Identification of common edible and medicinal mushrooms by DNA barcoding

Yan-Yan Su; Huan Tang; Xi-Wen Li; Wei Sun; Zeng-Hua Chen; Li Xiang

doi:10.15806/j.issn.2311-8571.2016.0016

MODERN RESEARCH ON CHINESE MATERIA MEDICA

Year : 2016 | Volume : 2 | Issue : 3 | Page : 10-16

Identification of common edible and medicinal mushrooms by DNA barcoding

Yan-Yan Su¹, Huan Tang², Xi-Wen Li², Wei Sun², Zeng-Hua Chen³, Li Xiang²
¹ Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing 100700; Huaibei Normal University, Huaibei 235000, China
² Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing 100700, China
³ Beijing Qianjunfang Academy of Sciences, China

Date of Submission	30-May-2016
Date of Acceptance	29-Sep-2016
Date of Web Publication	10-Sep-2020

Correspondence Address:
Li Xiang
Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences, No. 16 Nanxiaojie, Dongzhimennei Ave, Beijing, 100700
China

Source of Support: None, Conflict of Interest: None

DOI: 10.15806/j.issn.2311-8571.2016.0016

Abstract

Objective: To identify common edible and medicinal mushrooms using the internal transcribed spacer (ITS) region.
Method: A total of eighty-five samples belonging to forty-three species were used in this study. ITS regions were amplified and sequenced. All ITS regions were analyzed using BLAST and MEGA 5.10 methods.
Results: Forty species were identified correctly via BLAST method whereas forty-one species were distinguished via Neighbor joining (NJ) tree. All the species could be distinguished by BLAST combined with NJ tree and ITS sequence characteristics.
Conclusion: Forty-three common species of edible and medicinal mushrooms were effectively identified using DNA barcoding technology based on ITS region.

Keywords: Edible mushroom, Medicinal mushroom, ITS, DNA Barcoding

How to cite this article:
Su YY, Tang H, Li XW, Sun W, Chen ZH, Xiang L. Identification of common edible and medicinal mushrooms by DNA barcoding. World J Tradit Chin Med 2016;2:10-6

How to cite this URL:
Su YY, Tang H, Li XW, Sun W, Chen ZH, Xiang L. Identification of common edible and medicinal mushrooms by DNA barcoding. World J Tradit Chin Med [serial online] 2016 [cited 2023 Jun 2];2:10-6. Available from: https://www.wjtcm.net/text.asp?2016/2/3/10/294656

Introduction

Edible and medicinal mushrooms, whose history can be traced back to 4000 years, are treasured as a health food because of their unique edible and medicinal values^[1]. Studies have shown that edible mushrooms are low in calories and fat, but rich in proteins, amino acids, poly-saccharides, dietary fiber, and mineral elements^[2],[3],[4]. Edible mushrooms also have anti-tumor, anti-viral, blood lipid reducing, and immunity promoting effects^{[5],[6],[7],[8]}. In recent years, increasing attention has been given to edible and medicinal mushrooms because of their special nutritional and medicinal attributes. The United Nations Food and Agriculture Organization proposed that a rational diet should be “meat, vegetable, and mushroom every meal”. China has abundant edible and medicinal mushrooms resources. Reports have stated that more than 1000 wild species have been found in China. More than 100 species have also been cultivated and more than 80 species can be bought in markets as common edible and medicinal mushrooms^[9]. Common edible and medicinal mushrooms include the Lentinula edodes, Flammulina velutipes, Auricularia auricula-judae, Hericium erinaceum, Tricholoma matsutake, and so on. The special nutritional value and growing market demand for mushrooms, has resulted in higher prices, particularly for rare and wild species. Therefore, other fungi species are usually used to adulterate wild rare edible mushrooms. Unfortunately, species cannot be identified solely on morphological characteristics and fruiting body. Synonyms and homonyms are also so widespread that it could confuse consumers and disrupt the market. For example, adulterants of Ophiocordyceps sinensis included Cordyceps gunnii, Cordyceps militaris, Cordyceps cicadae, and so on^[10]. In addition, a significant number of poisoning through the accidental ingestion of wild mushroom pose a significant threat to food and drug safety^{[9],[11],[13]}. Currently, studies on fungal identification have focused mainly on traditional morphological identification. Molecular marker methods, such as Restriction Fragment Length Polymorphism and random amplified polymorphism DNA, are used mostly in strain breeding and identification. Unfortunately, these methods fail to achieve accurate and efficient identification results^[14],[15]. Hence, exploring a novel and powerful identification method for common edible and medicinal mushrooms to ensure food and drug safety is necessary.

