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.