Restriction analysis of internal transcribed spacers and the small subunit gene of ribosomal DNA among four Gaeumannomyces species

Abstract

The 18S rDNA and the region spanning the two internal transcribed spacers (ITS), which includes the 5.8S rDNA, from 36 isolates of four species of Gaeumannomyces (G. graminis, G. incrustans, G. cylindrosporous, and G. leptosporous) were amplified using the polymerase chain reaction. The amplified products were subjected to restriction enzyme digestion. The restriction patterns in the ITS regions separated both the species of Gaeumannomyces and the three varieties of G. graminis. Specific variation in restriction patterns of the ITS regions was observed within the isolates of G. g. var. graminis. Physical maps of the restriction sites in the ITS region were constructed. Variation among isolates of G. g. var. tritici and G. g. var. avenae was minimal. The 3′ end and 5′ end of the 18S rDNA are conserved among varieties of G. graminis and isolates of G. incrustans. The middle region of the nuclear 18S rDNA amplified with NS3/NS6 primers showed four different lengths, and based on these data the species and varieties of Gaeumannomyces were arranged into five different groups. Group 1 had a length of 1.6 Kb and included isolates of G. graminis var. tritici, G. graminis var. avenae, and G. graminis var. graminis isolate WF9463; group 2 (1.4 Kb) included G. graminis var. graminis isolates WF9238 and WF9469; group 3 (1.2 Kb) included isolates of G. incrustans and G. graminis var. graminis isolates WF9236, WF9237, and WF9454; group 4(1.0 Kb) included G. graminis var. graminis isolates WF9014, WF921, WF9452, WF9455, WF9457, WF9462, WF9470, and WF9471; and finally group 5 included G. cylindrosporous and G. leptosporous with a 1.1 Kb length.

Key words:

• Ascomycota
• Magnaporthaceae
• PCR
• phylogeny
• rDNA