The nuclear genomes of fungi have a number of particular features. They are relatively small (approximately 13-93 million nucleotide base pairs), and in comparison to higher plants and animals they have a much lower percentage of redundant DNA (about 10-20%) (Lu 1996). Around 30% of the entire fungal genome consists of duplicated regions and genes (Mewes 1997). These repetitive sequences provide potential targets for molecular markers due to their high copy number.
Ribosomal DNA (rDNA), specifically the regions coding for the rRNA subunits and their associated spacers, is one of the most commonly used DNA regions for fungal molecular markers [see Bridge and Arora 1998; Brans et al. 1991; www.mendel.berkeley.edu/boletus/boletus.html; www. biology.duke.edu/fungi/]. The nuclear-encoded rRNA genes (rDNA) and spacers occur as a gene cluster (typically of 8-12kb) that is multiply repeated (see Figure 1). The basic unit consists of the genes for the small ribosomal subunit, the 5.8S subunit, and the large subunit. The three genes are separated by two internally transcribed spacers (ITS), and the repeated gene clusters are separated by an intergenic spacer (IGS) that in many, but not all fungi, also contains the gene for the 5S subunit. [see Hillis and Dixon (1991)]. Several restrictions sites are conserved in fungal rDNA, and this makes them convenient sequences for cloning [see other reviews by Gargas and DePriest (1996); Hibbett (1992)]. The rRNA cluster has proved to be a good region for deriving molecular markers for many fungi [see Brans et al. (1991); Hibbett (1992)]. The subunit genes have both conserved and variable domains, and can be used for comparisons of genera, the spacer regions are considerably more variable and can be used for comparisons of species or in some cases specific pathogenic forms (see Table 1). As the cluster is universal and multiply repeatedly, it is a good target for molecular studies. Originally these studies involved obtaining RFLPs with probes hybridized to total genomic DNA digests, and many
Ribosomal RNA gene cluster
Intergenlc ¿pacer (iGS) region^--
Small subunit 5.83 Large subunit gene subunit gene
Multiply repeated rRNA gene clusters (5S gene not shown)
studies of populations and species of plant pathogens were carried out in this way [e.g., Jabaji-Hare et al. (1990), Manicom et al. (1990)]. More recently these studies have largely been replaced by polymerase chain reaction (PCR) based studies, particularly as the rRNA cluster can often be detected in old or contaminated environmental samples [see Brans et al. (1990)]. The varying levels of specificity of the different DNA regions in the gene cluster also mean that it is possible to amplify fungal DNA directly from samples of infected plant material (Bridge et al. 2000; Gardes and Brans 1993).
Both the ITS and the IGS regions have been used to develop species-specific primers for plant pathogen detection in plant material [e.g., Brown et al. (1993); Moukhamedov et al. (1993)]. It is becoming increasingly common in rRNA cluster studies to obtain sequences of all the regions of interest. Although knowledge of the complete sequences provides a large amount of information, useful information may be obtained from simple restriction digestions of rRNA amplification products. This approach generally produces relatively simple patterns containing 1-4 bands, and in certain cases these patterns, or individual bands, may be specific for particular pathogens [e.g., Chen (1992)]. It is not possible to list all the work done using ITS and IGS regions to develop molecular markers. One example of this was the study of Mazzola et al. (1996) who developed an oligonucleotide primer set that consistently and selectively amplified a 511 bp fragment in the ITS region that could be used to differentiate between Rhizoctonia solani and R. oryzae. It should, however, be remembered that RFLP analysis is essentially a one-tailed analysis of variation; and although different patterns indicate that two organisms are different, a common pattern is based on only the position of a few restriction sites. Therefore, identical RFLP band patterns do not imply that the rest of the sequence is identical.
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