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VARIATION OF RAMS MARKERS WITHIN THE INTERSTERILITY GROUPS OFHETEROBASIDION ANNOSUM IN EUROPE.
VARIATION OF RAMS MARKERS WITHIN THE INTERSTERILITY GROUPS OF HETEROBASIDION ANNOSUM IN EUROPE. EEVA J. VAINIO & JARKKO HANTULA Forest Research Institute, P.O. Box 18, FIN-01301, Vantaa, Finland. Background and objectives Heterobasidion annosum is a pathogenic basidiomycete causing root and butt rot primarily in conifers of northern temperate and boreal forests. In Europe this pathogen has been divided into three intersterility groups (IS groups) according to host tree: S (spruce), P (pine) and F (fir). The degree of genetic variation and geographical differentiation among the intersterility groups have been addressed by several techniques, but due to variable results the situation remains unclear. Random amplified microsatellite (RAMS) technique combines several characteristics of RAPD and microsatellite analysis and is applicable for studies of intraspecific variation in fungi. RAMS markers were used to test two hypotheses based on results of previous studies on the population structure of H. annosum: (i) the degree of genetic variation is higher within IS groups S and F than within IS group P, and (ii) genetic differentiation is more pronounced within IS group S than within IS group P. Materials and methods The 67 homokaryotic single spore isolates belonging to IS groups S, P or F originated from three separate regions: (i) Finland, (ii) Lithuania and Byelorussia, and (iii) Italy, Switzerland, Germany and France. Total DNA of the fungal isolates was isolated from mycelia cultivated on PTS-agar plates. Three RAMS primers (CGA, CCA and CT) were used for the amplification of fungal DNA. Amplification products were analyzed by agarose gel electrophoresis and photographic prints were used for scoring of the amplification products. The band sharing indices between isolates was calculated and used to construct Neighbor Joining (NJ) dendrograms for the three IS groups. Analysis of genetic diversity within IS groups was carried out using AMOVA software. Results and conclusions The analysis of 77 markers produced by three RAMS primers revealed banding patterns with several markers unique to intersterility groups S, P and F. In total 65 different haplotypes (marker combinations) were observed among the 67 isolates studied. The highest number of polymorphic markers and the lowest number of fixed markers was observed within IS group P. This suggests a higher level of genetic variation among IS group P than IS groups S and F. Thus the first hypothesis was not supported. The AMOVA analysis indicated that only 7% and 6% of the diversity observed within IS groups S and P, respectively, resulted from variation among different populations (geographical regions). The overall differentiation between the populations was low also according to the NJ clustering analysis. Therefore, neither the second hypothesis was supported as the level of gene flow within IS groups S and P seemed to be similar and relatively high.