Soil Microbial Community Co-occurrence Network Structure and Its Drivers in Arid Mountainous Areas
Description
In August 2021, eight typical vegetation zones—Desert Steppe (DS), Prunus mongolica (PM), Ulmus glaucescens (UG), Pinus tabuliformis (PT), aspen×juniper mixed forest (AJMF), spruce×juniper mixed forest (SJMF), Picea crassifolia (PC), and Alpine Meadow (AM) —were selected at altitudes between 1800-3000 m on the western slopes of the Helan Mountains in Ningxia. Six standard plots were established for desert grassland and alpine meadow zones, while 12 standard plots were set up for each of the other vegetation zones as replicates, following the contour lines. The plot sizes were 20 m × 20 m for forest plots, 5 m × 5 m for shrub plots, and 1 m × 1 m for grass plots.
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Steps to reproduce
Soil DNA was extracted from each sample using the E.Z.N.A. Soil DNA Kit (OMEGA, USA) following the manufacturer's instructions. Briefly, the process involved treating 0.5 g of soil with a detergent-containing buffer, followed by a heating and freezing step to precipitate contaminants. The DNA was then purified by elution in water or a low-ionic-strength buffer. Duplicate DNA samples from the same soil were combined, and quantification was performed using a spectrophotometer (NanoDrop ND-1000, Thermo Fisher Scientific). Extracted soil DNA was stored at -80 °C until PCR amplification and sequencing analysis of the 16S rRNA gene and ITS region. The 16S rRNA gene V3-V4 region was amplified using specific primers with unique barcodes: 341F (5'-CCTAYGGGRBGCASCAG-3') and 806R (5'-GGACTACNNGGGTATCTAAT-3'). For PCR reactions, 15 μL of Phusion® High-Fidelity PCR Master Mix, 2 μM forward and reverse primers, and approximately 10 ng of template DNA were used. The PCR procedure consisted of an initial denaturation at 98 ℃ for 1 min, followed by 30 cycles of denaturation at 98 ℃ for 10 s, annealing at 50 ℃ for 30 s, and extension at 72 ℃ for 30 s. The final extension was carried out at 72 ℃ for 5 min. The ITS rRNA gene in the ITS1 region was amplified using primers with specific barcodes: ITS3-2024-F (5'-GCATCGATGAAGAACGCAGC-3') and ITS4-2049-R (5'-TCCTCCGCTTATTGATATGC-3'). The PCR protocol consisted of an initial denaturation at 94 ℃ for 3 min, followed by 35 cycles of denaturation at 94 ℃ for 45 s, annealing at 60 ℃ for 45 s, and extension at 72 ℃ for 1 min. The final extension was performed at 72 ℃ for 7 min, with a ramp rate of 3 ℃/s. For gel detection, PCR products were mixed with an equal volume of loading buffer containing SYBR Green 1X and electrophoresed on a 2% agarose gel. To reduce redundancy, PCR products were mixed in equal proportions. The PCR product mixture was then purified using the Qiagen Gel Extraction Kit (Qiagen, Germany). Sequencing libraries were generated using the TruSeq® DNA PCR-Free Sample Preparation Kit (Illumina), and index codes were added according to the manufacturer's recommendations. Library quality was assessed using a Qubit® 2.0 fluorometer and an Agilent Bioanalyzer 2100 system. Subsequent assays were performed at Meggie Bioinformatics.
Institutions
- Ningxia University