2 Macau Institute for Applied Research in Medicine and Health, Avenida Wailong, Taipa, Macau Special Administrative Region, China;
3 Faculty of Chinese Medicine, Macau University of Science and Technology, Avenida Wailong, Taipa, Macau Special Administrative Region, China;
4 Sunshine Lake Pharma Co., Ltd., Dongguan 523808, China
Ophiocordyceps sinensis (syn. Cordyceps sinensis) is commonly known as the Chinese caterpillar fungus, which has been widely used as a tonic food and traditional Chinese medicine (TCM) to treat asthma and bronchial infection, strengthen lung and kidneys, and increase energy while recovering from illness, according to TCM theory [1-2]. It is mainly distributed in the Tibetan Plateau at an altitude of 3000 to 5000 m, with a small distribution in Nepal, India, and Bhutan , and its production in China accounts for more than 90 percent of the total production worldwide. Overexploitation of the wild O. sinensis has caused serious damage to its natural living environment, which further aggravates its endangered status , and it has been listed in the national key protection of wild plants in China.
Artificial cultivation of O. sinensis has always been a hot spot of scientific research and business; not only many universities and research institutes of China have tried very hard to explore it under the support of Chinese government recently, such as the Major State Basic Research Development Program of China (973 Program) and the National Natural Science Foundation of China, but also some industrial entities have invested heavily to develop this technology [5-6]. Although SHEN Nan-Ying cultivated the stroma completely similar to natural O. sinensis in the Erlenmeyer flask as early as 1983 , there still are lots of problems to cultivate complete O. sinensis artificially, especially in a large scale. Now several institutions have successively reported they have reproducibly completed the life cycle of O. sinensis from anamorph to teleomorph in their hosts, such as Prof. HAN Ri-Chou group from Guangdong Institute of Applied Biological Resources and Prof. QIN Qi-Lian group from Institute of Zoology in Chinese of Academy of Sciences [8-9]. Further realizing industrialization and commercialization of artificial cultivation can help save the endangered status of its wild resources. In 2016, Guangdong Sunshine Lake Pharma Co., Ltd. and its partners announced that they had successfully established a developing and producing base to simulate the original ecological condition of O. sinensis and completed its industrialization of large-scale cultivation after more than 10 years of research . And these artificial cultivated O. sinensis products have already been marketed, which are identified to be same with O. sinensis by the Institute of Microbiology Chinese Academy of Sciences . Nevertheless, the genetic background of these industrially cultivated O. sinensis remains unknown. The understanding of the genetic background is very important to efficiently authenticate these products, helping its sustainable breeding and development in the future.
With the rapid development of molecular technology in recent years, molecular identification, especially DNA barcoding, has made a great progress in identifying Chinese medicine, including medicinal plants, fungi, and animals [12-16], which has attracted a wide range of attention and recognition. DNA barcoding is a technique to identify species by analyzing a standard DNA sequence, and now it has successfully recognized the identification of plants, animals, and fungi [12, 17]. Owing to rapid concerted evolution, the internal transcribed spacer (ITS) of nuclear rDNA has many variable sites and potential informative sites among related species, which has been proven to be an important molecular marker in phylogenetic and evolutionary studies . In 2013, Prof. CHEN Shi-Lin reported the molecular identification method of O. sinensis and its counterfeit products based on ITS sequences, and this method can also be applied for identifying other fungi , so that the ITS sequence is regarded as a standard DNA barcode for identifying medicinal fungi. O. sinensis can be divided into different haplotypes by ITS sequence, and the selection of the appropriate fungi has a great significance for the successfully artificial cultivation of O. sinensis . According to the clustering results of different haplotypes of ITS sequences, the haplotypes that the cultivated O. sinensis samples belong to can be effectively identified. More than that, ITS sequence can also be used for the evolution analysis and phytogeography study. As we know the detailed origins of O. sinensis, combing its evolution analysis, the distribution of different haplotypes can help us find and identify the preponderant and specific fungi strains to optimize artificial cultivation of O. sinensis, which has not been reported thus far.
