Efficiency of ITS1-5.8S-ITS2 region in identifying cordyceps species

Journal of Biotechnology 16(4): 705-712, 2018 705 EFFICIENCY OF ITS1-5.8S-ITS2 REGION IN IDENTIFYING CORDYCEPS SPECIES Le Thi Thu Hien1,2,*, Ha Hong Hanh1 1Institute of Genome Research, Vietnam Academy of Science and Technology 2Graduate University of Science and Technology, Vietnam Academy of Science and Technology * To whom correspondence should be addressed. E-mail: hienlethu@igr.ac.vn Received: 18.10.2018 Accepted: 15.11.2018 SUMMARY Cordyceps genus is a well-known traditional medicine worldwide. It contains abundant physiological active compounds that were demonstrated to perform benefit in reducing progression of cancer as well as protecting human health. Accurately classifying species in this genus is essential in order to prevent commercial counterfeit medicines. Nowadays, a taxonomic classification of species based on DNA sequences can overcome the existed limitation in identifying by using only morphological characteristics of this genus. DNA barcodes are standard short genomic regions that are universally present in target lineages and has sufficient sequence variation to discriminate species in the genus. A variety of loci has been suggested as DNA barcodes for plants, including genes and non-coding regions in the nuclear and plastid genomes such as psbA- trnH, matK, rbcL, and ITS. Thus, the objective of this study was to identify selected species of Cordyceps genus using DNA barcodes. Seven strains of Cordyceps were collected. Total DNA extraction and purification, PCR amplification and DNA sequencing were performed with standard chemicals and kits. The candidate ITS1-5.8S-ITS2 region was amplified and sequenced. Data were analyzed using Bioedit 7.2.6 and MEGA 7 softwares. Analysis of seven obtained DNA barcode sequences of collected samples revealed that the ITS1- 5.8S-ITS2 region provided high species discriminating power for Cordyceps genus. Accordingly, phylogenetic trees based on this DNA barcode exhibited six samples had closed relationship to Cordyceps militaris, while another specimen was the nearest neighbor to Cordyceps sinensis with average similarities at 99.82% and 99.81%, respectively. Our results support the identification of valuable medicinal plant species within Cordyceps genus. Keywords: Cordyceps militaris, Cordyceps sinensis, ITS1-5.8S-ITS2 region, DNA barcode, species discrimination INTRODUCTION Genus Cordyceps belongs to division Ascomycota, class Sordariomycetes, order Hypocreales, family Clavicipitaceae and is entomopathogenic and endoparasitic fungi (Sung et al., 2007). Cordyceps species mainly parasitize in arthropod from lepidopteran larva and pupae to imago. They are distributed predominantly in higher than 4000 meter mountainous areas of Asia such as the Tibetan Plateau and Himalayan Mountain Range. They thrive vigorously in hot and humid subtropical climate and temperate forests. Currently, approximate 400 species of this genus have been described (Mains, 1957; Kobayasi, 1982; Stensrud et al., 2005). Many species of Cordyceps have been used as health food or medicine in China and South- East Asia since it contains abundant physiologically active compounds (Masuda et al., 2007; Hur, 2008). According to modern medicine, there are seven groups of basic organic compounds of Cordyceps: (1) Proteins, peptides, essential amino acids; (2) Polysaccharides; (3) Sterol including ergosterol, Delta-3 ergosterol, ergosterol peroxide, 3-sitosterol, daucosterol and campeasterol; (4) Nucleosides (also known as cordycepins) include adenine, uracil, uridine, guanosine, thymidine; (5) Saturated and unsaturated fatty acids; (6) Vitamins (B1, B12, E, K); (7) Minerals (K, Na, Ca, Mg, Fe, Cu, Mn, Zn, Pi, Se, Al, Si, Ni, Sr, Ti, Cr, Ga, V, and Zr). The main uses of Cordyceps have been demonstrated through a range of studies. Crude extracts of Cordyceps sinensis enable to inhibit the growth of several cancer cell linages such as K562, Vero, Wish, Calu-1 Le Thi Thu Hien & Ha Hong Hanh 706 and Raji (Kuo et al., 1994). Cordyceps acts on the immune system in two different ways, both boosting the immune system of people with immunodeficiency in cancer, diabetes, cardiovascular disease, while reducing unwanted immune responses for people with increased immunity (leukemia and organ transplantation, for instance) (Feng et al., 2008; Jordan et al., 2008; Ding et al., 2009). They also enhanced digestion and absorption by acting on the intestinal microflora (Koh et al., 2003). For glucose regulation, Lo et al. (2004) observed that diabetic mice fed with C. sinensis in 28 days gained weight and decreased blood glucose level compared to the mice using placebo (Lo et al., 2004). Adenosine in Cordyceps showed useful in protecting heart health, increase circulation and preventing cardiac arrhythmia (Mei et al., 1989), ischemia, and stroke (Liu et al., 2010). Cordyceps has a positive influence in sperm formation and restore sexual dysfunction in both genders (Zhu et al., 1998; Zhang et al., 2005). By reducing risk of Alzheimer disease due to β-amyloid Cordyceps proved to improve concentration and memory (Ji et al., 2009; Jin et al., 2004). Function of Cordyceps in improving liver function were showed in increasing ALT in hepatitis B virus infection patients (Zhu et al., 1992; Gong et al., 2000). Cordyceps also help to inhibit renal fibrosis, protect the kidneys from the effects of cyclosporine A (Wu et al., 2000). Such effects made Cordyceps to become a valuable tradition medicine not only in Asia but also in Western countries and at risk of commecial fraud due to very high price. Currently, besides the morphological classification, molecular techniques had been applied to increase accuracy and reliability identification of mushroom Cordyceps in order to control trading activities of this genus. DNA molecular barcodes based on short DNA fragments in mitochondrial genome for animals or in chloroplast and nuclear genomes for plants were developed to discriminate species. Mitochondrial DNA is an extranuclear genetic material which is capable of replicating independently with the nuclear DNA. Since mitochondrial DNA evolves five times faster than nuclear genetic markers, it becomes an effective tool for evaluating genetic diversity in phylogenetics and evolutionary biology. However, the rate of evolution of the mitochondrial genome in plants is not as fast as that of animals. Therefore, chloroplast DNA in plant cells is considered to be DNA barcodes for classifying species. In addition, it is possible to combine with nuclear DNA in order to increase identifiable ability (Kress et al., 2005). Almost all DNA barcodes utilized for classification in plants belong Internal Transcribed Spacers (ITS) regions in nuclear genome, and trnH-psbA (Kress et al., 2005), matK, rbcL (CBOL Plant Working Group, 2009), rpoC1, rpoB (Chase et al., 2007) regions in chloroplast genome. DNA molecular marker and DNA barcode techniques have been applied to study genetic relationship and classification of Cordyceps genus. Kuo et al. (2005) had built a phylogenetic tree of 17 species of Cordyceps genus based on ITS1-18S rRNA-ITS2 region. In 2007, Sung’s study demonstrated that molecular markers nrSSU, nrLSU, tef1, tub, atp6, rpb1 and rpb2 work well in identifying 162 different strains of this genus (Sung et al., 2007). In this study, seven Cordyceps samples were investigated based on the analysis of ITS1-5.8S-ITS2 region. The obtained results would contribute an additional DNA database for specifically identifying species in Cordyceps genus in Vietnam. MATERIALS AND METHODS Materials Seven fresh fungal strains of Cordyceps genus were provided by Agricutural Genetics Institute, Ministry of Agriculture and Rural Development (C1, C2), market (C3), Institute for Regional Research and Development, Ministry of Science and Technology (C4, C5, C6); THD Company (C7) (Figure 1). Methods Total DNA extraction and purification Total DNA of seven of Cordyceps specimens were extracted following CTAB method described by Doyle and Doyle (1990). Briefly, 50 mg of each sample was well-grounded in liquid nitrogen. Cell wall and membrane were degraded by suspending in 65oC buffer composed of 1.4 M NaCl (Scharlau, Spain), 0.1 M Tris - HCl (Biobasic, Canada) pH 8.0, 20 mM EDTA (Merck, Germany) pH 8.0, 2% CTAB (Affymetrix, USA), 1% PVP (Sigma- Aldrich, USA), and 1% β-mercaptoethanol (Sigma- Aldrich, USA) for 60 min. The mixture was then hold at room temperature for 5 min to cool down. Cell fragments were removed by adding one volume of phenol:chloroform:isoamyl alcohol (25:24:1) and centrifuged at 12000 rpm for 20 min at 4oC. The supernatants were transferred to new Journal of Biotechnology 16(4): 705-712, 2018 707 tubes, added 1 µL RNAse (Thermo Fisher Scientific, USA) and incubated for 15 min at 37oC. The supernatants were purified by adding cloroform:isoamy alcohol (24:1, v:v), centrifuging at 12000 rpm for 20 min at 4oC, transferring to clean tubes. DNA was then precipitated by two volume of absolute ethanol and one tenth of 0.3 M CH3COONa for 20 min at 4oC, centrifuged at 12000 rpm for 15 min at 4oC. The precipitated DNA was washed with 70% ethanol and air-dried. Finally, the DNA was resuspended in sterilized deionized water for further uses. Figure 1. Selected samples used for molecular study. Samples C1, C2, C3, C5, C6, C7 were marked as number 