Possible consequences of fungal contamination on the RAPD fingerprinting in Miscanthus (Poaceae)

Abstract

Fungal contamination has been frequently reported in higher plants. In Miscanthus species, a wide range of fungal flora has also been recorded previously, including an investigation based on nrITS amplification. In order to understand the effects of the fungal genomes on the random amplified polymorphic DNA (RAPD) fingerprinting, callus specimens were obtained from the tissue culture of shoot apices of Miscanthus. RAPD fingerprinting with 60 oligoprimers was conducted with genomic DNA extracted from leaf tissue collected in the field and from the greenhouse, as well as callus derived from the same individuals. Extra bands were detected in the RAPD fingerprints amplified with 44 primers (84.6%) from the genomic DNA of both the field and greenhouse leaf tissue of most Miscanthus taxa examined, except for M. sinensis var. condensatus. Positive PCR amplification of organelle DNA non-coding spacers with both leaf and callus DNA ruled out the possibility that such DNA fingerprinting discrepancies were due to loss of organelles in the callus after consecutive subcultures. Among the 44 primers, one yielded no amplified fragments from the callus DNA, indicating that the amplified DNA fragments from leaf-tissue DNA were likely to be derived from fungi. The contaminating fungal DNA not only caused the overestimation of genetic diversity in the host plants, but also interfered with the phylogenetic inference. Systematic inconsistency occurred between the UPGMA dendrograms of leaf and callus DNA fingerprints. The detection of contaminating fungal DNA suggested that precautions are required for PCR-based fingerprinting when field materials are used for DNA resources. A method for quick screening of the contaminating fungal DNA with universal primers for the nrITS (internal transcribed spacer) region is suggested.