In the present study, we downloaded and analyzed the ITS sequences of O. sinensis from NCBI website and supplemented the wild samples from different origins having geologic significances which had not been reported. We then amplified the ITS sequences by PCR and conducted the phylogenetic analysis. According to the different degree of genetic differentiation, we divided all the O. sinensis into seven phylogenetic groups, cast the different group of O. sinensis on the map to observe the distribution and occurrence frequency, and deduced the evolution process within the groups in conjunction with the geographical information. Meanwhile, we also tested the ITS sequences of representative samples from 3 batches of cultivated O. sinensis products with high yield, and carried out sequence alignment, phylogenetic analysis, and clustering with those ITS sequences from wild O. sinensis with known origins. We not only identified the cultivated O. sinensis at the molecular level for the first time, but also classified all the O. sinensis into seven groups by analyzing ITS sequences, so that we could deduce the evolution process within the different groups. At the same time, we analyzed which groups the cultivated samples were clustered into and which evolution level the cultivated samples were located at, to provide the basis and research strategy for the artificial cultivation of O. sinensis in the future.Materials and Methods Sampling
A total of 19 O. sinensis samples and one counterfeit sample were collected and morphologically authenticated, including 16 complete and wild samples with specific origins provided by Prof. JIANG Zhi-Hong from the state key laboratory of quality research in Chinese Medicine of Macao University of Science and Technology University (Numbered CSW081901-CSW081916), 3 HEC artificial cultivated O. sinensis product samples with high yield provided by Sunshine Lake Pharma Co., Ltd. (Numbered CSC080901- CSC080903, the 3 samples were representatives from those 3 batches of samples) and 1 counterfeit sample provided by the Faculty of Chinese Medicine of Macao University of Science and Technology (Numbered CSA090901). All the 16 wild samples were identified by Dr. ZHANG Zhi-Feng, an experienced researcher of pharmacognosy in Macao University of Science and Technology University.DNA extraction, amplification and sequencing
The nrDNA was isolated by DNeasy Plant Mini Kit (Qiagen, Hilden, Germany), and the specimens were divided into stromata and insects. In brief, the contaminating debris was removed by brush and the surface was scrubbed by 75% ethanol, and then the samples were dried by hair dryer. Single dry stroma (25 mg) of different samples was placed into a 2.0-mL Eppendorf tube and ground into powder by Tissuelyser Ⅱ instrument (Qiagen GmbH, Hilden, Germany) under the environment of liquid nitrogen. The concentrations and optical density ratios of these extracted DNA were measured by NanoDrop 2000c spectrophotometer (Thermo Scientific, Waltham, USA). The ITS regions of nrDNA were amplified by PCR reaction using a TaKaRa PrimeSTAR HS DNA Polymerase (TaKaRa Bio, Ohtsu, Japan) with a forward primer of ITS5F: 5'-GGA AGTAAAAGTCGTAACAAGG-3' and a reverse primer of ITS4R: 5'-TCCTCCGCTTATTGATATGC-3' [13, 20].
