amplicon sequencing from wheat roots_conventional farming_future climate_Replica10_third sampling_fungi
Genomic DNA were extracted from wheat straw.The ITS2 region of fungi was amplified using the following primers: fITS7 (5-GTGARTCATCGAATCTTTG-3)   and ITS4 (5-TCCTCCGCTTATTGATATGC-3). The amplification was performed in a two-step process. First amplifications were performed in 25 L reactions with a Qiagen HotStar Hi Fidelity Polymerase Kit (Qiagen Inc., Valencia, CA, USA), 1 L of each 5 M primer, and 1 L of template. Reactions were performed on ABI Veriti thermocyclers (Applied Biosytems, Carlsbad, CA, USA). The first PCR conditions were 95 C for 5 min, then 35 cycles of 94 C for 15 s, 54 C for 60 s, 72 C for 1 min, followed by one cycle of 72 C for 10 min and 4 C hold. Amplicons from the first stage amplification were diluted 1:10 and then used as a template in the second PCR. During the second PCR, dual indexes were attached using the Nextera XT Index Kit with the same amplification conditions as the first stage, except for 10 cycles.