2.4 | Molecular analyses protocol
The metabarcoding diet analyses based on sheep feces (n = 24) were used
to estimate dietary herbage items in different grazing treatments. All
aspects of the laboratory molecular protocol consisted of DNA
extraction, amplification of the ITS2 fragment, and amplicon sequencing
on a MiSeq run.
Total DNA was extracted from lamb feces samples using an E.Z.N.A. Stool
DNA Kit (Omega Bio-tek, Norcross, GA, U.S.) according to the
manufacturer’s instructions. Concrete operations were conducted as
described by Guo et al. (2018). The DNA extract
was checked on 2% agarose gels, and
DNA concentration and purity were determined using a NanoDrop 2000
UV-vis spectrophotometer (Thermo Scientific, Wilmington, USA).
The ITS2 region of the nuclear rDNA (350 bp) was amplified with primer
pairs rD5-ITS2
(5′-barcode-TCCTCCGCTTATTGATATGC-3′)
and rb1-ITS2F (5′- CGATACTTGGTGTGAATTGCAG-3′) (Bradley et al., 2007) by
an ABI GeneAmp® 9700 PCR thermocycler (ABI, CA, USA).
The PCR amplification of the ITS2 gene was performed as follows: initial
denaturation at 94℃ for 5 min followed by 45 cycles of denaturing at 94℃
for 30 s, annealing at 59℃ for 60 s and extension at 72℃ for 60 s, and
single extension at 72℃ for 10 min ending at 4℃. The PCR mixtures
contained 4 μL of 5 × FastPfu buffer, 2 μL of 2.5 mM dNTPs, 0.8 μL of
forward primer (5 μM), 0.8 μL of reverse primer (5 μM), 0.4 μL ofTransStart FastPfu DNA Polymerase, 10 ng of template DNA, and
finally ddH2O up to 20 μL. PCR reactions were performed
in triplicate. The PCR product was extracted from 2% agarose gel and
purified using the AxyPrep DNA Gel Extraction Kit (Axygen Biosciences,
Union City, CA, USA) according to the manufacturer’s instructions and
quantified using a Quantus™ Fluorometer (Promega, USA).
Purified ITS2 amplicons were pooled in equimolar volumes and paired-end
sequenced on an Illumina MiSeq PE300 platform (Illumina, San Diego, USA)
according to standard protocols by Majorbio Bio-Pharm Technology Co.
Ltd. (Shanghai, China). These
sequence data have been submitted to the GenBank databases under
accession
number
PRJNA660588.