The PCR reaction mixture contained 25.0 µL of PrimeSTAR HS Premix (Takara, Japan), 1.0 µL of ITS5F primer (10 µmol·L–1), 1.0 µL of ITS4R primer (10 µmol·L–1), 1.0 µL of DNA template and 23.0 µL of sterilized distilled water in a total volume of 50.0 µL. The samples were amplified using a Veriti 96-Well Fast Thermal Cycler (Applied Biosystems, Foster City, CA, USA) under the following conditions: initial denaturation at 94 ℃ for 2 min, followed by 30 cycles of denaturation at 98 ℃ for 10 s, annealing at 62 ℃ for 10 s, extension at 72 ℃ for 30 s, and a final elongation step at 72 ℃ for 7 min. The PCR products were confirmed by 1.0% agarose gel electrophoresis in 1 × TAE buffer to detect whether the ITS sequences were cloned successfully. And then the target fragments of 550 bp were sent to BGI (Shenzhen, China) for bidirectional sequencing.Phylogenetic analysis
In order to obtain high quality of ITS sequences, the forward and reverse sequencing results were assembled together after cutting off both ends of the primer area, and then the ITS sequences were uploaded to the "Chinese herbal medicines DNA barcoding identification system" (http://www.tcmbarcode.cn/china/) for online identification of species. The multiple sequence alignment was done to find the different sites and haplotypes of ITS sequences from O. sinensis by BioEdit software (Ibis Therapeutics, Carlsbad, USA). Furthermore, the reported ITS sequences of O. sinensis with specific origin information were downloaded from NCBI website and added to the ITS sequences we obtained in the present study, and all of them were analyzed with MEGA 7.0 software (MEGA, PA, USA) to construct the phylogenetic tree by calculating interspecific K2P evolutionary distance and using neighbor-joining method (bootstrap = 1000). These ITS sequences were then classified into different groups based on the phylogenetic tree result.Distribution analysis of wild O. sinensis
According to the classification of phylogenetic tree and information on origins of O. sinensis, the latitude, longitude and altitude of origins were found and recorded by Google Earth 7.1.2. The locations were projected to the provincial boundary map of China based on the latitude and longitude data to observe and analyze the distribution of different groups by ArcGIS10.2 software, using ArcMap tool and Lambert projection method, and the results were saved as TIF/PNG format.Results Morphological characterization of O. sinensis
Wild O. sinensis samples CSW081901-CSW081916 and artificially cultivated product samples CSC080901-CSC080903 had similar appearances (Fig. 1). The samples were consisted of insects and stromata, and they smelled with fragrance. The surface colors of insects were light yellow or dark yellow. In the insect section, there were eight pairs of feet, four obvious pairs of feet on the abdomen, three vestigial pairs of feet on the head, and one vestigial pair of feet on the end. The rings on the back were clear, and the color of the neck was brighter than that of insect. The surface colors of stromata were light brown or dark brown, and their sectional color was white. All these characters were consistent with those of authentic O. sinensis, which suggested that they should be authentic under the morphological identification.
The characteristics of counterfeit sample, CSA090901, were different from that of authentic samples (Fig. 1). The surface color of insect was light yellow and the feet on insect section were not obvious. Its stroma was thick and hard, liked wooden materials, so it was difficult to break. And in the process of wiping by ethanol, the yellow color of the surface was easy to erase. The characterization results did not meet the characters of authentic O. sinensis, proving that it should be a counterfeit.Molecular identification of O. sinensis
DNA was successfully extracted from the insects and stromata from 16 wild samples, 3 cultivated product samples and 1 counterfeit sample of O. sinensis. Their concentrations were from 6.1 to 72.5 ng·μL–1 and their optical density (OD260 : 280 nm) ratios were from 1.64 to 2.04. The PCR products were detected by agarose gel electrophoresis and the authentic samples showed a single band of about 570 bp. But the counterfeit sample showed differently: its stroma section had no amplified band, while its insect section showed a single band of about 550 bp (Fig. S1). The result showed that the ITS sequences were cloned and amplified efficiently, and the size of the ITS sequences from our wild and cultivated samples were consistent with the reported ITS sequences size of O. sinensis before. The forward and reverse sequencing results were assembled together with cutting off both 5'-ends and 3'-ends. The ITS sequences of insects and stromata were the same, which proved that this method was reliable for identifying the O. sinensis. All the ITS sequences were uploaded to the website of Chinese herbal medicines DNA barcoding identification system (http://www.tcmbarcode.cn/china/) for identification. The identification results showed the samples of CSW081901-CSW081916 and CSC080901-CSC080903 had more than 99.2% similarity with the species of O. sinensis, and 16 samples from them even had 100% similarity with the species of O. sinensis. But the identification result of CSA090901 revealed that it had up to 99.6% similarity with Metarhizium anisopliae.Haplotype analysis of ITS sequence of O. sinensis
The sequence length of ITS sequences from O. sinensis obtained from the present study was 492 bp. As there were different sites on the sequences, every different sequence was regarded as a haplotype. Those sequences were divided into 6 haplotypes, named H1–H6. Haplotype H1 included samples CSW081902-081907, CSW081909-081913, CSW081915- 081916, and CSC080903. Haplotype H2 included sample CSC080901. Haplotype H3 included sample CSC080902. Haplotype H4 included sample CSW081901. Haplotype H5 included sample CSW081908. And haplotype H6 included sample CSW081914. The detailed information on different haplotype is shown in Table 1.
