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%.