Subtractive Cloning of DNA from Polymyxa graminis- an Obligate Parasitic Plasmodiophorid

Abstract

A simple subtractive hybridization was applied for cloning of Polymyxa graminis deoxyribonucleic acid (DNA). Total DNA preparations from concentrated P. graminis resting spores and from roots of non-infested (healthy) barley were digested with different restriction enzymes and batch hybridized, followed by cloning in a plasmid vector. Sequencing and blast analysis of coincidentally selected clones enabled construction of polymerase chain reaction primers specific to P. graminis DNA. Four Polymyxa-specific primers showed different affinities to DNA of type I and type II P. graminis and to DNA of P. betae.