Ezup column animal genomic dna purification kit

DNA was extracted using an Ezup Column Animal Genomic DNA Purification Kit (Sangon Biotech, Shanghai, China) following the manufacturer’s standard protocols. Specimens were kept in 75% ethanol for further morphological examination after the 1h-lysis buffer was transferred to the pipette containing silica column. Extractions were performed non-destructively (small tissue biopsy) allowing further morphological examination of the specimens. Primers were LCO1490/HCO2198 commonly used for metazoa^{73 (link)}. Amplification volume and procedure were listed in Zhang et al.^{63 (link)}. All PCR products were checked on a 1% agarose gel. Successful products were purified and sequenced in both directions by Majorbio (Shanghai, China) on the ABI 3730XL DNA Analyser (Applied Biosystems). Sequences were assembled in Sequencher 4.5 (Gene Codes Corporation, Ann Arbor, USA), and deposited in GenBank (Supplementary Table S3). Sequences were blasted in GenBank and checked for possible errors, then were preliminarily aligned using MAFFT v7.149 by the Q-INS-I strategy^{74 (link)}. Alignments were checked and corrected manually, with a final 658 bp for COI.

Total DNA was extracted from the toe-clips samples of A. americanus or Bufotes pewzowi using an Ezup Column Animal Genomic DNA Purification Kit (Sangon Biotech Company, Shanghai, China). The 11 universal primers for standard polymerase chain reaction (PCR) amplification of mitogenomes were slightly modified according to Yu et al. (2015) (link) and Zhang et al. (2013) (link) and 15 specific primers were designed based on the sequenced fragments from universal primers using Primer Premier 5.0 (PREMIER Biosoft International, CA, USA) (Tables S1 and S2). All PCR amplifications were carried out in a 50 µL reaction mixture and the procedures were performed using an Eppendorf Thermal Cycler (Mastercycle® nexus GSX1, Hamburg, Germany). We used both standard PCR and Long-PCR methods with TaKaRa Ex-Taq and TaKaRa LA-Taq Kits (Takara Biomedical, Dalian, China). These two methods were slightly modified from Yu et al. (2015) (link) and Zhou et al. (2009) (link). All PCR products were detected by electrophoresis on 1% agarose gels, and sequences were obtained in an automated DNA sequencer (ABI 3730) by Sangon Biotech Company (Shanghai, China).

Genomic DNA (gDNA) was extracted from FFPE using the Ezup Column Animal Genomic DNA Purification Kit (Sangon Biotech (Shanghai) Co., Ltd.). gDNA was amplified with the forward and reverse primers. After purified PCR products were detected by agarose gel electrophoresis (150 V, 100 mA, 10–20 min), DNA was extracted from the agarose gel by a SanPrep Column DNA Gel Extraction Kit (Sangon Biotech (Shanghai) Co., Ltd.), and then Sanger-sequenced with the BigDye terminator v1.1 sequencing kit and a 3730xl automated sequencer (Applied Biosystems, Foster City, CA, USA). The results were analyzed by Variant reporter software version 2.1 (Applied Biosystems).

DNA was extracted using an Ezup Column Animal Genomic DNA Purification Kit (Sangon Biotech, Shanghai, China) following the manufacturer’s standard protocols. Primers used were LCO1490/HCO2198, which are commonly used for metazoans (Folmer et al. 1994 (link)). PCR amplification of mitochondrial COI was performed in 25 μL volumes containing 12.5 μL of Premix Taq (TaKaRa Taq v. 2.0 plus dye), 1.25 μL of each primer, 8 μL of ddH₂O, 2 μL of template DNA, with PCR programs following Zhang et al. (2014) (link). All PCR products were checked on a 1% agarose gel. Successful products were purified and sequenced in both directions by Majorbio (Shanghai, China) on an ABI 3730XL DNA Analyser (Applied Biosystems). COI sequences for the remaining species were obtained from the NCBI (https://www.ncbi.nlm.nih.gov/). Sequences were preliminarily aligned using MAFFT v. 7.450 by the L-INS-I strategy (Katoh and Standley 2013 (link)) and corrected manually, with a final 658-bp alignment. Neighbour-joining (NJ) tree and Kimura 2-parameter (K2P; Kimura 1980 (link)) distances were calculated in MEGA v. 7.0 (Kumar et al. 2016 (link)). Node supports were evaluated through 1,000 bootstrap replications.

Total genomic DNA was extracted from a clipped toe using the Ezup Column Animal Genomic DNA purification Kit (Sangon Biotech Company, Shanghai, China) in compliance with the manufacturer’s standard protocols for tissue. We amplified 10 overlapping genes via normal PCR and long-accurate PCR methods slightly modified from Zhang et al. [45 (link)]. Additionally, the ND5 fragments were successfully obtained using primers designed by Cai et al. [46 (link)]. After obtaining the ND5 gene fragment, four sets of primers were designed to sequence adjacent gene fragments, including the region from ND5₍₁₎ to ND5₍₂₎ genes, the region from Cytb to ND5₍₁₎ genes, and the region from ND5₍₂₎ to tRNA^Pro genes (Figure 1). An overview of all fragments is given in Table 1 and Table S2. Unexpectedly, the amplicons generated from Cytb to ND5₍₁₎, ND5₍₁₎ to ND5₍₂₎, and ND5₍₂₎ to tRNA^Pro fragments were found to have lengths of approximately 3400 bp, 3300 bp, and 4100 bp, respectively. These PCR products underwent bidirectional sequencing utilizing the primer-walking method from Sangon Biotech Company (Shanghai, China).

423 muscle specimens were collected from 43 localities in northeastern QTP (Fig 1, Table 1 and S1 Table) during the period 2014 to 2016. For each locality, at least 4 individuals were randomly sampled. After trapped, the marmots were anaesthetized to death by 100% diethyl ether. And then, muscle tissues were removed and stored in 95% ethanol immediately. Total genomic DNA was extracted using the commercial kit (Ezup Column Animal Genomic DNA Purification Kit, Sangon Biotech).

Total genomic DNA from hind-leg muscle tissue of each specimen was extracted using the Ezup Column Animal Genomic DNA Purification Kit (Sangon Biotech Company, Shanghai, China). The experimental design was approved by the Animal Research Ethics Committees of Zhejiang Normal University.
Several fragments were amplified using universal primers [6 (link)]. We then used Primer Premier 5.0 to design specific primers based on sequences previously obtained from universal primers [46 (link)]. The reaction conditions for normal PCR (product length <3000 bp) and long PCR (product length >3000 bp) were performed as described in Zhang et al. [47 (link)]. After we performed electrophoresis and gel purification of PCR products, all PCR products were sequenced at Sangon Biotech Company (Shanghai, China) using bidirectional sequencing.