TABLE 1.

Primers and protocols used for the quantification of functional genes encoding nitrate reductases (napA and narG), nitrite reductases (nirK and nirS), and bacterial 16S rRNA

Gene	Primer	Amplicon length (bp)	Reference	Reaction mixture	Thermal profile
napA	V17 m TGGACVATGGGYTTYAAYC	152	46	5 μl SsoAdvanced Universal SYBR, 0.5 μl V17 m (5 μM), 0.5 μl napA4r (5 μM), 3 μl PCR water, 1 μl template	15 s at 98°C, 15 s at 55°C, 15 s at 72°C × 45 cycles
	napA4r ACYTCRCGHGCVGTRCCRCA
narG	narG-f TCGCCSATYCCGGCSATGTC	173	46	5 μl SsoAdvanced Universal SYBR, 0.5 μl narG-f (5 μM), 0.5 μl narG-r (5 μM), 3 μl PCR water, 1 μl template	10 s at 98°C, 20 s at 62°C × 40 cycles
	narG-r GAGTTGTACCAGTCRGCSGAYTCSG
nirK	nirK876c ATYGGCGGVCAYGGCGA	164	47	5 μl SsoAdvanced Universal SYBR, 0.5 μl nirK876c (5 μM), 0.5 μl nirK1040 (5 μM), 3 μl PCR water, 1 μl template	10 s at 98°C, 20 s at 58°C × 40 cycles
	nirK1040 GCCTCGATCAGRTTRTGGTT (modified)
nirS	nirSCd3aF AACGYSAAGGARACSGG	407	48	5 μl SsoAdvanced Universal SYBR, 1 μl nirSCd3aF (5 μM), 1 μl nirSRcd (5 μM), 2 μl PCR water, 1 μl template	30 s at 98°C, 30 s at 57°C, 30 s at 72°C × 40 cycles
	nirSRcd GASTTCGGRTGSGTCTTSAYGAA
16S rRNA gene	341f CCTACGGGAGGCAGCAG	456	49, 50	5 μl SsoAdvanced Universal SYBR, 1 μl 341f (5 μM), 1 μl 797r (5 μM), 2 μl PCR water, 1 μl template	5 s at 98°C, 12 s at 60°C, 1 min at 95°C × 40 cycles
	797r GGACTACCAGGGTATCTAATCCTGTT