Abstract
As our understanding of soil biology deepens, there is a growing demand for investigations addressing microbial processes in the earth beneath the topsoil layer, called subsoil. High clay content in subsoils often hinders the recovery of sufficient quantities of DNA as clay particles bind nucleic acids. Here, an efficient and reproducible DNA extraction method for 200 mg dried soil based on sodium dodecyl sulfate (SDS) lysis in the presence of phosphate buffer has been developed. The extraction protocol was optimized by quantifying bacterial 16S and fungal 18S rRNA genes amplified from extracts obtained by different combinations of lysis methods and phosphate buffer washes. The combination of one minute of bead beating, followed by ten min incubation at 65°C in the presence of 1 M phosphate buffer with 0.5% SDS, was found to produce the best results. The optimized protocol was compared with a commonly used cetyltrimethylammonium bromide (CTAB) method, using Phaeozem soil collected from 60 cm depth at a conventional agricultural field and validated on five subsoils. The reproducibility and robustness of the protocol was corroborated by an interlaboratory comparison. The DNA extraction protocol offers a reproducible and cost-effective tool for DNA-based studies of subsoil biology.
Highlights
In agricultural systems, the distinction between top- and subsoil is made based on the present or historical tillage depth, which is commonly around 20 to 30 cm [1,2]
Extraction from 50 and 200 mg subsoil using a cetyltrimethylammonium bromide (CTAB)-based protocol with polyethylene glycol (PEG)-NaCl precipitation resulted in low total DNA yield (Figure 3 A)
We observed that the extraction of 200 mg soil resulted in lower recovery of bacteria (p = 0.0072) than 50 mg soil (Figure 3B), and fungal DNA was not detectable when DNA was extracted from 200 mg soil (Figure 3C)
Summary
The distinction between top- and subsoil is made based on the present or historical tillage depth, which is commonly around 20 to 30 cm [1,2]. The number of scientific studies investigating both top- and subsoils is steadily increasing, as the methodologies for such studies become more refined and reliably reproducible. These investigations have broadened the understanding of the role of soil microbes in carbon and nutrient cycling, as well as the fate of soil pollutants [4]. Alongside with absolute changes with increasing soil depth, compositional differences between top- and subsoil microbial communities have been reported using molecular tools [17,18]. Given the low abundance of microorganisms inhabiting subsoil environments, the extraction of sufficient amounts of nucleic acids for molecular methods like real-time PCR (qPCR) and generation sequencing is challenging, in clay-rich soil [16,19,20,21,22]
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