PLANT DISEASE DIAGNOSIS - The Epidemiology of Plant Diseases - page 21

thus enabling the amount of target DNA in the original sample to be determined by
titration.
In random amplified polymorphic DNA (RAPD), short oligonucleotides of about
10 oligomers are used as primers which should be complementary to sequences in
the target genome (Williams et al ., 1990). Although no prior knowledge of the
actual sequences in the target genome is necessary, the pathogens must be purified -
for example, by culturing - before fingerprinting (Crowhurst et al., 1991; Guthrie
et al., 1992; Welsh et al., 1992). Therefore, this method is unsuitable for the direct
detection of pathogens in situ in plants or soil. This procedure has also been termed
arbitarily primed PCR (AP-PCR) by Welsh and McClelland (1990) and produces
relatively simple fingerprints. DNA amplification fingerprinting (DAF) uses DNA
silver stains and polyacrylamide gel electrophoresis (PAGE) to produce more
complex fingerprints (Cactano-Anolles et al . , 1991).
Inter Simple Sequence Repeats (ISSRs) are an anonymous, RAPDs-like
approach that access variation around the numerous microsatellite regions dispersed
throughout almost all genomes (Zietkiewicz et al., 1994). Simple sequence repeats
(SSRs) (Tautz, 1989), also known as microsatellites (Litt and Luty, 1989) are
repeats of 1-6 nucleotides that are distributed ubiquitously in the genomes of higher
organisms (Toth et al., 2000). Almost synonymous terms are variable number of
tandem repeats (VNTR) or short tandem repeats (STR) (Edwards et al., 1991). They
can be found anywhere in the genome including protein coding and non-coding
regions and are characterized by mono-, di- or trinucleotide repeats, eg., GG…, or
GC…, AGT…, repeat units side by side. ISSR is a PCR-based method that involves
amplification of DNA present between such repeats (hence the name inter-SSR).
Mononucleotide repeats are characteristic of the chloroplast genome whereas di- and
trinucleotide repeats are characteristic of the nuclear genome and hence ISSRs
specifically target the di- and trinucleotide repeats of microsatellites.
Typically a single primer consisting of SSRs plus a 1-3 base pair randomly
selected anchor is used for the generation of a multiband pattern on gel. Di-
nucleotide, tri-nucleotide, tetra-nucleotide and penta-nucleotide repeats have been
used as primers. The primers can be anchored or non-anchored. Because the length
of the primers used is long enough (16-25 bp), ISSRs have high reproducibility as
compared with RAPD markers (Zietkiewicz et al., 1994). In fact, these markers
combine the universality of RAPD with benefits of SSRs and AFLP (Reddy et al.,
2002). When the primer successfully locates two microsatellite regions within an
amplifiable distance away on the DNA sample, the PCR reaction will generate a
band of particular molecular size for that locus. Usually several such paired
microsatellite regions exist in the nuclear genome, thereby resulting in many bands
of different molecular sizes. Studies of ISSR locus heritability have demonstrated an
exceedingly close approximation to classic Mendelian ratios (Tsumura et al., 1996).
The existence of high variability and high mapping density when compared to
RAPD and RFLP data make these new dominant molecular markers ideal for
producing genetic maps of individual species (Nagaoka and Ogihara, 1997).
Microsatellites and ISSR markers are useful in species identification (Niesters et al.,
1993; Graser et al., 1998) and also in the characterization of fungal strains due to
their high levels of polymorphism (Schonian et al., 1993; Longato and Bonfante,