DNA Barcoding of Economically Important Mushrooms: A Case Study on Lethal Amanitas from China

doi:10.3724/SP.J.1143.2012.12140

Abstract

Abstract:

Some species of the genus Amanita are economically important gourmet mushrooms, while others cause dramatic poisonings or even deaths every year in China and in many other countries. A DNA barcode is a short segment or a combination of short segments of DNA sequences that can distinguish species rapidly and accurately. To establish a standard DNA barcode for poisonous species of Amanita in China, three candidate markers, the large subunit nuclear ribosomal RNA (nLSU), the internal transcribed spacer (ITS), and the translation elongation factor 1alpha (tef1α) were tested using the eukaryotic general primers for their feasibility as barcodes to identify seven species of lethal fungi and two species of edible ones which can easily be confused with the lethal ones known from China. In addition, A.phalloides—a European and North American species closely related to one of the seven taxa, A.subjunquillea was also included. PCR amplification and sequencing success rate, intra and interspecific variation and rate of species identification were considered as main criteria for evaluation of the candidate DNA barcodes. Although the nLSU had high PCR and sequencing success rates (100% and 100% respectively), occasional overlapping occurred between the intra and interspecific variations. The PCR amplification and sequencing success rates of ITS were 100% and 85.7% respectively. ITS showed high sequence variation among species group and low variation within a given species. There was a relatively high PCR amplification and sequencing success rate for tef1α (85.7% and 100% respectively), and its intra and interspecific variation was higher than that of ITS or nLSU. All three candidate markers showed hight species resolution. ITS and tef1α had a more clearly defined barcode gap than nLSU. Our study showed that the tef1α and nLSU can be proposed as supplementary barcodes for the genus Amanita, while ITS can be used as a primary barcode marker considering that the ITS region may become a universal barcode marker for the fungal kingdom.

Key words: DNA barcoding, Edible wild mushrooms, Fungi, Poisonous mushrooms, Species recognition

摘要：

鹅膏属（Amanita）部分物种为重要食用真菌，而另外部分物种则是剧毒的，在我国及其他许多国家，每年都有因误食剧毒鹅膏而导致中毒甚至死亡的事件发生。DNA条形码是用一段或几段短的DNA序列来对物种进行快速、准确鉴定的方法。本研究选取三个候选片段，即核糖体大亚基（nLSU）、内转录间隔区（ITS）和翻译延长因子1-α（tef1-α），使用真核生物通用引物，测试我国已知的7种剧毒鹅膏及2种易混的可食鹅膏，并将欧美分布的但与黄盖鹅膏（A.subjunquillea）亲缘关系密切的绿盖鹅膏（A.phalloides）纳入分析中。nLSU的PCR扩增和测序成功率均为100%，但种内和种间遗传变异偶有重叠。ITS的PCR扩增和测序成功率分别达到100%和85.7%，且具有高的种间变异和低的种内变异。tef1-α的PCR扩增和测序成功率分别达到85.7%和100%，种间和种内遗传分化均高于ITS和nLSU。三个片段的物种分辨率均较高，但与nLSU相比，ITS和tef1-α具有更为明显的barcode gap。鉴于ITS可能会成为真菌界的通用条码，故建议将ITS作为鹅膏属的核心条形码，tef1α和nLSU作为该属的辅助条形码。

关键词: DNA条形码, 食用野生蘑菇, 真菌, 毒蘑菇, 物种识别

CLC Number:

Q 523
Q 949

CA Qing, TANG Li-Ping, YANG Zhu-Liang. DNA Barcoding of Economically Important Mushrooms: A Case Study on Lethal Amanitas from China[J]. Plant Diversity, 2012, 34(6): 614-622.

蔡箐, 唐丽萍, 杨祝良. 大型经济真菌的DNA条形码研究——以我国剧毒鹅膏为例[J]. Plant Diversity, 2012, 34(6): 614-622.

