Supplementary Table S1. Sequences of the primers for nirK and nirS in each cluster and the 16S rRNA gene and the optimal PCR conditions.
Primers
nirKC1F/ nirKC1R
Sequence a
ATGGCGCCATCatggtnytncc/
Conventional PCR b
Real-time QPCR c
Annealing temperature (○C)/time (second)
Annealing temperature (○C)
Tm (○C)
R2/Efficiency (%)
I
54/30
54
88.4
0.999/86
II
56/30
56
89.0
0.992/97
III
58/30
58
92.5
0.991/91
IV
60/30
60
87.4
0.996/84
Cluster
TCGAAGGCCTCGatnarrttrtg
nirKC2F/nirKC2R
TGCACATCGCCAACggnatgtwygg/
GGCGCGGAAGATGshrtgrtcnaca
nirKC3F/nirKC3R
CATCGGCAACGGCatgyayggngc/
CGACCATGGCCGTGGswnacraangg
nirKC4F/nirKC4R
TACGGTGTGATCatcrtsgatcc/
GCATCACGCATGgaatgatysac
F1aCu/R3Cu
(Hallin & Lindgren, 1999)
I
57/35
58
88.0
0.998/87
nirSC1F/ nirSC1R
ATCGTCAACGTCaargaracvgg/
I
56/30
56
90.2
0.999/80
II
56/30
56
86.8
0.993/73
III
－d
－
－
－
TTCGGGTGCGTCttsabgaasag
nirSC2F/ nirSC2R
TGGAGAACGCCggncargtntgg/
GATGATGTCCACGgcnacrtangg
nirSC3F/ nirSC3R
TTCGCCCTGaargayggngg/
AGGTGCCCACGaanarnccncc
cd3aF/R3cd
(Michotey et al., 2000; Throbäck et al., 2004)
I
57/30
57
90.4
0.996/95
357F/520R
(Itoh et al., 2013)
16S rRNA
58/30
58
83.2
0.999/91
a
the sequences with capital and lowercase letters denote the clamp and core region of the primer, respectively.
b
Thermal cycling conditions were initially set to 10 min at 95 ○C for the denaturation step, followed by 30 cycles of 95 ○C for 30 s, different annealing temperatures shown in this table for 30 s,
a 72 ○C extension for 30 s and a final extension at 72 ○C for 10 min.
Final concentration of PCR mixture (25 μl): 0.5U BIOTaq HS DNA polymerase (Bioline, London, UK), 4 mM MgCl2, 0.2 mM dNTP Mix, 0.5 µg·µl-1 bovine serum albumin, 0.2 µM for each
primer and 25-50 ng of genomic DNA. In addition, DMSO was used in the PCR amplification of nirK in Cluster III with a final concentration of 5% (v/v) because of the high guanine-cytosine
(GC) content of the sequences of Actinobacteria.
c
The thermal cycling conditions consisted of an initial denaturation step of 98 ○C for 2 min, followed by 40 cycles of 98 ○C for 10 s, different annealing temperatures shown in this table for 10 s
and 68○C for 30 s.
Final concentration of PCR mixture (20 μl): 10 µl of KOD SYBR qPCR Mix (ToYoBo, Osaka, Japan), 0.4 µl of 50×ROX reference dye, 0.2 µM of primers and 10 ng of environmental DNA.
d
not detected in the test strains and environmental samples.