Efficiency of RAPD and ISSR Markers in Differentiation of Homo- and Heterokaryotic Protoclones of Agaricus bisporus

Abstract

Morphologically, nine different slow-growing protoclones were screened from regenerated protoplast of heterokaryotic Agaricus bisporus. The present study is the first report of fingerprinting on differentiating homo- and heterokaryotic protoclones using random amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) markers. Among 80 primers tested, the seven ISSR and seven RAPD primers selected for the analysis generated a total of 94 ISSR and 52 RAPD fragments, respectively. ISSR fingerprinting detected more polymorphic loci (38.29%) than RAPD fingerprinting (34.61%). Principal coordinate analysis (PCA) was employed to evaluate the resolving power of the markers to differentiate protoclones. The mean polymorphism information content (PIC) for each of these marker systems (i.e., 0.787 for RAPD and 0.916 for ISSR, respectively) suggests that the ISSR marker system was more effective in determining polymorphisms. The dendrograms constructed using RAPD, ISSR, and integrated RAPD, and ISSR marker systems were highly correlated with each other as revealed by the high Mantel correlation (r = 0.98). Pair-wise similarity index values ranged from 0.64 to 0.95 (RAPD), 0.67 to 0.98 (ISSR), and 0.67 to 0.98 (RAPD and ISSR), and mean similarity index values of 0.82, 0.81, and 0.84 for RAPD, ISSR, and combined data, respectively, were obtained. As there was a good correspondence between RAPD and ISSR similarity matrices, ISSR may be used as an alternative to replace RAPD in the genetic diversity assessment and accurate differentiation of homo- and heterokaryotic protoclones of A. bisporus.