References

Bresinsky A, Besl H, 1985. Giftpilze, mit einer Einfuehrung in die Pilzbestimmung［M］. Stuttgart: Wissenschaftliche Verlagsgesellschaft
Cao J (曹杰), Zhang ZG (张志国), Liu D (刘弟) et al., 2009. ITS sequence analysis of 8 Amanita species from Tibetan and Yunnan［J］. Journal of Yunnan University, Natural Science Edition (云南大学学报, 自然科学版), 31: 90—96
CBOL Plant Working Group, 2009. A DNA barcode for land plants［J］. Proceedings of the National Academy of Sciences of the United States of America, 106: 12794—12797
Dentinger BTM, Didukh MY, Moncalvo JM, 2011. Comparing COI and ITS as DNA barcode markers for mushrooms and allies (Agaricomycotina)［J］. PLoS One, 6: e25081
Doyle JJ, Doyle JL, 1987. A rapid DNA isolation procedure for small quantities of fresh leaf material［J］. Phytochemistry Bulletin, 19: 11—15
Du XH, Zhao Q, Yang ZL et al., 2012. How well do ITS rDNA sequences differentiate species of true morels (Morchella) ［J］. Mycologia, 104 (6), doi: 10.3852/12056
Dupuis JR, Roe AD, Sperling FAH, 2012. Multilocus species delimitation in closely related animals and fungi: one marker is not enough［J］. Molecular Ecology, 21: 4422—4436
Eason RG, Pourmand N, Tongprasit W et al., 2004. Characterization of synthetic DNA barcodes in Saccharomyces cerevisiae genedeletion strains［J］. Proceedings of the National Academy of Sciences of the United States of America, 101: 11046—11051
Edgar RC, 2004. MUSCLE: a multiple sequences aligment method with reduced time and space complexity［J］. BMC Bioinformatics, 5: 1—19
Hall TA, 1999. Bioedit: a userfriendly biological sequences alignment editor and analysis program for Windows 95/98/NT［J］. Nucleic Acids Symposium Series, 41: 95—98
Hebert PDN,Cywinska A,Ball SL et al.,2003a.Biological identifications through DNA barcodes［J］. Proceedings of the Royal Society of London, Series B, Biological Sciences,270:313—321
Hebert PDN,Stoeckle MY,Zemlak TS et al.,2004.Identification of birds through DNA barcodes［J］. PLoS Biology, 2 (10): 1657—1663
Hollingsworth ML, Clark AA, Forrest LL et al., 2009. Selecting barcoding loci for plants: evaluation of seven candidate loci with specieslevel sampling in three divergent groups of land plants［J］. Moleculer Ecology Resources, 9: 439—457
Hollingsworth PM, 2011. Refining the DNA barcode for land plants［J］. Proceedings of the National Academy of Sciences of the United States of America, 108: 19451—19452
Kiss L, 2012. Limits of nuclear ribosomal DNA internal transcribed spacer (ITS) sequences as species barcodes for Fungi［J］. Proceedings of the National Academy of Sciences of the United States of America, 109: E1811
Kress WJ, Erickson DL, 2007. A twolocus global DNA barcode for land plants: the coding rbcL gene complements the noncoding trnHpsbA spacer region［J］. PLoS One, 2: e508
Kress WJ, Wurdack KJ, Zimmer EA et al., 2005. Use of DNA barcodes to identify flowering plants［J］. Proceedings of the National Academy of Sciences of the United States of America, 102: 8369—8374
Lahaye R, van der Bank M, Bogarin D et al., 2008. DNA barcoding the floras of biodiversity hotspots［J］. Proceedings of the National Academy of Sciences of the United States of America, 105: 2923—2928
Li DZ, Gao LM, Li HT et al., 2011. Comparative analysis of a large dataset indicates that internal transcribed spacer (ITS) should be incorporated into the core barcode for seed plants［J］. Proceedings of the National Academy of Sciences of the United States of America, 108: 19641—19646
Li HB (李海波), Wu XQ (吴学谦), WeiHL (魏海龙) et al., 2007. A primary studies on classification and identification of seven Amanita species based on morphological characteristics and ITS sequences of rDNA［J］. Journal of Fungal Research (菌物研究), 5: 14—19, 50
