USE OF RFLP AND SSCP ANALYSIS TO DIFFERENTIATE THE PINE RUSTS CRONARTIUM-FLACCIDUM AND PERIDERMIUM-PINI

Two rapid, independent molecular assays have been developed for discri minating between the closely related rusts of hard pines, Cronartium f laccidum and Peridermium pini. A portion of the ITS region and a porti on of the IGS region from the ribosomal RNA operon of the two organism s were amplified with the PCR. Amplifications were made using DNA extr acted from aeciospores collected from unopened aecia, taking a small n umber of spores as a source of template DNA. Amplified fragments were subjected to RFLP or to SSCP analysis. Digests of the amplified produc ts from the IGS1 region were electrophoresed on polyacrylamide gel and stained with ethidium bromide. Hinf I digestion of these fragments cr eated polymorphic restriction profiles which allowed differentiation o f the autoecious P. pini from the heteroecious C. flaccidum. Similar-s ized DNA fragments representing the ITS2 region of the two rusts were denatured, subjected to electrophoresis as single strands on polyacryl amide gel under non-denaturing conditions and 'silver stained'. The di fferent mobility displayed by these short fragments revealed sequence polymorphism in the examined portion of the ITS region. This technique therefore represents an accurate and sensitive method for detection o f base changes in given sequences of genomic DNA. A high level of homo logy was found between the two biotroph organisms in the loci screened . Results obtained in this trial indicate that PCR-RFLP and PCR-SSCP c an be used as simple, speedy taxonomic tools for elucidating relations hips among related organisms.