Total genomic DNA was extracted from each rhizosphere soil or root samples using a Power Soil® DNA Isolation Kit (MoBio Laboratories, Carlsbad, CA, USA) according to the manufacturer’s instructions. To assess DNA concentration and purity, the DNA extracts were run on 1% agarose gels at 110 V for 30 min and quantified using a NanoDrop 2000 spectrophotometer (Thermo Scientific). The extracted total genomic DNA samples were stored at 20 °C until subjected to high-throughput sequencing.
Approximately 400-bp DNA fragments of the bacterial 16S rRNA gene targeting the hypervariable region V3-V4 were amplified using barcoded universal primer pair 341F (5′-CCTACGGGNGGCWGCAG-3′) and 805R (5′-GACTACHVGGGTATCTAATCC-3′) in the bacterial community analysis for the five plant species [22 (link)]. To minimize the effect of chloroplast DNA of host plant on microbiota analyses, another barcoded universal primer pair 799F (5′-AACMGGATTAGATACCCKG-3′) and 1193R (5′-ACGTCATCCCCACCTTC C-3′), spanning ~ 450 bp of the V5-V7 regions of the 16S rRNA gene, was used in the subsequent community analysis, including the analysis of tomato microbiota at different developmental stages, and of tomato amended with different nitrogen sources [35 (link), 43 (link), 72 (link)]. Amplified PCR products in each experiment were separately processed to purify, combined in equimolar ratios, and subjected to high-throughput sequencing on an Illumina Mi-Seq sequencing platform, and paired 250-nucleotide reads were produced at Sangon Biotech (Shanghai, China).
Approximately 400-bp DNA fragments of the bacterial 16S rRNA gene targeting the hypervariable region V3-V4 were amplified using barcoded universal primer pair 341F (5′-CCTACGGGNGGCWGCAG-3′) and 805R (5′-GACTACHVGGGTATCTAATCC-3′) in the bacterial community analysis for the five plant species [22 (link)]. To minimize the effect of chloroplast DNA of host plant on microbiota analyses, another barcoded universal primer pair 799F (5′-AACMGGATTAGATACCCKG-3′) and 1193R (5′-ACGTCATCCCCACCTTC C-3′), spanning ~ 450 bp of the V5-V7 regions of the 16S rRNA gene, was used in the subsequent community analysis, including the analysis of tomato microbiota at different developmental stages, and of tomato amended with different nitrogen sources [35 (link), 43 (link), 72 (link)]. Amplified PCR products in each experiment were separately processed to purify, combined in equimolar ratios, and subjected to high-throughput sequencing on an Illumina Mi-Seq sequencing platform, and paired 250-nucleotide reads were produced at Sangon Biotech (Shanghai, China).
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