Liu J, Mller M, Gao LM et al., 2011. DNA barcoding for the discrimination of Eurasian yews (Taxus L., Taxaceae) and the discovery of cryptic species［J］. Moleculer Ecology Resouces, 11: 89—100
Liu SY (刘淑艳), Zhang A (张傲), Li Y (李玉), 2012. Principle and procedure of fungal DNA barcoding［J］. Journal of Fungal Research (菌物研究), 10: 205—209
Meyer CP, Paulay G, 2005. DNA barcoding: Error rates based on comprehensive sampling［J］. PLoS Biology, 3: e422
Mouhamadou B, Carriconde F, Gryta H et al., 2008. Molecular evolution of mitochondrial ribosomal DNA in the fungal genus Tricholoma: barcoding implications［J］. Fungal Genetics and Biology, 45: 1219—1226
Newmaster SG, Fazekas AJ, Steeves RAD et al., 2008. Testing candidate plant barcode regions in the Myristicaceae［J］. Moleculer Ecology Resources, 8: 480—490
Rehner S, 2001. Primers for elongation factor 1α (tef1α). http://www.aftol.org/pdfs/EF1primer.pdf.
Rossman A, 2007. Report of the planning workshop for All Fungi DNA Barcoding［J］. Supplement to Mycologia, 58: 1—5
Schoch CL, Seifert KA, Huhndorf S et al., 2012. Nuclear ribosomal internal transcribed spacer (ITS) region as a universal DNA barcode marker for Fungi［J］. Proceedings of the National Academy of Sciences of the United States of America, 109: 6241—6246
Seifert KA, Samson RA, Dewaard JR et al., 2007. Prospects for fungus identification using CO1 DNA barcodes, with Penicillium as a test case［J］. Proceedings of the National Academy of Sciences of the United States of America, 104: 3901—3906
Seifert KA, 2009. Progress towards DNA barcoding of fungi［J］. Molecular Ecology Resources, 9: 83—89
Tamura K, Dudley J, Nei M et al., 2007. MEGA4: Molecular evolutionary genetics analysis (MEGA) software version 4.0. Molecular Biology and Evolution, 24: 1596—1599
Tang MZ (谭铭雄), Li KY (李宽银), Ma L (马林) et al., 2002. Investigation of a case led to 8 persons death by mushroom poisoning［J］. South China Journal of Preventive Medicine (华南预防医学), 28 (2): 51—52
Valentini A, Pompanon F, Taberlet P, 2009. DNA barcoding for ecologists［J］. Trends in Ecology & Evolution, 24: 110—117
Vilgalys R, Hester M, 1990. Rapid genetic identification and mapping of enzymatically amplified ribosomal DNA from several Cryptococcus species［J］. Journal of Bacteriology, 172: 4238—4246
Wang WJ (王文婧), Wang XL (王晓亮), Wang XC (王新存) et al., 2009. Application of DNA barcoding in fungal research［J］. Frontier Science (前沿科学), 3: 4—12
White TJ, Bruns T, Lee S et al., 1990. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenies［A］. In: Innis MA, Gelfand DH, Sninsky JJ, White TJ (eds.), PCR Protocols: A Guide to Methods and Applications［M］. New York: Academic Press, 315—322
Xu JP, 2010. DNA barcoding, fungal diversity, and authentication of wild gourmet mushrooms［J］. Acta Agriculturae Universitatis Jiangxiensis, 32: 1010—1017
Yang ZL (杨祝良), 2005. Flora Fungorum Sinicorum, Vol.27, Amaniataceae［M］. Beijing: Science Press, 1—258
Yang ZL, Li TH, 2001. Notes on three white Amanita of section Phalloideae (Amanitaceae) from China［J］. Mycotaxon, 78: 439—448
Zeng ZQ, Zhao P, Lou J et al., 2012. Selection of a DNA barcode for Nectriaceae from fungal wholegenomes［J］. Science ChinaLife Sciences, 55: 80—88
Zhang P, Chen ZH, Xiao B et al., 2010. Lethal amanitas of East Asia characterized by morphological and molecular data［J］. Fungal Diversity, 42: 119—13
Zhao P, Luo J, Zhuang WY et al., 2011. DNA barcoding of the fungal genus Neonectria and the discovery of two new species［J］. Science ChinaLife Sciences, 54: 664—674

