Core Areas:
Keywords:
Konza Keywords:
Date Range:
Saturday, November 1, 2014 to Saturday, December 12, 2015
Publication Date:
Monday, January 30, 2023
To address these predictions, soils were collected from the Belowground Plot Experiment (BGPE) at Konza Prairie Biological Station: a 30-y factorial field manipulation of N fertilization and burning. Surface soils (0-15 cm) were sampled monthly between Nov 2014 – Dec 2015, including one week post-fire (April) and post-fertilization (June). Genomic DNA was extracted from each sample for qPCR and PCR for Illumina MiSeq library sequencing of the prokaryotic 16S rRNA gene and fungal ITS, to estimate population and community dynamics of soil microbes. Soil environmental characteristics and plant communities were measured in July 2015 to evaluate correlations between plant and microbial communities, and environmental variability.
Bacterial 16S rRNA gene and fungal ITS copy number:
Population sizes of bacteria and fungi were estimated using quantitative PCR (qPCR) assays using a Bio-Rad CFX CONNECT system with Bio-Rad SsoAdvanced Universal SYBR Green Supermix (Bio-Rad Laboratories, Hercules, CA, USA). For 16S rRNA gene and ITS assays, respectively, ~1 ng (exact amount calculated on a per sample basis) template gDNA was added to a 10 μL PCR volume, with 0.02% and 0.04% final BSA concentration, 100 nM and 500 nM final primer concentrations, using established primer sequences and thermal cycler programs (Fierer et al. 2005, AEM 71: 4117-4120). Standard curves for 16S assays were prepared using E. Coli ATCC 25922 at 5x100 - 5x10-6 ng ul-1 DNA concentrations, and ITS standard curves were prepared using Candidia albicans SC5314 at 5x100 - 5x10-6 ng ul-1 concentrations; successful curves were accepted at 100 +/- 15% efficiency and R2 > 0.99. All assays included no-template controls and melting curves, which confirmed that only gene copies from template soil gDNA were quantified, and 3 technical replicates were run per sample. Soil gDNA yield (µg DNA g-1 dry soil) was used to normalize marker gene copy number per g dry soil.
complete