1, 2, 3, 5, 6, 7 Amplification of ITS1-5.8S-ITS2 region and PCR product purification The target DNA region was amplified using primers designed based on the GenBank sequences as following 5’-TCCGTAGGTGAACCTGCGGT-3’ (Cor-ITS1-F) and 5’-TCCTCCGCTTAT TGATATGC-3’ (Cor-ITS4-R). The reaction was carried out in a volume of 20 µL containing 1X DreamTaq buffer, 200 mM of each dNTP, 2.5 µM of each primer, 1.5 mM MgCl2, 0.75 units of Dream Taq DNA polymerase (Thermo Fisher Scientific, USA) and 50 ng of template DNA. The PCR was performed by IBM Veriti (Applied Biosystems, USA) for 2 min at 95°C denaturation, 35 amplification cycles (30 s at 95°C denaturation, 30 s at 56°C annealing, and 1 min at 72°C extension), 5 min at 72°C extension, then hold at 4°C. The amplified products were purified using GeneJET™ PCR Purification Kit (ThermoFisher Scientific, USA) as described by the manufacturer, then screened on 0.8% agarose gel. Sequence analysis and alignment Purified DNA was sequenced by ABI 3500 Genetic Analyzer applying BigDye® Terminator v3.1 Cycle Sequencing Kit (Thermo Fisher Scientific, USA). The sequencing mixture was prepared in total volume of 15 µL containing 1X BigDye buffer, BigDye, 3.2 pM primer, and 200 ng purified DNA. The PCR was performed by IBM Veriti (Applied Biosystems, USA) for 1 min at 96°C denaturation, 25 amplification cycles (10 s at 96°C, 5 s at 50°C, Le Thi Thu Hien & Ha Hong Hanh 708 and 4 min at 60°C), then hold at 4°C. After amplification, PCR product was purified by ethanol/EDTA method. Briefly, 5 µL of 125 mM EDTA and 60 µL of absolute ethanol were added to the tube of PCR product and kept at room temperature for 15 min. The supernatant was discarded after centrifuging at 12000 rpm for 15 min. Next, 60 µL of 70% ethanol was added and centrifuged again at 10000 rpm for 10 min. The acquired pellet contained precipitated DNA was dried. The purified DNA was then denatured by adding 10 µL of Hi-Di Formamide at 95oC for 5 min. The samples were loaded to each well of a sample tray, and performed electrophoresis in 80 cm × 50 µm capillary tubes with POP-4 polymer (ABI, USA). The nucleotide sequence of each sample was identified both forward and reverse directions. All raw obtained sequences were analysed using Bioedit 7.2.6 softwares (Hall, 1999). Pairwise distance was performed using MEGA 7.0 (Kumar et al., 2016). The phylogenetic trees were constructed using Maximum Likelihood methods with bootstrap value equal 1000. RESULTS Efficiency of DNA extraction and PCR amplification of target region Total DNA of all samples were extracted by CTAB method. The obtained genomic DNA in each lane performed an explicit band without any smear in agarose gel (Figure 2A). Additionally, the total amount DNA of these specimens ranged from 6.8 to 7.4 µg, and the purity of extracted DNA was excellent as evident by A260/A280 ratio ranging from 1.72 to 1.97 and A260/A230 ratio was >2. They all indicate that obtained genomic DNA of samples meet quality and quantity requirements for molecular analyses. Amplicons acquired with specific primers for ITS1-5.8S-ITS2 region represented precise length as expected (650 bp). The success rates of PCR amplification were high for all samples (Figure 2B). Figure 2. Electrophoresis of genomic DNA extraction (A) and PCR products of amplified target region (B). Journal of Biotechnology 16(4): 705-712, 2018 709 Assessment of barcoding gap and authentication ability To estimate the identification ability using DNA barcoding, BLAST tool was used for determining the identity of a sample based on the best hit of the query sequence, and the E-value for the match must be less than the cut-off value. Accordingly, sequences of typical representatives of Cordyceps genus were retrieved to construct a reference sequence library for alignment with obtained sequences in this study. The results showed that the target DNA region was successfully sequenced. In this analysis, the sequences of six samples C1, C2, C3, C5, C6, C7 were completely similar to each other, while there was a pretty difference from C4 specimen. Comparison of the target region between reference sequences and examined ones revealed that the variability of ITS1 and ITS2 regions is higher than that of 5.8S region. Using three ITS1-5.8S-ITS2 sequences of C1-C3 samples as a query yielded the best matches which were 99.82% identical to ITS1- 5.8S-ITS2 sequences of known species C. militaris. These results indicated that the samples C1-C3, C5- C7 were most closely related to C. militaris. Likewise, alignment of the ITS1-5.8S-ITS2 sequence of C4 exhibited a high degree of homology (99.81%) to C. sinensis, which could be implied that they had the close phylogenetic