DNA analysis for differentiation of the shitake mushroom isolates of Lentinus edodes (Berk.) sing

Abstract

Studies on differentiation of the 8 shitake mushroom isolates of Lentinus edodes (Berk.) Sing strains Mul 2, Mul 4, Mul5, Mul9/2 , Mul11 and Mul12 obtained from mushroom Research Unit, Chulalongkorn University , and a Japanee cultivar were carried out. First the development of cultivation system was established to obtain mycella used for DNA preparation. The experiment was conducted in Split Plot Design with 4 repllcations. The development of a simple and rapid DNA extraction method suitable for DNA analysis used in combination with the appllcation of the RAPD analysis. Results revealed that among 12 media conditions, the liquld culture formulation 11 provided better mycelia growth to L. edodes within a short period of time than those of other formulations significantly. This formulation was used to obtain suitable mycella for DNA preparation and RAPD analysis in further step. Several DNA extraction methods were performed and DNA quality and quantity were compared. Results showed that the CTAB method, the NaOH method, the Chelex method did not give good results. However, standard method, SDS method, and Glass bead method provided better quality and yield of DNA. Thus these later three methods were appropriate for simultaneous assessing of several samples in a single working day due to their less operating time, The resulting DNA could be a template for PCR amplification and differentiation with RAPD analysis using random primers. Only 4 primers were applicable for RAPD analysis giving DNA profile scorable in form of matrix file and analyzable with jaccard’s simllarity coefficient and UPGMA method. The RAPD analysis classified the relationship of L. edodes isolates into two groups. First group was cluster A having members of subcluster A1 Mul4, Mul9/2, Mul9/4: subcluster A2 Mul 2 and japan: subcluster A3 Mul11 and subcluster A4 Mul5 and second group is a cluster B having a member of Mul12.The combination of DNA extraction methods and RAPD analysis that enabled the prediction of the differentiation of L. edodes and subsequent phylogenetic relationship (can be predicted) within 4 days indicated its simpllcity, rapidness and its non-labor-intensiveness. The method not only provided basic information for the improvement program of this mushroom but also for other mushroom isolate differentiation.