Length heterogeneity in ITS 2 and the methylation status of CCGG and GCGC sites in the rRNA genes of the genus Peronosclerospora

Abstract

The polymerase chain reaction (PCR) was used with primers complementary to conserved flanking sequences to amplify the internal transcribed spacer 2 (ITS 2) of the rDNA repeat units of five Peronoscleropora isolates, one each of P. sorghi, P. maydis, P. sacchari and two of P. zeae. In contrast to the situation found in most-fungi that have been examined, length heterogeneity was evident in each sample. The rDNA composition of the amplified bands was confirmed by Southern hybridizations using an ITS 2 amplified from P. sorghi and cloned rDNA from Neurospora crassa as probes. Length heterogeneity was also detected in genomic DNA digests using the same probes. In addition to one dominant fragment for each isolate, there were several less frequent fragments of different sizes, and the isolate(s) for each species had a unique banding pattern for ITS 2. The absence of 5-methylcytosine residues in CCGG and GCGC sequences in the ribosomal genes of these four Peronosclerospora species was demonstrated by the production of identical banding patterns with ribosomal DNA probes following digestion of genomic DNA with MspI and HpaII, and by complete digestion with CfoI.