title: Novel PCR primers for the archaeal phylum Thaumarchaeota designed based on the comparative analysis of 16S rRNA gene sequences.
abstract: Based on comparative phylogenetic analysis of 16S rRNA gene sequences deposited in an RDP database, we constructed a local database of thaumarchaeotal 16S rRNA gene sequences and developed a novel PCR primer specific for the archaeal phylum Thaumarchaeota. Among 9,727 quality-filtered (chimeral-checked, size >1.2 kb) archaeal sequences downloaded from the RDP database, 1,549 thaumarchaeotal sequences were identified and included in our local database. In our study, Thaumarchaeota included archaeal groups MG-I, SAGMCG-I, SCG, FSCG, RC, and HWCG-III, forming a monophyletic group in the phylogenetic tree. Cluster analysis revealed 114 phylotypes for Thaumarchaeota. The majority of the phylotypes (66.7%) belonged to the MG-I and SCG, which together contained most (93.9%) of the thaumarchaeotal sequences in our local database. A phylum-directed primer was designed from a consensus sequence of the phylotype sequences, and the primer's specificity was evaluated for coverage and tolerance both in silico and empirically. The phylum-directed primer, designated THAUM-494, showed >90% coverage for Thaumarchaeota and <1% tolerance to non-target taxa, indicating high specificity. To validate this result experimentally, PCRs were performed with THAUM-494 in combination with a universal archaeal primer (ARC917R or 1017FAR) and DNAs from five environmental samples to construct clone libraries. THAUM-494 showed a satisfactory specificity in empirical studies, as expected from the in silico results. Phylogenetic analysis of 859 cloned sequences obtained from 10 clone libraries revealed that >95% of the amplified sequences belonged to Thaumarchaeota. The most frequently sampled thaumarchaeotal subgroups in our samples were SCG, MG-I, and SAGMCG-I. To our knowledge, THAUM-494 is the first phylum-level primer for Thaumarchaeota. Furthermore, the high coverage and low tolerance of THAUM-494 will make it a potentially valuable tool in understanding the phylogenetic diversity and ecological niche of Thaumarchaeota.
authors.affiliation: Institute of Environmental Sciences and Department of Environmental Sciences, Hankuk University of Foreign Studies, Yong-In, Korea.
authors.forename: Jin-Kyung
authors.initials: JK
authors.lastname: Hong
authors.affiliation: Institute of Environmental Sciences and Department of Environmental Sciences, Hankuk University of Foreign Studies, Yong-In, Korea.
authors.forename: Hye-Jin
authors.initials: HJ
authors.lastname: Kim
authors.affiliation: Institute of Environmental Sciences and Department of Environmental Sciences, Hankuk University of Foreign Studies, Yong-In, Korea.
authors.forename: Jae-Chang
authors.initials: JC
authors.lastname: Cho
mesh_terms: D001105:Archaea / Q000187:drug effects / Q000235:genetics*; D017931:DNA Primers / Q000235:genetics*; D010802:Phylogeny; D016133:Polymerase Chain Reaction / Q000379:methods*; D012336:RNA, Ribosomal, 16S / Q000235:genetics*
chemical_list: D017931:DNA Primers; D012336:RNA, Ribosomal, 16S