2.3 High-throughput amplicon sequencing
Amplicon sequencing of the bacterial and fungal rhizosphere populations for 20 soil samples was conducted with the V4 fragment of the 16S rRNA gene and the ITS1 gene fragment, respectively, using the Illumina HiSeq platform (Illumina, San Diego, CA, USA). The broadly conserved primers, 515F, 5′-GTGCCAGCMGCCGCGG-3′, and 806R, 5′-GGACTACHVGGGTWTCTAAT-3′, were used for PCR to amplify the sequencing region of the 16S rRNA gene. PCR primers for amplifying fungal ITS1-2 are: ITS1-1737F, 5′-GGAAGTAAAAGTCGTAACAAGG-3′; ITS2-2043R, 5′-GCTGCGTTCTTCATCGATG C-3′.
PCR reactions were carried out in 30 µL reactions with 15 µL of Phusion® High-Fidelity PCR Master Mix (New England Biolabs, USA), 0.2 µM of forward and reverse primers, and about 10 ng of template DNA. Thermal cycling consisted of initial denaturation at 98 °C for 1 min, followed by 30 cycles of denaturation at 98 °C for 10 s, annealing at 50 °C for 30 s, elongation at 72 °C for 30 s, and finally 72 °C for 5 min. The same PCR conditions were used for ITS, except that the second stage had 35 cycles. PCR products were subsequently subjected to electrophoresis on a 2% agarose gel, stained with ethidium bromide, and the targeted fragment size purified with an AxyPrepDNA gel extraction kit (Axygen, China). Sequencing libraries were generated using Ion Plus Fragment Library Kit 48 rxns (Thermo Scientific, USA), following the manufacturer’s recommendations. The library quality was assessed on the QuantiFluorTM-ST fluorometer (Promega) and Agilent 2100 Bioanalyzer system (Agilent, USA). Lastly, the library was sequenced on an Ion S5TM XL platform, and 400 bp/600 bp single-end reads were generated. All the high-throughput sequencing was done by Novogene Technology Co, Ltd. (Beijing, China).