Quantifying Microbial N Cycling Genes

We used qPCR to quantity the absolute abundance of bacterial and archaeal 16S rRNA genes as well as the key genes involved in N cycle pathways, including denitrification (nirS, nirK, nosZ clade I, and nosZ clade II), N fixation (nifH), dissimilatory nitrate reduction to ammonia (DNRA; nrfA), ammonia oxidation (bacterial amoA, archaeal amoA, comammox amoA), and anammox- and n-damo-specific 16S rRNA genes (Supplementary Fig. 7). The qPCR assays were performed using RotorGene^® Q equipment (Qiagen, Valencia, CA, USA). The qPCR method was performed following ref. ^{34 (link)}. Briefly, the qPCR reactions were performed in 10 μL volume containing 5 μL Maxima SYBR Green Master Mix (Thermo Fisher Scientific Inc., Waltham, MA, USA), an optimized concentration of forward and reverse primers, 1 μL of template DNA and sterile distilled water. The gene-specific primer sets, optimized primer concentrations and thermal cycling conditions for each target gene are shown in Supplementary Data 8. The quantification data were analyzed with RotorGene Series Software (version 2.0.2; Qiagen, Hilden, Germany) and LinRegPCR program (version 2020.0)^{42 (link)}. The gene abundances were calculated as a mean of fold differences between a sample and each 10-fold standard dilution in respective standard as recommended by ref. ^{42 (link)}; gene abundances were reported as gene copy numbers per gram of dry soil.