For the purposes of this chapter DNA fingerprinting methods will be limited to those that have been used with plant
Ribosomal RNA gene cluster
Small subunit 5.83 Large subunit gene subunit gene
Table 1 Features of commonly used molecular markers
Taxonomic level resolved
Affected by meiosisa
Simple repetitive PCR sequences; micro- and mini-satellite probes and primers AFLPs mtDNA RFLPs
ITS/IGS region RFLPs
ITS region sequencing rRNA gene sequences
Major structural/functional protein genes
Individuals, subspecific groups Individuals, subspecific groups
Individuals, subspecific groups, some closely related species
Subspecific groups, closely related species Closely related species, some subspecific groups Some subspecific groups, closely related species Species, genera, families, phyla Species, genera, families, phyla
Not generallyb Not generally No No a For many fungi the effects of meiosis on markers have not been specifically considered. Yes and no entries refer to general assumptions. b One reason for the selection of rRNA gene cluster was that it was resistant to crossover. However, there is at least one report of presumed recombination in the rDNA of fungi (see text).
pathogenic fungi to generate largely random PCR fragments from the total genome. These techniques have also been referred to as total genome profiling. One of the oldest and most widely used of such PCR methods is random amplified polymorphic DNA (RAPD) analysis (Welsh and McClelland 1990; Williams et al. 1990). Essentially RAPD analysis relies on the reduction in specificity of the PCR process at reduced temperatures. Total genome DNA extractions are used, and these are amplified with single, short (usually decamer) primers at a reduced annealing temperature. These conditions result in less stringent binding of the primer to the target DNA, and allow the amplification of a number of generally small regions of DNA. These are separated by electrophoresis to give a profile of bands. The RAPD analysis has been criticized as it is not always entirely reproducible, but it has been used extensively for profiling populations of plant pathogens [e.g., Bentley et al. (1995); Cooke et al. (1996)]. In many cases these studies have shown correlations between band patterns and host, disease type or geographical origin, and band patterns have also been used to differentiate between forms of the same fungus causing different disease symptoms [e.g., Pei et al. (1997)]. Another common PCR fingerprinting method that has been used for plant pathogenic fungi is amplification of sequences based on simple repetitive primers. In this method, single short repetitive primers are used at moderate annealing temperatures in order to amplify largely repetitive fragments of the genome. One target site for this method is the flanking region of genes that can contain variable numbers of such repeats. Repeat motifs that have been used for primers have varied from simple 2-3 base repeats such as (CA)8 and (CAG)5 (Freeman and Rodriguez 1995; Latge et al. 1998) to more complex sequences including the M13 bacteriophage universal sequencing primer [see Bridge et al. (1997)]. Another method of fingerprinting that has been used with some fungi is based on a group of short repetitive DNA sequences that have been found dispersed throughout the genome of diverse bacterial species [see van Belkum et al. (1998)]. Primers specific to these repetitive sequences produce multiple products in PCR with fungal genomic DNA, and these can then be separated to provide simple fingerprints. Three particular unrelated families of such repetitive DNA sequences, BOX (54 bp), ERIC (124 bp), and REP, (35-40 bp) have also been used to characterize subspecific populations of different plant pathogenic fungi [see Arora et al. (1996); Toda et al. (1999)].
A relatively recent development in fingerprinting fungi has been the introduction of amplified fragment length polymorphism (AFLP) analysis [see Vos and Kuipper (1997); Vos et al. (1995)]. In this technique, total DNA is digested with restriction enzymes, and then short artificial oligonucleotides (linkers) are ligated to the restriction enzyme sites. Specific primers are then designed that show a particular degree of specificity to the linker sequences, and large fractions of the total DNA can then be amplified as fragments. The AFLP analysis generates many bands, and electrophoresis is usually undertaken in large polyacrylamide gels, it is however, possible to undertake more restricted studies that generate fewer bands and that can be analyzed in smaller electrophoresis systems [e.g., Mueller et al. (1996)]. At the conclusion of RAPD and AFLP analyses PCR bands of interest can be extracted, purified, and sequenced to produce sequence characterized amplified regions (SCARs). The sequence information obtained from SCARs can then be used to develop more specific PCR primers for detection methods [e.g., Dobrowolski and O'Brien (1993); Leclerc-Potvin et al. (1999)].
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