PCR-SSCP Analysis of the Ribosomal DNA ITS Regions of the Willow Rust Fungi in Japan

Abstract

Single-strand DNA conformation polymorphism (SSCP) analysis using internal transcribed spacer (ITS) regions of ribosomal DNA (rDNA) amplified by polymerase chain reaction (PCR) was performed to detect DNA polymorphism of nine species of Melampsora rust fungi on willows. The rDNA ITS 1 region (approximately 300-320bp in size), was amplified from 49 collections. The SSCP patterns in the amplified ITS 1 region of five species, M. capraearum, M. epiphylla, M. larici-urbaniana, M. microsora and M. yezoensis were species-specific. On the other hand, no distinction was found in the SSCP patterns between M. chelidonii-pierotii and M. coleosporioides, and between M. epitea and M. humilis. In improved PCR-SSCP analysis using restriction fragments of the amplified ITS 1 and ITS 2 regions, no difference was detected between M. chelidonii-pierotii and M. coleosporioides, and between M. epitea and M. humilis. These results suggest the respective two species have a close genetic and taxonomical relationship. We revealed the PCR-SSCP analysis may be beneficial as a tool for distinguishing species of Melampsora because the analysis is simple and sensitive.