Detection of methanogenic Archaea in peat: comparison of PCR primers targeting the mcrA gene.
Methanogens (domain Archaea) have a unique role in the carbon cycle as producers of the greenhouse gas methane (CH(4)). Methyl-coenzyme M reductase (MCR) is a vital enzyme in CH(4) production, and the mcrA gene coding for a subunit of MCR has been employed as a specific marker for the detection and differentiation of methanogen communities. A critical step in assessing environmental mcrA diversity is the selection of PCR primers. The objective of this study was to compare the diversity coverage of three published mcrA primer sets MCR, ME and ML (also known as MCR and Luton-mcrA) and their ability to discern methanogen communities in a drained peatland. The primers were applied to DNA extracts from unfertilised and ash-fertilised peat from two different depths. Amplified mcrA communities were cloned and sequenced, and the sequences were divided into operational taxonomic units (OTUs) by restriction fragment length polymorphism (RFLP) and sequence analysis. All primers recovered characteristic OTUs associated with the peat depths and treatments and confirmed a previous observation of low methanogen diversity. The minor differences in OTU ranges of the primers did not greatly affect the observed community composition. However, as the proportions of several OTUs varied strongly, the primers provided different quantitative representations of mcrA communities. We concluded that the ML and MCR primers had better amplification ranges than the ME set, but the use of MCR with peat samples was problematic due to poor amplification. Consequently, the ML primers were best suited for mcrA analysis of peatland methanogen communities.
Juottonen
Heli
H
Department of Biological and Environmental Sciences, General Microbiology, 00014 University of Helsinki, Finland.
Galand
Pierre E
PE
Yrjälä
Kim
K
D000595:Amino Acid Sequence; D001105:Archaea / Q000235:genetics / Q000302:isolation & purification*; D044822:Biodiversity; D017931:DNA Primers; D008969:Molecular Sequence Data; D010088:Oxidoreductases / Q000235:genetics*; D010802:Phylogeny; D016133:Polymerase Chain Reaction / Q000379:methods; D012150:Polymorphism, Restriction Fragment Length; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D012988:Soil Microbiology
D017931:DNA Primers; D012336:RNA, Ribosomal, 16S; D010088:Oxidoreductases; C021572:methyl coenzyme M reductase