relationship (Table 1). Table 1. Average similarity of ITS1-5.8S-ITS2 region. Reference species Average similarity (%) C1-C3, C5-C7 C4 Cordyceps brongniartii 91.53 82.47 Cordyceps cardinalis 86.30 81.88 Cordyceps cicadae 88.69 86.35 Cordyceps confragosa 90.07 83.00 Cordyceps cylindrica 85.93 88.07 Cordyceps emeiensis 85.85 85.46 Cordyceps gunnii 82.86 89.56 Cordyceps militaris 99.82 84.41 Cordyceps morakotii 88.45 86.16 Cordyceps ninchukispora 89.56 87.97 Cordyceps ningxiaensis 95.93 84.39 Cordyceps ochraceostromata 88.43 87.21 Cordyceps pleuricapitata 83.25 83.49 Cordyceps pruinosa 89.62 87.10 Cordyceps pseudomilitaris 87.99 83.61 Cordyceps scarabaeicola 91.68 82.99 Cordyceps scarabaeucika 91.78 82.50 Cordyceps spegazzinii 88.95 82.56 Cordyceps sinensis 84.24 99.81 Cordyceps takaomontana 88.43 86.29 Phylogenetic reconstruction Sequence analyses of the ITS1-5.8S-ITS2 region were used for reconstructing phylogenetic tree based on Maximum Likelihood method. Twenty selected reference sequences belonged to the Cordyceps genus served as representative and Akanthomyces cinereus was an outgroup. Corresponding to the phylogeny analysis, samples C1-C3, C5-C7 obviously revealed the closest relationships to C. militaris. A relevant bootstrap value at 99 by Maximum Likelihood method indicated that a confidence interval was eligible for genetic correlation of these species. For the sample C4, C. sinensis was inferred to be the nearest neighbors with the average similarities at 99.81%. This evaluation was confirmed evidently by the result presented in the phylogenetic tree with extremely high bootstrap value (Figure 3). Le Thi Thu Hien & Ha Hong Hanh 710 Figure 3. Phylogenetic tree based on ITS1-5.8S-ITS2 of the Cordyceps genus imaged by Maximum Likelihood methods. Bootstrap values are indicated below the node. DISCUSSION A number of studies which aim to find suitable DNA markers for species identification have been conducted over many decades. Different regions as well as combination thereof have been examined and proposed as preferred plant barcodes. In this study, selected species of Cordyceps genus could be identified using the ITS1-5.8S-ITS2 region. The findings on ITS regions are congruent with similar previous study (Kuo et al., 2005). Due to quick evolution and considerable variation, ITS sequences are suitable for utilizing as standard DNA barcodes for taxonomy classification at both the infrageneric and intergeneric levels (Liu et al., 2009; Tripathi et al., 2013; Keskin et al., 2017). Based on ITS1- 18S rRNA- ITS2 region, 17 distinct strains belonged to the Cordyceps genus were classified into 5 major group and C. sinensis speciments generated a seperate group (Kuo et al., 2005). In 2007, Sung et al. built phylogeny of 162 different species of Cordyceps genus by using 5 to 7 DNA markers including nrSSU (nuclear ribosomal small subunits), nrLSU (nuclear ribosomal large subunits), tef1 (the elongation factor 1α), tub (β-tubulin), atp6 (mitochondrial ATP), rpb1 and rpb2 (the largest and the second largest subunits of RNA polymerase ІІ) and suggested that the discrimination at species level need to combine multiple DNA barcodes and morphology characteristics (Sung et al., 2007). Journal of Biotechnology 16(4): 705-712, 2018 711 In the genus of Cordyceps, C. militaris and especially C. sinensis are highly commercially valuable species, therefore, it is special important to control and accurately determine these species in trade transactions. In the current study, samples were discriminated pronouncedly based on a combination of morphology of plants and average similarities’ assessment between query and reference sequences. The results indicated the ITS1-5.8S-ITS2 region was efficient in distinguishing C. militaris and C. sinensis species that contributes additional reference sequences for Cordyceps species in Vietnam as well as confirms molecular discrimination ability of ITS regions. CONCLUSION The results obtained in this study indicated that similarity and phylogenetic analysis based on ITS1- 5.8S-ITS2 sequence is the potential DNA barcode to discriminate species of Cordyceps genus. These findings can support morphological classification method and generate a foundation for conservation and commercialization of the important traditional medicines in Vietnam. Acknowledgement: This work was partially supported by the Institute of Genome Research. The authors would like to thank Nguyen Tien Dung from the Institute for Regional Research and Development for providing samples; colleagues at the Genome Biodiversity Laboratory, Institute of Genome Research for their assistance with this project. 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