA Sequences

Ryuji J Machida,Nancy Knowlton,Jack Anthony Gilbert

doi:10.1371/journal.pone.0046180

Abstract

BackgroundMetagenetic analyses, which amplify and sequence target marker DNA regions from environmental samples, are increasingly employed to assess the biodiversity of communities of small organisms. Using this approach, our understanding of microbial diversity has expanded greatly. In contrast, only a few studies using this approach to characterize metazoan diversity have been reported, despite the fact that many metazoan species are small and difficult to identify or are undescribed. One of the reasons for this discrepancy is the availability of universal primers for the target taxa. In microbial studies, analysis of the 16S ribosomal DNA is standard. In contrast, the best gene for metazoan metagenetics is less clear. In the present study, we have designed primers that amplify the nuclear 18S and 28S ribosomal DNA sequences of most metazoan species with the goal of providing effective approaches for metagenetic analyses of metazoan diversity in environmental samples, with a particular emphasis on marine biodiversity.Methodology/Principal FindingsConserved regions suitable for designing PCR primers were identified using 14,503 and 1,072 metazoan sequences of the nuclear 18S and 28S rDNA regions, respectively. The sequence similarity of both these newly designed and the previously reported primers to the target regions of these primers were compared for each phylum to determine the expected amplification efficacy. The nucleotide diversity of the flanking regions of the primers was also estimated for genera or higher taxonomic groups of 11 phyla to determine the variable regions within the genes.Conclusions/SignificanceThe identified nuclear ribosomal DNA primers (five primer pairs for 18S and eleven for 28S) and the results of the nucleotide diversity analyses provide options for primer combinations for metazoan metagenetic analyses. Additionally, advantages and disadvantages of not only the 18S and 28S ribosomal DNA, but also other marker regions as targets for metazoan metagenetic analyses, are discussed.

Highlights

Human activities pose severe threats to planetary biodiversity e.g. [1,2,3], and it is critically important to be able to rapidly estimate biodiversity across space and through time
Analyses based on second-generation sequencing have many advantages in this regard, as they can produce very large numbers of sequences from single samples, either by targeting single or multiple genes using PCR or by targeting entire genomes
By integrating results obtained from these analyses, we propose combinations of primers that are likely to retrieve a more complete representation of the taxonomic diversity of metazoans present in environmental samples

Summary

Introduction

Human activities pose severe threats to planetary biodiversity e.g. [1,2,3], and it is critically important to be able to rapidly estimate biodiversity across space and through time. Species that comprise the majority of metazoan biodiversity are often small and difficult to sample and analyze individually (for example Nematoda, Copepoda, Ostracoda, Rotifera, Kinorhyncha, Loricifera, and Tardigrada). Metagenetic analyses, which amplify and sequence target marker DNA regions from environmental samples, are increasingly employed to assess the biodiversity of communities of small organisms. Using this approach, our understanding of microbial diversity has expanded greatly. We have designed primers that amplify the nuclear 18S and 28S ribosomal DNA sequences of most metazoan species with the goal of providing effective approaches for metagenetic analyses of metazoan diversity in environmental samples, with a particular emphasis on marine biodiversity

Objectives

Methods

Results