1997 Volume 27 Issue 2 Pages 67-75
The polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis of the two internal transcribed spacer regions (ITS 1 and ITS 2), including the 5.8S gene and small portions of the 18S and 28S genes of the ribosomal DNA, was performed to distinguish Globodera rostochiensis, Heterodera elachista, H. trifolii, H. glycines and Heterodera sp. The single second-stage juveniles of each species were ruptured individually in a drop of lysis buffer (DNA extraction buffer), heat-treated, and added directly to a PCR reaction mixture. The size of amplified product was about 1.0 kb in G. rostochiensis, and about 1.1 kb in H. elachista, and about 1.05 kb in H. trifolii, H. glycines and Heterodera sp. The two restriction patterns of the amplified products with Rsa I and with one of Alu I, Mse I and Tha I were suitable for discrimination of these five species. No intraspecific variation in the two restriction patterns with AluI and RsaI was found in 17 isolates of H. elachista and H. glycines from Kanto district of Japan.