2.4 Soil microbial community analysis
The whole genome DNA of soil was extracted using CTAB method. The
concentration and purity of DNA were detected on 1% agarose gel. DNA
was diluted to 1ng/µL using double distilled water according to the
concentration.
16S rRNA genes from V4-V5 hypervariable region has been amplified with a
primary (515F/806R) with barcode for bacteria. The internal transcribed
spacer two regions (ITS2) were amplified using the primer set ITS7/ITS4.
All PCR reactions have been carried out with 15 µL of Phusion®
High-Fidelity PCR Master Mix, two µM of forward and reverse primers, and
about ten ng template DNA. The program consisted of initial denaturation
at 95 ℃ for one minute, followed by 27 cycles of 95 ℃ for ten seconds,
57 ℃ for 30 seconds, and elongation at 72 ℃ for 30 seconds, final 72 ℃
for 10 minutes.
The same quantity of 1XTAE buffer was mixed with PCR products, and
electrophoresis detection was operated on 2% agarose gel. PCR products
were mixed in EU density ratios. Then, mixture PCR products were
purified using DNA Purification Kit (Novo, Beijing). The
methods for Illumina sequencing
and Bioinformatics analysis see supplementary materials X1 .