Computational protocol: Nitrous Oxide Emissions from Ephemeral Wetland Soils are Correlated with Microbial Community Composition

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Protocol publication

[…] At our study site there is no landform difference in bacterial amoA and nosZ community composition (Ma et al., ). Therefore, we only evaluated community composition in water-accumulating landforms (i.e., the CD and BC elements). Community composition analysis was also limited to samples from the start (June 1) and end (September 12) of the sampling season, because the greatest difference in gene abundance and measured activity occurred between these two dates.Fragments of amoA and nosZ were amplified, cloned, and analyzed for restriction fragment length polymorphism (RFLP). Procedures and conditions for amplifying amoA and nosZ fragments follow Ma et al., . PCR products of the expected size (490 bp for bacterial amoA and 700 bp for nosZ) were excised after agarose gel electrophoresis, purified using QIAquick® Gel Extraction Kit (Qiagen), and cloned using TOPO® TA Cloning Kit (Invitrogen). Forty-eight clones were selected for each sample and gene combination. Clones were screened for the proper inserted fragment by PCR product size. The PCR product for each clone was then used in three separate reactions with the endonucleases AluI, HhaI, and RsaI (Invitrogen) and visualized by agarose gel electrophoresis (3% w/v gel; 80 V for 90 min). Clones were classified into operational taxonomic units (OTUs) based on the combination of the three separate RFLP patterns.Based on indicator species analysis (McCune and Mefford, ), five clones from each indicator OTU – clones that differed significantly between land-use and time for each gene – were sequenced at the National Research Council Plant Biotechnology Institute (Saskatoon, SK, Canada) using the amoA-1F or nosZ-F primer. A consensus sequence for each OTU was generated by alignment in ClustalX (v1.81) and edited with GeneDoc (v2.6). Phylogenetic trees using the cloned sequences were created using the programs DNADIST (Jukes–Cantor model), NEIGHBOR (neighbor-joining method; out-group = Nitrosomonas europaea accession L08050 for amoA and Ralstonia eutropha accession X65278 for nosZ), and SEQBOOT available in the PHYLIP (v3.5c) computer package (Felsenstein, ). […]

Pipeline specifications

Software tools Clustal W, PHYLIP
Application Phylogenetics
Chemicals Ammonia, Nitrous Oxide