These six haplotypes were also found in the article published by Prof. CHEN Shi-Lin , and the haplotypes of H1, H2 and H3 in our study were also the haplotypes having the top 3 highest frequency of occurrence in that paper. Haplotypes H2 and H3 only had a C/T difference at the position of 358 bp.Evolutionary analysis of O. sinensis
The ITS sequences results from the present study, combining reported ITS sequences of O. sinensis with known origins downloaded from the NCBI website, were analyzed to conduct a phylogenetic tree by neighbor-joining method and the ITS sequence (GenBank No. JX402630) were used as the outer group reference sequence. O. sinensis were classified into seven groups based on the evolutionary level, the evolution trend was deduced among the different groups, and which stage and group the cultivated samples could be clustered into were analyzed as well (Fig. 2).
According to the results of this phylogenetic analysis, these samples were divided into seven phylogenetic groups, named G1–G7, as shown in Fig. 2. The detailed information of O. sinensis could be found in Table S1. Based on the evolutionary distance of the phylogenetic tree, the group G7 was composed of FJ654148 and FJ654149, which was the closest branch to the outer group, and the remarkable difference between G7 and other groups was a 4-bp deletion within the ITS2 region. This result also implied G7 might be the most ancient group of O. sinensis in this phylogenetic analysis. Based on the G7, O. sinensis were evolved to G4, G5 and G6 for the first generation, and further evolution occurred to generate more modern groups of G3, G2, and G1. G1 was the biggest group, which included cultivated sample (CSC080903), wild samples (CSW081901- 081907, CSW081908-081913, and CSW081915-081916) and other 41 reported O. sinensis samples. G1 was proven to be the most suitable group to the modern environment with highest frequency of occurrence and the most widely distribution. G6 was the second largest group, whose frequency of occurrence and distribution were only less than G1, including cultivated samples (CSC080901 and 080902), wild sample (CSW081914), and other 21 reported O. sinensis samples. Although G6 located in the early evolutionary stage of O. sinensis, it was still the most suitable group to the modern environment among the primary groups G4, G5, and G6.Distribution of different groups of O. sinensis
The phylogenetic analysis of ITS sequences was carried out to find the significant gene differentiation among O. sinensis. Combining their geographical distribution information, they could be divided into two main populations: southern and northern populations. Northern population included G1, G2, and G3, and southern population included G4, G5, G6, and G7. The southern population was mainly distributed in Yunnan, southern Tibet and southern Sichuan. The northern population was mainly distributed in Qinghai, Gansu, northern Tibet and northern Sichuan, as shown in Fig. 3.
G1 was distributed in four provinces in China, i.e., Tibet, Qinghai, Gansu, Yunnan, and Sichuan, which was the group with highest frequency of occurrence, and its distribution range was the most extensive of all groups. G6 was distributed in three provinces, i.e., Tibet, Sichuan, and Yunnan, with the range of distribution and frequency of occurrence just less than those of G1. The other five groups appeared only in few provinces and their frequency of occurrence was also not very high. So G1 and G6 were the most dominant groups from northern and southern populations, respectively.
The results also showed that one group might include samples from different geographical locations, such as G1 (spread in four provinces) and G6 (spread in three provinces). And the samples from the same location might also belong to different groups; for example, the samples from three branches of G4, G6 and G7 were located in Tibet Nyingchi.Discussion
In the present study, we used DNA barcoding technology to identify the authentic, cultivated, and counterfeit samples of O. sinensis. The method can not only identify the authenticity of O. sinensis quickly and accurately, but also find out that the cultivated and authentic O. sinensis can be classified into the same cluster. This was the first report to prove that the cultivated O. sinensis product which realized industrialization belonged to authentic O. sinensis at the molecular level. Then, the wild authentic and cultivated samples were classified into seven groups G1–G7, which were divided into northern and southern populations. Diversification of southern population was greater than that of northern population. And among all the regions, Tibet had the greatest diversification, 6 groups could be found in Tibet, which should result from its unique geographic feature and evolution. In other 4 provinces, Qinghai, Gansu, Sichuan, and Yunnan, there were no more than 2 groups existing in the same province. The reason why there were less groups in other regions might be that their geographic differences were not as wide as Tibet, or the groups finally spread to had already been evolved into those which were similar and more adaptable to modern environment.