DNA barcoding is a novel and powerful identification method, which has been applied widely to species identification^{[16],[17],[18],[19],[20]}. Cytochrome c oxidase subunit 1 (COI) region has been used as a universal barcode for animals^[21]. Internal transcribed spacer 2 region complemented with psbA-trnH has been proposed as a candidate DNA barcode for identification of medicinal plants^[22],[23]. Internal transcribed spacer (ITS) region has also been suggested as a candidate barcode for fungal identification and has been used in the successful identification of many edible and medicinal mushrooms at the species level, such as O. sinensis, Boletus roseoflavus, and B. brunneissimus^{[10],[24],[25]}. Therefore, in this paper, ITS region was used as a universal barcode to identify common edible and medicinal mushrooms.

Materials and Methods

1. Materials

Eighty-five samples representing forty-three species from five orders were collected from wild areas and markets [Table 1]. The fifteen representative species of common edible and medicinal mushrooms are shown in [Supplemental Figure S1].

Table 1: Information on the samples

Click here to view

All samples were identified by morphology by Professor Xiaolan Mao from the Institute of Microbiology of the Chinese Academy of Sciences. Voucher samples were deposited at the Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences. All ITS sequences were submitted to GenBank [Table 1].

2. Methods

2.1 DNA extraction

Total genomic DNA was extracted from 20-40 mg dry internal tissue of eighty-five samples following the protocol of the Plant Genomic DNA Kit (Tiangen Biotech Co.) and previous studies^[26]. The materials were mashed for 200 s at 50 HZ by a DNA Extraction Grinder (Sceintz Biotech Co., China). Then, 800 μL nucleus separation liquid (100 mmol·L^-1 Tris-HCl (pH 8.0), 20 mmol·L^-1 EDTA (pH 8.0), 0.7 mol·L^-1 NaCl, 2% PVP- 40, 0.4% β-mercaptoethanol) was added once or several times to clean the materials until they were colorless.

2.2 PCR amplification and sequencing

Primers of ITS region: ITS5F (5′-GGAAGTAAAAGTCGTAA CAAGG-3′) ITS4R (5′-TCCTCCGCTTATTGATATGC-3′). PCRs were performed in 25 μL reaction mixtures containing 4 μL of genomic DNA, 12.5 μL of 2×Taq PCR Master Mix (Aidlab Biotech Co., China), 1 μL of each primer (2.5 μmol·L^-1). The mixtures were adjusted to 25 μL with distilled deionized water. Reaction conditions were as follows: 94 °C 5 min; 94 °C 1 min, 50 °C 1 min, 72 °C 90 s, 30 cycles; 72 °C 7 min. After obtaining the PCR products, electrophoresis (1.0% agarose gel in 0.5×TBE purified buffer), purification, and sequencing (3730 XL Sequencer, Applied Biosystems) were performed.

2.3 Data analysis

Trimming and assembling trace files were performed using CondonCode Aligner V 3.0 (CondonCode Co., USA). The ITS sequences were obtained by annotation based on the Hidden Markov model. All sequences were blasted in the NCBI database, then aligned through Muscle and ClustalW analysis and analyzed by MEGA 5.10 (Center for Evolutionary Medicine and Information, USA). The neighbor joining (NJ) trees were constructed based on the Kimura 2-Parame- ter model.

Results

1. DNA extraction and PCR amplification

Total genomic DNA of eighty-five samples was extracted by an optimal DNA extraction method. Eighty-five available ITS sequences were obtained after amplification and sequencing and subsequently submitted to GenBank [Table 1].

2. Sequence characteristics analysis

The sequence characteristics are shown in [Table 2]. All ITS sequences were more than 500 bp in length except for H. russula (464 bp). The longest sequence was that of A. tabescens (751 bp). The ITS sequences belonging to the same genus were similar in length, such as P. citrinopileatus (581 bp), P. eryngi (589 bp) and P. ostreatus (587 bp) of Pleurotus genus; T. populinum (618 bp), T. squarrulosum (608 bp) and T. virgatum (614 bp) of Tricholoma genus; A. auricula-judae (514 bp) and A. polytricha (520 bp) of Auricularia genus.

