Biofast soil genomic dna extraction kit

According to our previous study (Chen et al., 2017 (link)), after 5 months of planting R. pseudostellariae, its above-ground and underground biomass was significantly different (expanding period of root tubers). Therefore, we randomly collected soil samples from five different locations within each plot on 25th April, 2018 (Figure 1A). Moreover, for yield determination, we harvested the plants on 10th July, 2018 (Figure 1B). While taking soil samples, the rhizosphere soil clung to the root system of R. pseudostellariae was collected. DNA of soil (0.5 g) was extracted with BioFast Soil Genomic DNA Extraction Kit (BioFlux, Hangzhou, China) following the instructions. Furthermore, DNA concentration was measured using NanoDrop 2000C Spectrophotometer (Thermo Scientific, United States) and diluted to 20 ngμl^–1.

Rice roots from the pot were obtained for bacterial 16sRNA gene profiling. Root sampling was performed at the heading stage and washed until there were no visible soil particles. Next, 10 cm long roots from the ground were sliced into 2 mm sections and placed in a 2-ml tube. Three replicates were performed for each sample. Total DNA extraction from root bacteria was performed using the BioFast soil Genomic DNA Extraction kit (BioFlux, Hangzhou, China), as instructed by the manufacturer. Total DNA from root bacteria were detected by 1% agarose electrophoresis and their concentrations determined by the NanoDrop 2000 nucleic acid analyzer (Thermo Scientific, USA). The V5-V7 region of bacterial 16 S ribosomal RNA gene was amplified using 799 F (AACMGGATTAGATACCCKG) and 1193R (ACGTCATCCCCACCTTCC) primers. The PCR assay was performed using Trans Start Fastpfu DNA Polymerase (TransGen Biotech, China). After amplification, PCR products were identified by electrophoresis on 2% agarose gel and recovered using the AxyPrepDNA Gel Recovery Kit (Axygen Bioscience, China) after elution using Tris-HCl. The library was sequenced on a HiSeq 2500 platform (Illumina, San Diego, CA, USA).

Each sample was weighed 0.7 g of soil, and use BioFast Soil Genomic DNA Extraction kit (BioFlux Hangzhou, China) to extracted the DNA. Then use the Nanodrop 2000C Spectrophotometer (Thermo Fisher Scientific, United States) to detection concentration. The bacterial community region (V3-V4) and the fungi region (ITS1) were used to sequenced. The samples were then commissioned to be sequenced by Biomarker Technologies (Beijing, China) using the Illumina Novaseq2500 platform.
A quantitative PCR assay was performed on the CFX96 Real-Time system (Bio-RDA, United States) to determine the abundance of microorganisms in different rhizosphere soils, including total soil bacteria, total fungi, Actinomycetes, Firmicutes, Bacteroides, and Acidobacteria. The bacterial primers were Eub338/Eub518; Fungi: ITS1F/ITS4; Actinobacteria: Actino235/Eub518; Firmicutes: Lgc353/Eub518; Bacteroidetes: Cfb319/Eub518; Acidobacteria Acid31/Eub 518 (Supplementary Table 1). The plasmid vectors with the specific fragment were constructed through gene cloning and vector link transformation in the early stage of the laboratory. Based on the optimized PCR system, fluorescence quantitative PCR was used for amplification. Four independent quantitative PCR assays were performed for each treatment.

The above ground or below ground biomass of R. pseudostellariae become significantly different after 5 months of planting (Figure 1A), according to our previously study (Wu et al., 2016b (link)). Therefore, soil samples were randomly collected from five different points at each field on April 22nd, 2015. Additionally, we harvested the plants for yield determination on July 2nd, 2015 (Figure 1B).
Soil samples were collected after digging the plant samples. Firstly, the loosely adhering soil was shaken off, then scraping the soil that was still attached to the root as rhizosphere soil. DNA was immediately extracted from 0.5 g soil sample per treatment using Biofast Soil Genomic DNA Extraction Kit (BioFlux, Hangzhou, China) according to the manufacturer’s protocols. We further determined the DNA concentration using Nanodrop 2000C Spectrophotometer (Thermo Scientific, United States) and then diluted it to 20 ng μL^-1.