2. Materials and Methods

2.1. Production of qPCR standards

Synthetic standards were designed for qPCR assays targeting bacterial 16S rRNA genes, fungal ITS and a
broad range of genes involved in C and N cycling, mcr A,pmo A, nif H, nos Z, amo A, nir S,nir K, nxr B, nor B and pho D genes (Table 1). Synthetic DNA fragments were designed by aligning between 10 and 20 gene sequences per targeted gene with the software Geneious (version 9.1.8). The gene sequences were downloaded from the National Center for Biotechnology Information (NCBI). For each targeted gene, a consensus sequence was obtained from at least 10 downloaded sequences. The consensus sequences were created with most frequent nucleotide for each base of the aligned sequences, therefore not including degenerate nucleotides of A, T, C and G. Forward and reverse primer sequences were included in the consensus sequence to target the interest gene. The consensus sequences of synthetic DNA fragments for each gene are available in Supplementary Table S1. Double-stranded synthetic DNA fragments (between 250 and 650 bp) were then ordered from gBlocks gene fragments (Integrated DNA Technologies: IDT, Inc.), with 500 ng dry DNA in a tube for each target. Upon reception, the DNA was resuspended in nuclease-free water (H2O) and stored at −20 °C freezer for long-term use. The copy numbers of synthetic DNA per microliter were calculated using the formula according to Godornes et al. (2007):
\begin{equation} \frac{\text{gene\ copies}}{\text{μL}}=\frac{\text{DNA\ concentration\ }\left(\frac{\text{ng}}{\text{μL}}\right)\times 10^{-9}\times 6.022\ \times 10^{23}}{\left(\text{fragment\ size\ bp}\right)\times 660\ g/mol\ }\nonumber \\ \end{equation}
where the Avogadro number is 6.022 × 1023(molecules/mole), the fragment size is the length of the synthesized DNA (bp), and 660 is the average weight of a single DNA base pair (g/mol).
For several targeted genes, qPCR standards were also produced via plasmids. PCR products of the particular gene (bacterial 16S rRNA gene, fungal ITS region, mcr A, pmo A, nif H andnos Z) were cloned into the vector and competent cells using the pGEM-T Easy Vector System II Systems Kit according to the manufacturer’s instructions (Promega, Madison, WI, USA) (Frey et al., 2011; Henry et al., 2006). Briefly, PCR reactions were conducted to amplify the targeted gene from DNA extracted from soils. PCR products were then inserted to the Vector (Ligation), which was added to the E. coli JM109 competent cells (Transformation). Transformed E. coli were then spread on Luria-Bertani (LB) agar plates with appropriate antibiotics. After 16-24 hours of incubation at 37 °C, colonies were observed on the plates, and only white colonies were picked up and incubated in liquid LB medium to grow with shaking over 24 h. The plasmids were extracted by Plasmid Miniprep Kit (Promega, Madison, WI, USA) according to the manufacturer’s instructions. The plasmids were further verified by Sanger sequencing: Colony PCR products of the selected marker genes were sequenced on both strands (up to 960 bp), according to Frey et al. (2008). Cycle sequencing was carried out using the Big Dye-Terminator Cycle Sequencing Kit, version 1.3 (PE Applied Biosystems, Foster City, CA) according to the manufacturer’s recommendations. The copy numbers of plasmid DNA per microliter were calculated using the following formula:
\begin{equation} \frac{\text{gene\ copies}}{\text{μL}}=\frac{\text{DNA\ concentration\ }\left(\frac{\text{ng}}{\text{μL}}\right)\times 10^{-9}\times 6.022\ \times 10^{23}}{\left(3015\ bp+amplicon\ size\ bp\right)\times 660\ g/mol\ }\nonumber \\ \end{equation}
where the length of the pGEM-T Easy vector is 3015 bp.
Standards produced by synthetic DNA and plasmids were directly compared by qPCR (top bold six genes in Table 1). Synthetic DNA fragments for microbial functional genes amo A, nir S, nir K,nxr B, nor B and pho D, were not compared with homologous standards produced via cloning, but were tested and verified by qPCR assays.

2.2. qPCR of standards and DNA extracted from soils

The effectiveness of the standard fragments produced via synthetic DNA and plasmids clones were tested and compared in qPCR assays for different genes with soil DNA on a QuantStudio5 Real-Time PCR System (Thermo Fisher Scientific, Waltham, MA, USA) by SYBR Green. qPCR reactions (10 μL) were composed of 5 μL GoTaq® qPCR Master Mix (Promega, Madison, WI, USA), 0.1 μL 30 mg mL-1 bovine serum albumin (BSA), 0.5 μL 10 μM of each primer, 1.9 μL molecular-grade water and 2 μL DNA template. Soil DNA was diluted at a concentration of ~2 ng/µL to avoid potential PCR inhibition. The different primers used in the reactions are shown in Table 1 and details on qPCR thermocycling conditions are described in Table 2 and Supplementary Table S2. Three standard dilution series per target gene (for both synthetic DNA standard and plasmid DNA standard) were obtained from 10-fold serial dilutions of standards with molecular-grade H2O. The standard series ranged ranging from 101 to 108 copies per μL.
DNA was extracted from soils collected along an altitudinal gradient in Switzerland in August 2021 (soil samples from 530, 1159, 1844, 2336 and 2715 m.a.s.l, Supplementary Table S3). The soils varied in pH, total organic carbon, C and N content (Supplementary Table S3). Total DNA was extracted with the DNeasy Powersoil Pro Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. The DNA was quantified with PicoGreen (ThermoFisher Scientific, Cleveland, OH, USA), and subsequently diluted to 2.0 ng per µL by molecular-grade H2O and measured in triplicate.
A standard curve for each gene was generated by plotting cycle threshold (Ct) or quantification (Cq) of cycle values of each dilution step against the corresponding log10 transformed number of gene copies in the standard. The amplification efficiency (E) was estimated using the slope of the standard curve with the formula: E = (10−1/slope) − 1. The detection limit was 10 copies per μL according to the lowest concentration standard (101 copies per μL).