Optimal Eukaryotic 18S and Universal 16S/18S Ribosomal RNA Primers and Their Application in a Study of Symbiosis

Qian P-Y (2014) Optimal Eukaryotic 18S and Universal 16S/18S Ribosomal RNA Primers and Their Application in a Study of Symbiosis. PLoS ONE 9(3): e90053. doi:10.1371/journal.pone.0090053 Optimal Eukaryotic 18S and Universal 16S/18S Ribosomal RNA Primers and Their Application in a Study of Symbiosis Yong Wang 0 Ren Mao Tian 0 Zhao Ming Gao 0 Salim Bougouffa 0 Pei-Yuan Qian 0 Newton C. M. Gomes, University of Aveiro, Portugal 0 1 Division of Life Sciences, Hong Kong University of Science and Technology, Clear Water Bay, Hong Kong, 2 Sanya Institute of Deep Sea Science and Engineering, Chinese Academy of Sciences , San Ya, Hai Nan , China Eukaryotic 18S ribosomal RNA (rRNA) gene primers that feature a wide coverage are critical in detecting the composition of eukaryotic microscopic organisms in ecosystems. Here, we predicted 18S rRNA primers based on consecutive conserved sites and evaluated their coverage efficiency and scope of application to different eukaryotic groups. After evaluation, eight of them were considered as qualified 18S primers based on coverage rate. Next, we examined common conserved regions in prokaryotic 16S and eukaryotic 18S rRNA sequences to design 16S/18S universal primers. Three 16S/18S candidate primers, U515, U1390 and U1492, were then considered to be suitable for simultaneous amplification of the rRNA sequences in three domains. Eukaryotic 18S and prokaryotic 16S rRNA genes in a sponge were amplified simultaneously using universal primers U515 and U1390, and the subsequent sorting of pyrosequenced reads revealed some distinctive communities in different parts of the sample. The real difference in biodiversity between prokaryotic and eukaryotic symbionts could be discerned as the dissimilarity between OTUs was increased from 0.005 to 0.1. A network of the communities in external and internal parts of the sponge illustrated the co-variation of some unique microbes in certain parts of the sponge, suggesting that the universal primers are useful in simultaneous detection of prokaryotic and eukaryotic microbial communities. - Funding: This study was supported by the'Strategic Priority Research Program of the Chinese Academy of Sciences (CAS) (No. X XDB06010100 and XXDB06010200), National Basic Research Program of China (973 Program, No. 2012CB417304), the NatFure Science Foundation of China (U1301232) and awards from the Sany Institute of Deep Sea Science and Engineering, CAS (SIDSSE-201206, SIDSSE-201305) and SIDSSE-BR-201303) and from King Abdullah University of Science and Technology (SA-C0040/UK-C0016). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Eukaryotic microbes play important roles as organic degraders, predators, producers and parasites [13]; however, our knowledge regarding their taxonomy, evolution, ecology and diversity remains limited. Eukaryotic microbes are typically ignored in most ecological settings, perhaps due to technical difficulties and a poor understanding of the microscopic world at this scale [4,5]. As a result, a vital position for them in a variety of ecosystems has not been demonstrated. In some biospheres, there may be interplay between eukaryotic microbes and prokaryotic organisms [6], potentially mediating bioremediation and nutrient flow from autotrophic microbes to higher eukaryotes [7]. However, only prokaryotes have been reported in extreme environments; most studies have neglected to examine microbial eukaryotes [8,9]. To what extent these eukaryotic microbes can spread into extreme conditions and the factors that are most critical to their survival are questions that need to be answered. The effects of anoxic conditions on protistan microbial communities were recently examined using 18S ribosomal DNA (rDNA) cloning and nextgeneration pyrosequencing techniques [1012]. We have thus obtained a glimpse of the eukaryotic microbial communities present in environmental samples despite the difficulties encountered during sampling and data analysis [13]. However, eukaryotes have not been studied as extensively as prokaryotes in similar niches. For example, protist diversity was found to be much higher than originally estimated; it is believed that less than 10% of the rDNA sequences have been detected [14]. Despite a number of recent reports on biodiversity of eukaryotic microbes [1517], their fitness to the local environment and role in the ecosystems are not determined. Thus, estimates of species richness and diversity of microbial eukaryotes and their relationship with prokaryotic organisms are a high priority of current studies. The relationships between eukaryotes and prokaryotes can be inferred from co-variation of the corresponding species. Therefore, efficient, simultaneous amplification of 16S and 18S rDNA sequences from a given sample is critical. Small subunit ribosomal RNA (SSU rRNA) genes are the standard reference sequences for taxonomic classification of organisms. By calculating the similarity between rRNAs, Archaea was separated from Bacteria as an independent domain in 1977 [18]. Eukaryotic 18S rRNA primers have been applied in many studies investigating environmental communities [13,19,20]. In 2001, novel small eukaryotes were reported in an aphotic zone in the Antarctic polar front [21]. In addition, novel phylogenetic groups of fungal microbes have been defined using the same method [22]. Fungal diversity in indoor environments has been assessed using 18S rRNA amplicon pyrosequencing [23]. The eukaryotic primers A and B were designed in the 1980s and are still widely used at present [24]. Subsequently, several other eukaryote-specific and universal primers were developed, such as E82, E528, U1391 and U1492 [21,25]. Although the 18S rRNA amplicon pyrosequencing technique has been applied to study environmental microbial eukaryotic communities, the specificity and coverage of the primers have not been evaluated. The addition of more eukaryotic rRNA sequences to the databases has revealed polymorphisms in several primer target regions, suggesting that significant eukaryotic diversity may be escaping detection. Thus, there are currently questions regarding the validity of some primers; optimization is required by modifying the primers to cover the most conserved regions. The number of 18S rRNA sequences in the current SILVA rRNA database is increasing rapidly [26]. In the present study, we used sequences available in this database to predict suitable primers for eukaryotic 18S rRNA genes and assess their coverage. The prediction scheme used herein to predict and evaluate prokaryotic primers has been introduced in our previous work [27]. Eighteen candidate eukaryotic primers were designed using the same protocol coupled with the ARB package [28]. These primers were evaluated together with a list of published 18S primers. Next, common (...truncated)