【A,New,Protocol,for,Extracting,DNA,from,Agaricus,bisporus,Fruit,Bodies,without,Thermal,Incubation】 for from

　　College of Food Science, South China Agricultural University, Guangzhou, Guangdong 510642, China
　　A new, simple and rapid method for obtaining DNA from Agaricus bisporus fruit bodies is described and compared with the widely used CTAB and SDS methods in terms of yield, DNA quality and integrity, and duration of extraction time. Total DNA quality was estimated by restriction endonuclease digestion and by ITS, ISSR and RAPD analyses using standard protocols. Our data demonstrated that the new method renders improved yields of total DNA of high quality and integrity, and is less time-consuming than existing procedures.
　　Agaricus bisporus; DNA isolation; restriction endonuclease digestion
　　Agaricus bisporus, the white button mushroom, contains high levels of dietary fiber and antioxidants, including vitamins C, D and B 12 , folates and polyphenols, that are generally recognized as beneficial in preventing cardiovascular disease and diabetes［1-3］. A. bisporus is cultivated worldwide, and extensive research has been undertaken on the molecular biology of this important mushroom. This requires a simple and effective method for extracting genomic DNA and, although different methods are available, and the quality of DNA obtained is generally satisfactory, these procedures are time consuming and tedious. Extended incubation of fungal material in hot water (65 ℃)［4,5］ makes a method unsuitable for analyzing large number of samples and, furthermore, toxic chemicals such as phenol and chloroform are required. Loss and fragmentation of DNA can easily occur during these stages, which is particularly undesirable when attempting to isolate DNA from small amounts of starting material.
　　A sufficient quantity of DNA containing low levels of protein and other impurities is necessary for PCR amplification. When extracting DNA from mushroom tissue (fruit bodies, mycelium), efficient disruption of the cell walls is important to ensure a high recovery rate. Several methods have been employed for disrupting cell walls including intensive shaking with glass beads［6］, grinding with a mortar and pestle, mechanical homogenization［7,8］, sonication, enzymic digestion, the French press［9,10］, and microwave irradiation［5,11］. Although shaking with glass beads is widely used, it is not very effective for extracting DNA from mushroom fruit bodies. Freezing fruit bodies with liquid nitrogen is versatile but leads to severe DNA fragmentation, while enzymic digestion is relatively costly. Moreover, isolation of DNA from fungal mycelium by incubating at 65 ℃ in buffers containing either 3% SDS or 2% CTAB requires several steps, toxic chemicals, and is time consuming. Since there are disadvantages associated with all the available methods, we have developed a new protocol for isolating DNA from A. bisporus fruit bodies, and compared it with other procedures in terms of cost, rapidity, and the quality and quantity of the DNA obtained.