title: New PCR primers based on mcrA gene for retrieving more anaerobic methanotrophic archaea from coastal reedbed sediments.
abstract: Two pairs of PCR primes ANMEallF/R and ANME23F/R were designed by Codehop method based on sequences available to retrieve more anaerobic methanotrophic (ANME) archaea mcrA gene sequences and ANME 2 and 3 subtypes from reedbed in two seasons. Overall, the PCR primers showed slightly favor for ANME group mcrA gene sequences. Due to the predominance of methanogens mainly affiliated to Methanomicrobiales in the samples, a large portion of mcrA gene sequences amplified in the clone libraries belonged to methanogens. Differences in PCR primers and performance affected the mcrA gene-PCR-amplified community composition to a minor extent. PCR primers targeting ANME mcrA group g-h were designed to apply real-time PCR for quantifying more groups of mcrA gene-affiliated ANME archaea and tested with these same samples, and the most abundant group in the whole ANME mcrA community was ANME group g-h. In addition, a stable mcrA gene-harboring archaeal community pattern was detected in the reedbed sediment samples collected from two distinctively different seasons. The PCR and qPCR primers designed in this study can expand our knowledge on the distribution of ANME mcrA genes and community composition in the ecosystem to better understand the carbon cycle.
authors.affiliation: Laboratory of Environmental Microbiology and Toxicology, School of Biological Sciences, The University of Hong Kong, Pokfulam Road, Hong Kong SAR, Hong Kong, People's Republic of China.
authors.forename: Zhichao
authors.initials: Z
authors.lastname: Zhou
authors.forename: Ping
authors.initials: P
authors.lastname: Han
authors.forename: Ji-Dong
authors.initials: JD
authors.lastname: Gu
mesh_terms: D017931:DNA Primers / Q000235:genetics*; D019641:DNA, Archaeal / Q000737:chemistry / Q000235:genetics; D019015:Geologic Sediments / Q000382:microbiology*; D017018:Methanomicrobiales / Q000201:enzymology / Q000235:genetics* / Q000302:isolation & purification*; D008969:Molecular Sequence Data; D010088:Oxidoreductases / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods*; D017422:Sequence Analysis, DNA
chemical_list: D017931:DNA Primers; D019641:DNA, Archaeal; D010088:Oxidoreductases; C021572:methyl coenzyme M reductase