title: Development of 16S rRNA gene-targeted primers for detection of archaeal anaerobic methanotrophs (ANMEs).
abstract: Uncultured archaeal anaerobic methanotrophs (ANMEs) are known to operate the anaerobic oxidation of methane process, an important sink for the greenhouse gas methane in natural environments. In this study, we designed 16S rRNA gene-specific primers for each of the phylogenetic groups of ANMEs (ANME-1, Guaymas Basin hydrothermal sediment clones group within the ANME-1, ANME-2a, ANME-2b, ANME-2c and ANME-3) based on previously reported sequences. The newly designed primers were used for the detection of the various groups of ANMEs in the sulphate-limited anaerobic environmental samples, i.e. methanogenic sludges, rice field soils, lotus field sediments and natural gas fields. The ANME 16S rRNA gene sequences were detected only in a natural gas field sample among the environments examined in this study and were of the ANME-1 and -2c groups. In addition, the quantitative real-time PCR analysis using the designed primers showed that abundances of ANME-1 and -2c were estimated to be <0.02% of the total prokaryotic 16S rRNA gene community. The newly designed ANME group-specific primers in this study may be useful to survey the distribution and quantitative determination of ANMEs.
authors.affiliation: Subsurface Geobiology Advanced Research Team, Extremobiosphere Research Program, Institute of Biogeosciences, Japan Agency for Marine-Earth Science and Technology, Kanagawa, Japan.
authors.forename: Ai
authors.initials: A
authors.lastname: Miyashita
authors.forename: Hanako
authors.initials: H
authors.lastname: Mochimaru
authors.forename: Hiromi
authors.initials: H
authors.lastname: Kazama
authors.forename: Akiyoshi
authors.initials: A
authors.lastname: Ohashi
authors.forename: Takashi
authors.initials: T
authors.lastname: Yamaguchi
authors.forename: Takuro
authors.initials: T
authors.lastname: Nunoura
authors.forename: Koki
authors.initials: K
authors.lastname: Horikoshi
authors.forename: Ken
authors.initials: K
authors.lastname: Takai
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Imachi
mesh_terms: D000693:Anaerobiosis; D001105:Archaea / Q000145:classification / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D052818:Autotrophic Processes; D017931:DNA Primers / Q000235:genetics*; D019641:DNA, Archaeal / Q000235:genetics; D004275:DNA, Ribosomal / Q000235:genetics; D004783:Environmental Microbiology; D008697:Methane / Q000378:metabolism*; D008969:Molecular Sequence Data; D010802:Phylogeny; D016133:Polymerase Chain Reaction / Q000379:methods*; D012336:RNA, Ribosomal, 16S / Q000235:genetics*
chemical_list: D017931:DNA Primers; D019641:DNA, Archaeal; D004275:DNA, Ribosomal; D012336:RNA, Ribosomal, 16S; D008697:Methane