The Korean Journal of Mycology (한국균학회지)

The Korean Society of Mycology (한국균학회)
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A Rapid and Universal Direct PCR Method for Macrofungi

  • Park, Mi-Jeong (Division of Forest Microbiology, National Institute of Forest Science) ;
  • Lee, Hyorim (Division of Forest Microbiology, National Institute of Forest Science) ;
  • Ryoo, Rhim (Division of Forest Microbiology, National Institute of Forest Science) ;
  • Jang, Yeongseon (Division of Forest Microbiology, National Institute of Forest Science) ;
  • Ka, Kang-Hyeon (Division of Forest Microbiology, National Institute of Forest Science)
  • Received : 2021.08.12
  • Accepted : 2021.11.10
  • Published : 2021.12.31
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Abstract

Macrofungi are valuable resources as novel drug candidates, new biomaterials, and edible materials. Recently, genetic approaches pertaining to macrofungi have been continuously growing for their identification, molecular breeding, and genetic engineering. However, purification and amplification of fungal DNA is challenging because of the rigid cell wall and presence of PCR inhibitory metabolites. Here, we established a direct PCR method to provide a rapid and efficient method for PCR-grade macrofungal DNA preparation applicable to both conventional PCR and real-time PCR. We first optimized the procedure of lysis and PCR using the mycelia of Lentinula edodes, one of the most widely consumed macrofungal species. Lysates prepared by neutralizing with (NH4)2SO4 after heating the mycelia in a mixture of TE buffer and KOH at 65℃ for 10 min showed successful amplification in both conventional and real-time PCR. Moreover, the addition of bovine serum albumin to the PCR mixture enhanced the amplification in conventional PCR. Using this method, we successfully amplified not only internal transcribed spacer fragments but also low-copy genes ranging in length from 500 to 3,000 bp. Next, we applied this method to 62 different species (54 genera) of macrofungi, including edible mushrooms, such as Pleurotus ostreatus, and medicinal mushrooms such as Cordyceps militaris. It was found that our method is widely applicable to both ascomycetes and basidiomycetes. We expect that our method will contribute to accelerating PCR-based approaches, such as molecular identification, DNA marker typing, gene cloning, and transformant screening, in macrofungal studies.

Keywords

Acknowledgement

This work was supported by the Golden Seed Project of 'Breeding of new strains of shiitake for cultivar protection and substitution of import [213007-05-5-SBH10]' provided by the Ministry of Agriculture, Food and Rural Affairs, Ministry of Oceans and Fisheries, Rural Development Administration and Korea Forest Service.

