A Rapid and Universal Direct PCR Method for Macrofungi

Mi-Jeong Park¹ Hyorim Lee¹ Rhim Ryoo^1,* Yeongseon Jang¹ Kang-Hyeon Ka¹

¹Division of Forest Microbiology, National Institute of Forest Science, Suwon 16631, Korea

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.

Figures & Tables

Fig. 1. Effects of lysis conditions. Monokaryotic mycelia of Sanmaru 1ho were lysed with different methods. The resultant lysates were subjected to internal transcribed spacer (ITS) region amplification with conventional PCR (A, C, D) or real-time PCR (B, D, E). The relative quantities of DW, 50℃, and 5 min were set to 1 as reference. The letters, a-c, indicate statistically significant differences based on one-way ANOVA followed by Duncan’s multiple-range test (<0.05). Bars, S.E. (A, B) Effects of lysis solutions. SM, size marker; NC, negative control; PC, positive control; DW, distilled water; TE, Tris/EDTA; NEU, neutralization of KOH with (NH)SO; TE/NEU, neutralization of Tris/EDTA/KOH with (NH)SO. (C, D) Effects of lysis temperatures. (E, F) Effects of heat treatment times.