2.2 DNA extraction, PCR, and NGS sequencing
K . schrenkianum plants were divided in to aerial parts and
roots to analyze the endophytic microbes, and altogether 40 samples (5
individual × 4 treatments × 2 tissue types) were used for DNA
extraction. Each plant material (20 g) was surface sterilized with 75%
ethanol for 1 min, 3.25% sodium hypochlorite for 3 min, and 75%
ethanol for 30 s (Guo, Hyde, & Liew, 2000). Genomic DNA was extracted
following CTAB method (Guo et al., 2000). One gram of the
surface-sterilized plant material was freeze-dried using liquid
nitrogen, homogenized with a mortar and pestle, transferred to a tube
with 5 mL 2× cetyltrimethylammonium bromide (CTAB) extraction buffer
(2% (w/v) CTAB, 100 mM Tris-HCl, 1.4 M NaCl, 20 mM EDTA, 1.5%
polyvinyl-pyrolidone (PVP), 0.5% 2-mercaptoethanol; pH 8.0; preheated
to 65 °C). The concentration of DNA was measured using
a NanoDrop 1000 Spectrophotometer
(Thermo
Scientific, Wilmington, USA).
The V5-V7 hypervariable region of the bacterial 16S ribosomal RNA gene
was amplified using 799F (AACMGGATTAGATACCCKG) (Chelius & Triplett,
2001) and 1193R (ACGTCATCCCCACCTTCC) primers (Bodenhausen, Horton, &
Bergelson, 2013). The fungal internal transcribed spacer region 1 (ITS1
region) of ribosomal RNA was amplified using ITS1F
(CTTGGTCATTTAGAGGAAGTAA) (Gardes & Bruns, 1993) and ITS2
(GCTGCGTTCTTCATCGATGC) primers (White, Bruns, Lee, & Taylor, 1990). All
PCR reactions were carried out in 30 µL reactions with 15 µL of Phusion®
High-Fidelity PCR Master Mix (New England Biolabs, Ipswich, MA); 0.2 µM
of forward and reverse primers, and about 10 ng template DNA. Thermal
cycling consisted of initial denaturation at 98℃ for 1 min, 30 cycles of
denaturation at 98℃ for 10 s, annealing at 50℃ for 30 s, and elongation
at 72℃ for 30 s, followed by a final extension at 72℃ for 5 min. Mix
same volume of 1× loading buffer (contained SYB green) with PCR products
and operate electrophoresis on 2% agarose gel for detection. Then, PCR
products was purified with GeneJETTM Gel Extraction Kit (Thermo
Scientific, Waltham, MA). Sequencing libraries were generated using Ion
Plus Fragment Library Kit 48 rxns (Thermo Scientific, Waltham, MA)
following manufacturer’s recommendations. The library quality was
assessed on the Qubit@ 2.0 Fluorometer (Thermo Scientific, Waltham, MA).
At last, the library was sequenced on an Ion S5TM XL platform.