Real-time PCR Quantification and high-throughput sequencing
Soil total genomic DNA was extracted using a MoBio Power soil TMDNA isolation kit (San Diego, CA) according to the manufacturer’s instructions and was evaluated by a Nanodrop ND-2000 UV–vis spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA).
To amply the genes of bacterial and archaeal amoA, primer sets amoA-1F/amoA-2R (Rotthauwe et al. 1997) and Arch-amoAF/Arch-amoAR (Francis et al. 2005) were selected, respectively. The comaA-244F/comaA-659R and comaB-244F/comaB-659R (Pjevacet al., 2017) primers were applied to amplify the comammox Nitrospira CladeA and CladeB amoA genes, respectively. Ammonia oxidizer abundances were detected according to the gene copy numbers revealed by real-time quantitative PCR using the iCycler iQ 5 thermocycler (BioRad Laboratories, Hercules, CA, USA). Serial dilutions of the plasmid DNA were used for standard curves for qPCR assays following Di et al. 2009.
Soil ammonia-oxidizer community structures were characterized only in soil samples collected in September 2021 using the high-throughput sequencing method. Briefly, the PCR amplification of the amoA gene was conducted using the DNA extracted. The PCR products were gel purified, quantified using PicoGreen Kits (Invitrogen, Shanghai, China), and pyrosequenced on a GS FLX Titanium sequencer (Roche Diagnostics Corporation, Branford, CT, USA). Sequencing was performed using the Roche 454 Flx+ platform at Personalbio Company (Shanghai, China). The raw high-throughput sequencing data were first processed using the Quantitative Insights Into Microbial Ecology (QIIME) toolkit. The potentially similar sequences were clustered into operational taxonomic units (OTUs) at a similarity level of 97%.