Table 2: ITS sequence characteristics of common edible and medicinal mushrooms

Click here to view

The lowest GC content was 37.2% (H. russula) and the highest was 59.4% (V. volvacea). The GC contents of four species were lower than 40%, including H. russula (37.2%), L. giganteus (37.8%), L. edodes (38.4%), and I. gibba (39.0%). GC contents of six species were higher than 50%, including V. volvacea (59.4%), S. granulatus (53.4%), F. velutipes (50.2%), A. auricula-judae (50.1%), M. eximia, (51.5%) and L. sulphureus var. miniatus (51.7%). In addition, the GC contents of thirty-three species ranged from 40% to 50%. The average GC content of thirty-one species belonging to Agaricales order was 44%. Four species of Russulaceae order had GC contents in close range, including H. erinaceum (48.4%), R. rosea (49.5%), R. turci (48.2%) and R. vesca (49.5%).

The ITS sequences of mushrooms belonging to the same genus had similar lengths and GC contents. For example, Tricholoma genus: T. populinum (618 bp; 43.2%), T. squarrulosum (608 bp; 43.4%) and T. virgatum (614 bp; 42.0%); Auricularia genus: A. auricula-judae (514 bp; 50.1%) and A. polytricha (520 bp; 49.4%).

For ITS sequences, ten species (S. granulatus, C. claricolor, P. eryngi, P. nebrodensis, V. volvacea, I. gibba, T. indicum, M. eximia, H. erinaceum, R. rosea) had no variable site. Five species (C. utriformis, P. squarrosa, T. matsutake, A. auriculajudae and R. vesca) had one variable site. Three species (H. russula, S. commune and A. polytricha) had two variable sites. Three species (L. perlatum, P. citrinopileatus and T. populinum) had three variable sites. H. marmoreus had four variable sites and L. edodes had eight variable sites. On the whole, the ITS sequences of common edible and medicinal mushrooms were conservative.

3. Intra- and inter-specific distance analysis

The intra- and inter-specific K2P distances of ITS sequences are listed in [Table 2]. The maximum intra-specific K2P distances of twenty-three species were less than that of the minimum inter-specific distances, indicating that these twenty-three species could be distinguished from others by distance method. The other twenty species only had one sequence. The minimum inter-specific K2P distance of F. velutipes was 0.323, seven species (C. comatus, G. confluens, A. tabescens, R. turci, N. lepideus, P. umbellatus, L. sulphureus var. miniatus) were from 0.2 to 0.3, ten species (C. gigantea, L. shimeji, H. capnoides, P. microspora, S. rugosoannulata, L. proxima, L. nuda, L. giganteus, T. squarrulosum, T. virgatum) were from 0.1 to 0.2, L. connatum and P. ostreatus’s were less than 0.1.

4. BLAST analysis

In this study, the highest similarity species of forty species were the species themselves after being blasted in the NCBI database using ITS sequence. The sequence identities were higher than 99%. Therefore, forty species could be successfully identified at the species level by BLAST method in the NCBI website (http://blast.ncbi.nlm.nih.gov/). However, the highest similarity species of three species were not themselves after being blasted in NCBI database using ITS sequence. The highest similarity species of P. ostreatus were species in the same Pleurotus genus (P. cornucopiae, P. pulmonarius and P. sapidus) with 100% sequence identities. The highest similarity species of L. giganteus was L. paradoxus with 99% sequence identities, which was higher than its own 97% sequence identities. The highest similarity species of L. proxima was itself, with 83% sequence identities.

