6 Wood Discoloration

2.4 Identification 37
RAPD analysis does not require information of the target DNA and is fast
when starting from pure cultures. However, at least four primers should be used
to avoid spurious results, because the short primers imply a great sensitivity to
contamination. In addition, the technique is unsuitable for the identification
of unknown samples by comparison, because other not yet investigated fungi
by chance may share a similar banding pattern.
Use of ribosomal DNA
The investigation of the ribosomal DNA (rDNA) has become popular, because
the rRNA genes and spacers are assumed to evolve cohesively within a single
species, to exhibit only very little sequence divergence between rDNA copies
within single individuals, but to show normal levels of sequence divergence between
species. The repetitive units of the nuclear rDNA of Eukaryotes consists
of the conserved coding domains 18S and 28S rDNA. The conserved domains
are interrupted by the non-coding variable internal transcribed spacer ITS I
(between 18S and 5.8S) and ITS II (between 5.8S and 28S) which are informative
for differentiation. The intergenic spacer IGS is located between the 28S
andthe18SofthenextrDNAunit.Inthecaseofapresent5SrRNAgene,
the IGS consists of two parts, IGS I and IGS II (Fig. 2.20). The conserved
regions are preferentially used for phylogenetic analyses of genera, families,
and orders. The rapidly evolving ITS spacers have become a popular choice
for closely related species and at the subspecies level. After amplification by
PCR, the amplicons are either restricted by endonucleases providing restriction
fragments (RFLPs) which are subsequently separated according to size
using agarose or polyacrylamide gel electrophoresis, or the DNA sequence is
determined (“sequencing”).
In addition to the nuclear rDNA, also mitochondrial rDNA was used for
Basidiomycetes, e.g., by Bao et al. (2005a) in view of phylogenetic relationships
among closely related Pleurotus species.
Restriction fragment length polymorphism (RFLP) of rDNA
RFLP analysis based on the rDNA was also called amplified ribosomal DNA
restriction analysis (ARDRA). Depending on the intension, the RNA genes or
the spacers are used. For RFLP analysis of the ITS, the ITS is first amplified,
often using the “universal primers” ITS 1 and ITS 4 (White et al. 1990), which
anneal to the evolutionary stable 18S and 28S rRNA genes. This attachment
Fig.2.20. Schematic diagram of one rDNA unit. The number in the boxes is the size in base
pairs for Serpula lacrymans (supplemented from Moreth and Schmidt 2005)
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