References

  1. Lundell TK, Makela MR, Hilden K. Lignin-modifying enzymes in filamentous basidiomycetes-ecological, functional and phylogenetic review. J Basic Microbiol 2010;50:5-20. https://doi.org/10.1002/jobm.200900338
  2. Zotti M, Persiani AM, Ambrosio E, Vizzini A, Venturella G, Donnini D, Angelini P, Piazza SD, Pavarino M, Lunghini D, et al. Macrofungi as ecosystem resources: conservation versus exploitation. Plant Biosyst 2013;147:219-25. https://doi.org/10.1080/11263504.2012.753133
  3. Hyde KD, Xu J, Rapior S, Jeewon R, Lumyong S, Niego AGT, Abeywickrama PD, Aluthmuhandiram JVS, Brahamanage RS, Brooks S, et al. The amazing potential of fungi: 50 ways we can exploit fungi industrially. Fungal Divers 2019;97:1-136. https://doi.org/10.1007/s13225-019-00430-9
  4. Sun L, Zhang Z, Xin G, Sun B, Bao X, Wei Y, Zhao X, Xu H. Advances in umami taste and aroma of edible mushrooms. Trends Food Sci Tech 2020;96:176-87. https://doi.org/10.1016/j.tifs.2019.12.018
  5. Reis FS, Martins A, Vasconcelos MH, Morales P, Ferreira. ICFR. Functional foods based on extracts or compounds derived from mushrooms. Trends Food Sci Tech 2017;66:48-62. https://doi.org/10.1016/j.tifs.2017.05.010
  6. Lu H, Lou H, Hu J, Liu Z, Chen Q. Macrofungi: a review of cultivation strategies, bioactivity, and application of mushrooms. Compr Rev Food Sci Food Saf 2020;19:2333-56. https://doi.org/10.1111/1541-4337.12602
  7. Kozarski M, Klaus A, Jakovljevic D, Todorovic N, Vunduk J, Petrovic P, Niksic M, Vrvic MM, van Griensven L. Antioxidants of edible mushrooms. Molecules 2015;20:19489-525. https://doi.org/10.3390/molecules201019489
  8. Khonde PL, Jardine A. Improved synthesis of the super antioxidant, ergothioneine, and its biosynthetic pathway intermediates. Org Biomol Chem 2015;13:1415-9. https://doi.org/10.1039/C4OB02023E
  9. Kalaras MD, Richie JP, Calcagnotto A, Beelman RB. Mushrooms: a rich source of the antioxidants ergothioneine and glutathione. Food Chem 2017;233:429-33. https://doi.org/10.1016/j.foodchem.2017.04.109
  10. Yoon SY, Park SJ, Park YJ. The anticancer properties of cordycepin and their underlying mechanisms. Int J Mol Sci 2018;19:3027. https://doi.org/10.3390/ijms19103027
  11. Qin P, Li X, Yang H, Wang ZY, Lu D. Therapeutic potential and biological applications of cordycepin and metabolic mechanisms in cordycepin-producing fungi. Molecules 2019;24:2231. https://doi.org/10.3390/molecules24122231
  12. Lee S, Lee D, Lee JC, Kang KS, Ryoo R, Park HJ, Kim KH. Bioactivity-guided isolation of anti-inflammatory constituents of the rare mushroom Calvatia nipponica in LPS-stimulated RAW264.7 macrophages. Chem Biodivers 2018;15:e1800203.
  13. Lee SR, Seok S, Ryoo R, Choi SU, Kim KH. Macrocyclic trichothecene mycotoxins from a deadly poisonous mushroom, Podostroma cornu-damae. J Nat Prod 2019;82:122-8. https://doi.org/10.1021/acs.jnatprod.8b00823
  14. Yang SO, Sodaneath H, Lee JI, Jung H, Choi JH, Ryu HW, Cho KS. Decolorization of acid, disperse and reactive dyes by Trametes versicolor CBR43. J Environ Sci Health A Tox Hazard Subst Environ Eng 2017;52:862-72. https://doi.org/10.1080/10934529.2017.1316164
  15. da Luz JM, Paes SA, Nunes MD, da Silva MC, Kasuya MC. Degradation of oxobiodegradable plastic by Pleurotus ostreatus. PLoS One 2013;8:e69386. https://doi.org/10.1371/journal.pone.0069386
  16. Gomez-Mendez LD, Moreno-Bayona DA, Poutou-Pinales RA, Salcedo-Reyes JC, PedrozaRodriguez AM, Vargas A, Bogoya JM. Biodeterioration of plasma pretreated LDPE sheets by Pleurotus ostreatus. PLoS One 2018;13:e0203786. https://doi.org/10.1371/journal.pone.0203786
  17. Schoch CL, Seifert KA, Huhndorf S, Robert V, Spouge JL, Levesque CA, Chen W. Nuclear ribosomal internal transcribed spacer (ITS) region as a universal DNA barcode marker for fungi. Proc Natl Acad Sci USA 2012;109:6241-6. https://doi.org/10.1073/pnas.1117018109
  18. Xu, J. Fungal DNA barcoding. Genome 2016;59:913-32. https://doi.org/10.1139/gen-2016-0046
  19. Ben-Amar A, Oueslati S, Mliki A. Universal direct PCR amplification system: a time- and cost-effective tool for high-throughput applications. 3 Biotech 2017;7:246. https://doi.org/10.1007/s13205-017-0890-7
  20. Chen Y, Bi C, Tong S, Gong Z, Hou H. An improved and reliable method for microalgae direct PCR. J Appl Phycol 2019;31:2411-21. https://doi.org/10.1007/s10811-019-01768-y
  21. Li Y, Zhao H, Yan X, Li M, Chen P, Zhang S. A universal method for direct PCR amplification of plant tissues. Anal Methods 2017;9:1800-5. https://doi.org/10.1039/C6AY03156K
  22. Choudhary P, Das S, Chakdar H, Singh A, Goswami SK, Saxena AK. Rapid high throughput template preparation (rHTTP) method: a novel cost effective method of direct PCR for a wide range of plants. BMC Biotechnol 2019;19:69. https://doi.org/10.1186/s12896-019-0560-4
  23. Iotti M, Zambonelli A. A quick and precise technique for identifying ectomycorrhizas by PCR. Mycol Res 2006;110:60-5. https://doi.org/10.1016/j.mycres.2005.09.010