[1]	Hai-Su Hu, Jiu-Yang Mao, Xue Wang, Yu-Ze Liang, Bei Jiang, De-Quan Zhang. Plastid phylogenomics and species discrimination in the “Chinese” clade of Roscoea (Zingiberaceae) [J]. Plant Diversity, 2023, 45(05): 523-534.
[2]	Shi-Yu Lv, Xia-Ying Ye, Zhong-Hu Li, Peng-Fei Ma, De-Zhu Li. Testing complete plastomes and nuclear ribosomal DNA sequences for species identification in a taxonomically difficult bamboo genus Fargesia [J]. Plant Diversity, 2023, 45(02): 147-155.
[3]	Kai-Wen Jiang, Rong Zhang, Zhong-Fu Zhang, Bo Pan, Bin Tian. DNA barcoding and molecular phylogeny of Dumasia (Fabaceae: Phaseoleae) reveals a cryptic lineage [J]. Plant Diversity, 2020, 42(05): 376-385.
[4]	Jiao Qin, Wei Zhang, Shi-Bao Zhang, Ji-Hua Wang. Similar mycorrhizal fungal communities associated with epiphytic and lithophytic orchids of Coelogyne corymbosa [J]. Plant Diversity, 2020, 42(05): 362-369.
[5]	Xiangqin Yu, Chunlei Xiang, Hua Peng. Taxonomy in the Kunming Institute of Botany (KIB): Progress during the past decade (2008-2018) and perspectives on future development [J]. Plant Diversity, 2018, 40(04): 147-157.
[6]	Xue-Zhao Wang, Xiao-Lin Sui, Yan-Yan Liu, Lei Xiang, Ting Zhang, Juan-Juan Fu, Ai-Rong Li, Pei-Zhi Yang. N-P fertilization did not reduce AMF abundance or diversity but alter AMF composition in an alpine grassland infested by a root hemiparasitic plant [J]. Plant Diversity, 2018, 40(03): 117-126.
[7]	Tao Liu, Alex Greenslade, Shengchao Yang. Levels of rhizome endophytic fungi fluctuate in Paris polyphylla var. yunnanensis as plants age [J]. Plant Diversity, 2017, 39(01): 60-64.
[8]	JIANG Dai-Feng-, ZHAO Lu-, GUO Hong-, WANG Hai-Ying. New Lineages of Elaphocordyceps Harboring in Lichen Thalli [J]. Plant Diversity, 2015, 37(06): 746-750.
[9]	HE Wei-Ying-, PAN Yue-Zhi. Study on the DNA Barcoding of Genus Ligularia Cass. (Asteraceae) [J]. Plant Diversity, 2015, 37(06): 693-703.
[10]	WANG Qiu-Xia, YAN Ning, JI Da-Gan, LI Shu-Yun, HU Jiang-Miao, HU Hong. Mycorrhizal Fungi Promote Growth and Nitrogen Utilization by Dendrobium nobile (Orchidaceae) [J]. Plant Diversity, 2014, 36(03): 321-330.
[11]	JIN Xin, Yang-Ming-Zhi, LIU Fei-Hu. Effects of Endophytic Fungi Reinoculation on Physiological and Agronomic Characters of Hemp (Cannabis sativa) [J]. Plant Diversity, 2014, 36(01): 65-69.
[12]	TANG Min-, YI Ting-Shuang-, WANG Xin-, TAN Mei-Hua-, ZHOU Xin. The Application of Metabarcoding Technology in Identification of Plant Species Diversity [J]. Plant Diversity, 2013, 35(6): 769-773.
[13]	LUO Ya-Huang-, LIU Jie-, GAO Lian-Ming-**, LI De-Zhu. Applications and Advances of DNA Barcoding in Ecological Studies [J]. Plant Diversity, 2013, 35(6): 761-768.
[14]	LI Yan-, Wu Ding-, GAO Lian-Ming. Highly Universal DNA Barcoding Primers of ITS2 for Gymnosperms* [J]. Plant Diversity, 2013, 35(6): 751-760.
[15]	ZHANG Yu-Xiao-, HU Yu-Xing-, MA Peng-Fei-, ZHANG Li-Na-, LI De-Zhu. Selection of Potential Plastid DNA Barcodes for Bambusoideae (Poaceae) [J]. Plant Diversity, 2013, 35(6): 743-750.