amplicon of symbiotic bacteria:Curculio david
Amplification of the V3-V4 regions of the 16S rRNA gene was performed using the primers 338F 5-ACTCCTACGGGAGGCAGCA-3 and 806R 5-GGACTACHVGGGTWTCTAAT-3. The PCR reaction was performed in initial denaturation at 95 C for 3 min, followed by 30 cycles of 95 C for 30 s, 55 C for 30 s, 72 C for 45 s and a final extension step of 72 C for 10 min. All PCRs were performed in a total volume of 20 L using TransStart FastPfu DNA Polymerase according to the manufacturers instructions. PCR reactions were performed in triplicate. PCR products were purified, pooled with the equimolar concentrations, and sequenced on the Illumina MiSeq PE300 platform (Illumina, San Diego, CA, United States) at the Majorbio Bio-Pharm Technology Co., Ltd. (Shanghai, China). Paired-end sequences were merged to a single sequence with length of 300bp using FLASH 1.2.11.