Identification of RAPD molecular polymorphism and cloning of polymorphic bands in potato late blight (Phytophthora infestans)

摘要

Seven accessions of the potato late blight (PLB) fungus Phytophthora infestans, derived from different potato varieties and different Romanian locations, were isolated and multiplied in vitro on selective Zea medium with Vancocine (200 ppm) and Nystatine (100 ppm) antibiotics. The extracted DNA was amplified with several decamer primers. The amplification products were separated by electrophoresis in 1.4 % agarose gel and revealed in UV. Relying on molecular polymorphism characterization, genetic distances and phenetic relationship among the seven potato late blight accessions were established. It seems that the seven accessions belong to three major genotypes, with higher or lower levels of differentiation within them, according to location and potato variety. Thus, there are the A genotype, with two identical accessions isolated from one variety of potato and two different locations; the B and C genotypes, with three and two relatively different accessions isolated from different potato varietiesbut from relatively close locations. This differentiation could be a sign of an evolution process. DNA from polymorphic bands was purified using Gene Elute Agarose Spin Columns (Sigma 5-6500) and cloned by pGEM T Easy Vector Sistem II (Promega A 1380).The genetic transformation of host bacteria (JM 109 E. coli) with polymorphic bands integrated in pGEM T vector was confirmed by blue/white reaction. Thus, there were nine cloned polymorphic bands obtained from six PLB accessions with one primer; two polymorphic bands were obtained from two PLB accessions with other two primers and one polymorphic band from one PLB accession with another primer. Some of the cloned polymorphic bands were specific for a certain genotype and are to be sequenced in order toconvert the RAPD markers into more stable SCAR markers.