Legacy rhizosphere V3-V4 sequence of 16S rDNA
Total DNA was extracted from 500 mg rhizosphere soil samples using a Power Soil DNA Isolation kit (MoBio Laboratories, Inc) according to the manufacturers instructions. The purity and quality of the genomic DNA were checked on 1% agarose gels and a NanoDrop spectrophotometer (Thermo Scientific). The V3-V4 hypervariable region of the bacterial 16S rRNA gene was amplified with the primers 338F (5-ACTCCTACGGGAGGCAGCAG-3) and 806R (5-GGACTACNNGGGTATCTAAT-3). For each soil sample, an 8-digit barcode sequence was added to the 5 end of the forward and reverse primers. PCR was carried out on a Mastercycler Gradient thermalcycler (Eppendorf) using 25 l reaction solutions consisting of 12.5 l of KAPA 2G Robust Hot Start Ready Mix, 1 l of forward and reverse primers (5 M), 5 l of DNA (with a total template quantity of 30 ng), and 5.5 l of H2O. The cycling program was 95 C for 5 min; 28 cycles of 95 C for 45 s, 55 C for 50 s and 72 C for 45 s; and a final extension at 72 C for 10 min. The PCR products were subsequently purified using an Agencourt AMPure XP Kit. Deep sequencing was performed on the MiSeq platform at Allwegene Company (Beijing).