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Representative sequences were selected by aligning the normalized reads and clustering them at a 97% nucleotide identity cutoff in RDPipeline, and singletons were removed as described by Zhou et al. (2011). The representative sequences without singletons were aligned in RDPipeline and neighbor-joining (NJ) trees were constructed based on the Kimura two-parameter model using the Rucaparib clinical trial PHYLIP software (ver. 3.695) (Felsenstein, 2002). The NEXUS files of the NJ trees were used as input files for weighted hierarchical clustering and principal coordinate analysis (PCoA). Taxonomic classification was performed for all high-quality sequences of Bacteria and Archaea at the phylum, class, and genus levels based on the SILVA database (v.102), using the nearest-neighbor method within the mothur program (Pruesse et al., 2007). The absolute abundances of methanotrophic and methanogenic groups along a depth gradient in the bulk and rhizosphere soils of the rice paddy were estimated by multiplying the relative abundances of methanotrophic and methanogenic groups that were classified at the genus level and the gene copy numbers of bacterial and archaeal 16S rRNA genes that were obtained by the qPCR analysis. Nucleotide sequence accession number The pyrosequencing data of the bacterial and archaeal 16S rRNA genes are publicly available in the NCBI Short Read Archive (SRA) under accession no. SRP052852 (NCBI BioProject PRJNA273696). Results Vertical profiles of oxygen, methane, and TOC concentrations The concentration profiles of oxygen, methane, and TOC along a depth gradient in the flooded rice paddy are shown in Figure ?Figure1.1. The oxygen concentration in the surface floodwater of the paddy soil was approximately 8.8 ppm, but the concentration decreased very sharply to below the detection limit at 8-mm (Figure ?(Figure1A).1A). This result suggested that the oxic-anoxic interface representing the penetration limit of oxygen from the soil surface was located at the depth range of approximately 6�C10-mm, which was slightly different from previously reported results in which the oxic-anoxic interfaces were located at approximately 2-mm depth (Revsbech et al., 1999; Reim et al., 2012). This difference might be explained by the high oxygen concentration in the floodwater in this study. No clear difference was observed in the vertical oxygen profiles of the bulk and rhizosphere soils. The methane concentrations were extremely low in the aerobic surface soil (