接第三篇

4. Materials and Methods

4.1. Fungal Material, DNA Extraction, and Sequencing

　　Thirteen fungal samples (four of C. militaris, three of C. pruinosa, two of I. tenuipes, and four of I. cicadae) were provided by the Korean Agricultural Culture Collection (KACC) and the Korean Collection for Type Cultures (KCTC), as listed in Table 1. O. sinensis, the Cordyceps herbal medicine, were provided by Prof. Gi-Ho Sung; they were collected in their native habitat in Bhutan (Table 1).Mycelia of the 13 fungal samples were obtained after growth on medium as suggested by the providers, consisting of 2% dextrose, 0.5% peptone, 0.5% yeast extract, and 1.5% agar. Fungal gDNA was extracted using the DNeasy plant mini kit (Qiagen, Valencia, CA, USA), gDNA from Cordyceps herbal medicines and commercial products was extracted from the fruiting bodies and individual components,respectively, using the same method, after processing the samples into a fine powder with a grinder (Precellys™ Grinder, Bertin Technologies, Montigny-le-Bretonneux, France). The concentration and purity of the extracted gDNA were determined using the NanoDrop ND-1000 spectrophotometer(NanoDrop,Wilmington, DE, USA), and by electrophoresis on 1.5% agarose gels with known standards. The final DNA concentrations in the samples were adjusted to approximately 15 ng/uL with TE buffer.The samples were stored at -20 ℃ for further analysis.

4.2. Phylogenetic Analysis

　　A phylogenetic tree was constructed using 31 complete nrDNA-ITS sequences in the MEGA7 program (Version 7.0.26). In addition to the 18 sequences obtained in the current study, 13 previously reported nrDNA-ITS sequences were retrieved from the GenBank, as follows: three I. tenuipes sequences(AB086215, AB086224, and EF411223) and two sequences each from C. pruinosa (AJ039338 and AY491995), C. militaris (AB084156 and AB255603), I. cicadae (KX017277 and KP771871), Beauveria bassiana (KU702657 and GU233698), and O. sinensis (EU570943 and AB067713). A phylogenetic tree was constructed using the neighbor-joining method and Kimura’s two-parameter model, with pairwise deletion for gaps and/or missing data, and 1000 replications for bootstrapping. Nectria cinnabarina(AB237663) was used as an outgroup control [35,37].

4.3. PCR Amplification of nrDNA-ITS and Species Identification

　　The nrDNA-ITS regions, including the 5.8S rRNA gene, were amplified in a 50 ul PCR mixture containing approximately 15 ng of gDNA, 0.4 uMeach of the primers ITS1 and ITS4, and Solg™2 × Taq PCR Smart Premix 1 (Solgent, Daejeon, Korea), using a Pro Flex PCR system (Applied Biosystems,Waltham, MA, USA) as previously described [38]. The PCR products were separated on a 1.5% agarose gel. The target amplicons were then isolated using a gel extraction kit (Qiagen, Valencia, CA, USA)and sub-cloned into the pGEM™-T Easy vector system (Promega, Madison, WI, USA) following the manufacturer’s instructions. The inserted DNA fragments were sequenced using the primers SP6 and T7, using an ABI3730 DNA sequence analyzer (Applied Biosystems, Waltham, MA, USA). To check for the presence of PCR errors and misreads, five inserted PCR products were analyzed for each sample,and the sequences were manually edited after alignment, as previously described [17]. The identities of each species in the individual samples were confirmed using a BLAST-based comparison and similarity analysis involving the obtained sequences, and sequences were deposited in the NCBI GenBank and BOLD databases (Table 1). Species identity was further confirmed by a comparison of nucleotide sequences at the intra- and inter-species levels using the entire suite of nrDNA-ITS sequences listed in Table 1.

4.4. Analysis of the nrDNA-ITS Sequences and Development of SCAR Markers

　　To identify the species-specific nucleotide variants in the nrDNA-ITS region that could be used for SCAR marker development, 18 sample sequences representing six species, including B. bassiana (Bals.-Criv.) Vuill., were aligned and manually edited using the ClustalW algorithm in the BioEdit program (Version 7.2.5). The inter- and intraspecific variabilities were then analyzed using the MEGA7 program [33,37]. The resulting species-specific regions with distinct indels and nucleotide substitutions were selected as potential candidate SCAR primers, and they were synthesized to amplify the SCAR regions. A PCR was performed to confirm the specificities of individual primer sets, in 20 uL reaction mixtures containing approximately 15 ng of gDNA, and 0.4 uM of each species-specific forward and reverse primers. The amplification reactions were performed using the Pro Flex PCR system (Applied Biosystems,Waltham, MA, USA). The amplification conditions were as follows: initial denaturation at 95 ℃for 2 min, followed by 35 cycles of 95  ℃ for 30 s, 63  ℃for 30 s, and 72  ℃ for 30 s, and a final extension step at 72  ℃ for 5 min. To verify the PCR results, PCR products were resolved by 1.5% agarose gel electrophoresis, and the specificity and size of the DNA fragments were verified with a 100 bp DNA ladder (Solgent, Daejeon, Korea). To improve the stability and discriminability of the SCAR marker-based species identification method, two sets of SCAR primers were developed for each species, and the specificities of both SCAR markers were confirmed using the 18 samples listed in Table 1.