5. NJ tree analysis

Two NJ trees were constructed using eighty-five ITS sequences of the forty-three species. But the NJ tree based on Muscle analysis exhibited better topology than the NJ tree based on ClustalW analysis [Figure 1]. Forty-one species were formed into separate branches with high bootstrap scores. Moreover, species from the same family clustered together. Species of Lyophyllaceae (H. marmoreus, L. connatum and L. shimeji), Agaricaceae (C. Gigantea, C. utriformis, L. perlatum and C. comatus), Strophariaceae (H. capnoides, S. rugosoannulata and P. squarrosa) and Physalacriaceae (A. tabescens and F. velutipes) family clustered together with bootstrap values of 99%, 96%, 84% and 57%, respectively. Species from the same genus also clustered together. Species from Auricularia (A. auricula-judae and A. polytricha), Pleurotus (P. citrinopileatus, P. eryngi, P. nebrodensis and P. ostreatus), Russula (R. rosea, R. turci and R. vesca) and Tricholoma (T. matsutake, T. populinum, T. squarrulosum and T. virgatum) genus clustered together with high bootstrap values of 100%, 100%, 93%, and 84%, respectively. However, two ITS sequences of H. capnoides and S. rugosoannulata of the Strophariaceae family formed into a single branch with high bootstrap value of 99%, and hence requires combining with other methods for authentication.

Figure 1: Neighbor-joining (NJ) tree based on ITS sequences of common edible and medicinal mushrooms
(Note: The bootstrap values (1000 replicates) were showed (≥50%) for each branch)

Click here to view

Discussion

Edible and medicinal mushrooms play an important role in fungi resources because of its high economic value. Hence, accurately authenticating edible and medicinal mushrooms is highly important to ensure the safety in its use. DNA barcoding technology based on ITS region was used to identify common edible and medicinal mushrooms in our study.

Plant Genomic DNA kit was used to extract total genomic DNA. Samples were cleaned with 75% ethanol because the DNA is easy to pollute. The samples were then cleaned once or several times using the nucleus separation liquid (100 mmol·L^-1 Tris-HCl (pH 8.0), 20 mmol·L^-1 EDTA (pH 8.0), 0.7 mol·L^-1 NaCl, 2% PVP-40, 0.4% β-mercaptoethanol) until the liquid layer became lightly colored or colorless. The total genomic DNA of eighty-five samples was successfully extracted by the modification of a Plant Genomic DNA kit protocol.

Agaricales order, thirty-one species of which were included in this study, was an important taxon of edible and medicinal mushrooms. The results showed that the variable sites of most species were less than three, especially for cultivated mushrooms, such as A. polytricha (two variable sites), A. auricula-judae (one variable site), P. eryngi (no variable site) and V. volvacea (no variable site). Li Li also found that the A. auricula-judae originated from the same area were with a narrow genetic basis by analysing the ITS and IGS regions^[27]. The results further indicated that ITS sequences of cultivated mushrooms were conservative, because of mature cultivation techniques and stable strains^[28].

The sequence characteristics combined with BLAST and NJ tree method were used to estimate the capability of distinguishing edible and medicinal mushrooms using DNA barcoding based on ITS region. The results showed that the highest similarity species of forty species were themselves after being blasted in the NCBI database using ITS sequence, while three species, including P. ostreatus, L. giganteus, and L. proxima were not. Fortunately, P. ostreatus, L. giganteus, and L. proxima could be distinguished from the other forty species by NJ tree method. Furthermore, forty-one species were clearly identified by NJ tree. H. capnoides and S. rugosoannulata of Strophariaceae family formed into a single branch with high bootstrap value of 99%. This result indicated that the two species has a considerably close genetic relationship. These species could be successfully identified at the species level by BLAST method. Tian Enjing reported a close genetic relationship between Hypholoma and Stropharia using Bayesian analysis on the basis of rDNA-ITS sequences, which was consistent with our results^[29].

In this study, some species only had one ITS sequence respectively. However, despite the limited sequences, the common edible and medicinal mushrooms could still be identified using the combined methods with ITS sequence characteristics, NJ tree and BLAST methods. Both A. tabescens and F. velutipes, which had only one ITS sequence, were good cases. A. tabescens and F. velutipes formed into a single branch with a bootstrap value of 57%, but they could be identified by the BLAST method. Because of the difficulty in collecting abundant samples, enough ITS sequences could not be obtained in the present study. Further research on common edible and medicinal mushrooms identification using DNA barcoding could be performed after the supplement of samples.

In conclusion, forty-three common species of edible and medicinal mushrooms could be identified effectively using DNA barcoding technology based on the ITS region. The study also estimated the capability of DNA barcoding technology to authenticate fungi based on the ITS region and established a powerful tool for fungal identification.

Acknowledgements

This work was supported by the National Natural Science Foundation (81503192) and the Major Scientific and Technological Special Project for “Significant New Drug Creation” (No. 81503192).

Conflict of Interest

The authors declare that they have no competing interests.

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