According to the results of phylogenetic analysis, the O. sinensis of G7 from southern population was evolved into those of G4, G5 and G6, spreading eastward and westward. This spread direction of east and west might be initiated along two large and parallel mountain ranges, the Himalaya Mountains and the Nyainqentanglha Mountains. And then the second evolution occurred with spreading from south to north, which resulted in the generation of G1, G2, and G3. This spread might be achieved by air flow from the Indian Ocean through Yarlung Zangbo Grand Canyon . The G7 contained only two samples which were collected by digging out from Nyingchi of Tibetan Plateau by Prof. ZHANG Yong-Jie . In order to verify its credibility, we also took more ITS sequences into the phylogenetic tree, and the classification result was consistent (Fig. S2). And the samples from Nyingchi had greater within-population genetic diversity than other origins. The phylogenetic tree implied that Nyingchi acted as a very important and ancient site in O. sinensis evolution history, and O. sinensis might have been transmitted from Nyingchi to other sites. But whether Nyingchi is the most ancient site remain unclear. To answer this, more samples are needed.
The cultivated sample of CSC080903 was clustered into the G1, while CSC080901 and CSC080902 were clustered to G6. G1 and G6 were the top 2 groups with the widest distribution range and highest frequency of occurrence. In Nagqu of Tibet and Kangding of Sichuan, G1 and G6 could both existed at those places, which suggested the fungi strains of O. sinensis from G1 and G6 should be the strains with higher survival ability on this environment stage. As these 3 cultivated product samples were representatives of 3 batches with high yield, it revealed that the fungi strains with higher survival ability in wild were also easier to be cultivated. So, the selection of dominant strains is very important for achieving high yield of cultivated O. sinensis.
Future investigations should address the following questions: whether the fungi strains of cultivated O. sinensis in the company are all or most belonged to the G1 and G6 groups, whether these two kinds of O. sinensis fungi are the most suitable strain for artificial cultivation, whether these two kinds of O. sinensis fungi have a positive correlation with high yield of O. sinensis, whether the variable sites and potential informative sites we found on ITS sequence can play as stable molecular markers, and whether the chemical components of those two groups were higher than other groups, If these two groups of O. sinensis have the advantages of chemical components content and artificial cultivation, we can design specific primers based on the specific sites of their ITS sequences, in order to provide the research basis and strategy for quick breeding selection of dominant species, improving the success rate and production of industrially cultivated O. sinensis.Conclusion
Annual production of natural O. sinensis in China is just about 60–100 tons, and because of the warming climate and the deterioration of the environment, the yield of the wild O. sinensis is decreasing every year. As an important medicinal material for Tibetan medicine as well as Chinese medicine, artificial cultivation has been planned and promoted for a long time Almost 40 years later, nowadays we were surprised by the success of the artificial cultivation. However, the slow yield increase hinders the industrial development. Therefore, selection of dominant strains is very important to the next step of research for increasing yield of O. sinensis. With the development of molecular technology, developing molecular markers for further breeding has become the preferred strategy in industrial or agricultural breeding. So, the investigation of genetic background of the cultivated O. sinensis and the primordial distribution of dominant strains we reported in this paper, is of significance for further breeding work and also can directly guide the optimization of the production process, especially the process of selection of strain. Furthermore, evolutionary dominant haplotypes we found on ITS sequence in the present study are expected to provide reference for strain screening, which are conducive to further molecular biology, breeding, and identification on O. sinensis.Acknowledgement:
We thank Dr. ZHANG Zhi-Feng from Macao University of Science and Technology University for identifying the samples of O. sinensis.
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