  24. Walch G, Knapp M, Rainer G, Peintner U. Colony-PCR is a rapid method for DNA amplification of hyphomycetes. J Fungi 2016;2:12. https://doi.org/10.3390/jof2020012
  25. Bonito G. Fast DNA-based identification of the black truffle Tuber melanosporum with direct PCR and species-specific primers. FEMS Microbiol Lett 2009;301:171-5. https://doi.org/10.1111/j.1574-6968.2009.01812.x
  26. Izumitsu K, Hatoh K, Sumita T, Kitade Y, Morita A, Tanaka C, Gafur A, Ohta A, Kawai M, Yamanaka T, et al. Rapid and simple preparation of mushroom DNA directly from colonies and fruiting bodies for PCR. Mycoscience 2012;53:396-401. https://doi.org/10.1007/s10267-012-0182-3
  27. Wang Y, Xu D, Liu D, Sun X, Chen Y, Zheng L, Chen L, Ma A. A rapid and effective colony PCR procedure for screening transformants in several common mushrooms. Mycobiology 2019;47:350-4. https://doi.org/10.1080/12298093.2019.1628523
  28. Kreader CA. Relief of amplification inhibition in PCR with bovine serum albumin or T4 gene 32 protein. Appl Environ Microbiol 1996;62:1102-6. https://doi.org/10.1128/aem.62.3.1102-1106.1996
  29. Innis MA, Myambo KB, Gelfand DH, Brow MA. DNA sequencing with Thermus aquaticus DNA polymerase and direct sequencing of polymerase chain reaction-amplified DNA. Proc Natl Acad Sci USA 1988;85:9436-40. https://doi.org/10.1073/pnas.85.24.9436
  30. Nagai M, Yoshida A, Sato N. Additive effects of bovine serum albumin, dithiothreitol, and glycerol on PCR. IUBMB Life 1998;44:157-63. https://doi.org/10.1080/15216549800201172
  31. Musso M, Bocciardi R, Parodi S, Ravazzolo R, Ceccherini I. Betaine, dimethyl sulfoxide, and 7-deaza-dGTP, a powerful mixture for amplification of GC-rich DNA sequences. J Mol Diagn 2006;8:544-50. https://doi.org/10.2353/jmoldx.2006.060058
  32. Rees WA, Yager TD, Korte J, von Hippel PH. Betaine can eliminate the base pair composition dependence of DNA melting. Biochemistry 1993;32:137-44. https://doi.org/10.1021/bi00052a019
  33. Bachmann B, Luke W, Hunsmann G. Improvement of PCR amplified DNA sequencing with the aid of detergents. Nucleic Acids Res 1990;18:1309. https://doi.org/10.1093/nar/18.5.1309
  34. Ha B, Moon YJ, Song Y, Kim S, Kim M, Yoon CW, Ro HS. Molecular analysis of B mating type diversity in Lentinula edodes. Sci Hortic 2019;243:55-63. https://doi.org/10.1016/j.scienta.2018.08.009
  35. Au CH, Wong MC, Bao D, Zhang M, Song C, Song W, Law PTW, Kues U, Kwan HS. The genetic structure of the A mating-type locus of Lentinula edodes. Gene 2014;535:184-90. https://doi.org/10.1016/j.gene.2013.11.036
  36. Wong KS, Cheung MK, Au CH, Kwan HS. A novel Lentinula edodes laccase and its comparative enzymology suggest guaiacol-based laccase engineering for bioremediation. PLoS One 2013;8:e66426. https://doi.org/10.1371/journal.pone.0066426
  37. Sano H, Narikiyo T, Kaneko S, Yamazaki T, Shishido K. Sequence analysis and expression of a blue-light photoreceptor gene, Le.phrA from the basidiomycetous mushroom Lentinula edodes. Biosci Biotechnol Biochem 2007;71:2206-13. https://doi.org/10.1271/bbb.70170
  38. Carbonell LM, Kanetsuna F, Gil F. Chemical morphology of glucan and chitin in the cell wall of the yeast phase of Paracoccidioides brasiliensis. J Bacteriol 1970;101:636-42. https://doi.org/10.1128/jb.101.2.636-642.1970
  39. Ehren HL, Appels FVW, Houben K, Renault MAM, Wosten HAB, Baldus M. Characterization of the cell wall of a mushroom forming fungus at atomic resolution using solid-state NMR spectroscopy. Cell Surf 2020;6:100046. https://doi.org/10.1016/j.tcsw.2020.100046
  40. Olive DM, Simsek M, Al-Mufti S. Polymerase chain reaction assay for detection of human cytomegalovirus. J Clin Microbiol 1989;27:1238-42. https://doi.org/10.1128/jcm.27.6.1238-1242.1989
  41. Watanabe M, Abe K, Aoki M, Kameya T, Itoyama Y, Shoji M, Ikeda M, Iizuka T, Hirai S. A reproducible assay of polymerase chain reaction to detect trinucleotide repeat expansion of Huntington's disease and senile chorea. Neurol Res 1996;18:16-8. https://doi.org/10.1080/01616412.1996.11740370
  42. Ralser M, Querfurth R, Warnatz HJ, Lehrach H, Yaspo ML, Krobitsch S. An efficient and economic enhancer mix for PCR. Biochem Biophys Res Commun 2006;347:747-51. https://doi.org/10.1016/j.bbrc.2006.06.151
  43. Li FW, Kuo LY, Huang YM, Chiou WL, Wang CN. Tissue-direct PCR, a rapid and extraction-free method for barcoding of ferns. Mol Ecol Resour 2010;10:92-5. https://doi.org/10.1111/j.1755-0998.2009.02745.x
  44. Mok E, Wee E, Wang Y, Trau M. Comprehensive evaluation of molecular enhancers of the isothermal exponential amplification reaction. Sci Rep 2016;6:37837. https://doi.org/10.1038/srep37837
  45. Wang Y, Prosen DE, Mei L, Sullivan JC, Finney M, Vander Horn PB. A novel strategy to engineer DNA polymerases for enhanced processivity and improved performance in vitro. Nucleic Acids Res 2004;32:1197-1207. https://doi.org/10.1093/nar/gkh271