تنوع ژنتیکی جدایه های Rhizoctonia solani عامل شانکر ساقه و شوره سیاه سیب زمینی با استفاده از مارکرهای RAPD و ISSR

Potato, Solanum tuberosum L. is an important crop product which is grown commercially throughout the world. This product is also important in Iran. One of the most important diseases of potato is the stem canker and black scurf by a fungus Rhizoctonia solani Kühn, with a worldwide distribution. In this study, 30 isolates of R. solani were collected from the main potato-growing provinces of Iran, including Isfahan, Ardebil, Fars, Hamedan, Kurdestan and Kerman. The isolates were analyzed based on sequence analyses of random amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR). Total genomic DNA was extracted from all isolates using the CTAB method. Ten RAPD primers, including OPA-03, OPB-17, OPC-08, OPC-9, OPP-16, OPP-17, OPP-18, OPP-19, OPX-14 and OPF-10 were used for genetic analysis of polymorphisms among the 30 R. solani isolates. For the ISSR analysis, nine primers EZ1, EZ2, EZ7, EZ9, EZ11, EZ13, EZ18, EZ25, and EZ27 were used. RAPD analysis was performed in three replications to confirm the consistency of amplification and only repeatable bands were scored. Monomorphic and polymorphic bands were considered as binary characters and were scored as 1 for presence and 0 for absence of DNA bands. The scores were then entered into a matrix for analysis by numerical taxonomy and multivariate analysis system, NTSYS-pc 1.8 program. The similarity matrix was calculated using Jaccard’s similarity coefficient. Clustering was performed using the unweighted pair group method using arithmetic averages (UPGMA) to generate the dendrogram. A total of 116 consistently amplified DNA bands were generated from ten RAPD primers, of which 87.93% were polymorphic. The mean number of bands per primer was 11.6, which ranged in size from approximately 100 to 3,000 bp. The dendrogram produced from UPGMA analysis based on Jaccard's coefficient revealed that the variability was relatively high among the 30 R. solani isolates, and the similarity index ranged from 100 to 52%. This result also indicated that the isolates obtained from the main potato growing regions of Iran, (Ardebil, Hamadan, Fars, Isfahan, Kerman and Kurdestan provinces) were clustered into three main groups at a similarity index of 64%. Application of nine ISSR primers generated a total of 44 consistently amplified fragments (100-3,000 bp), of which 42 (95.45 %) fragments were polymorphic. The mean number of bands per primer was 4.89. The dendrogram showed that variability was relatively high among the isolates, and the similarity index ranged from 100 to 60. The result indicated that 30 R. solani isolates were also clustered into three main groups at similarity index of 78%. Cluster analysis using un-weighted pair group method and arithmetic averages (UPGMA) method for both RAPD and ISSR markers divided into three main groups, with no correlation to geographical regions of the isolates.