Agriculture Reference
In-Depth Information
1997). While RAPD analyses can provide good discrimination within and between
species, it is generally not considered a robust enough technique (e.g. for
comparisons at different times and in different laboratories) and has been slowly
abandoned due to its poor reproducibility. Since ISSR primers are longer than
RAPD primers, higher annealing temperatures can be used, which result in stable
and reproducible markers (Han et al., 2002).
ISSR markers are generally detected on agarose gels with ethidium bromide
staining. However, detection on acrylamide gels with silver-staining or radioactive
detection increases sensitivity. Recently, the introduction of fluorescently labelled
primers combined with fragment analysis on DNA sequencers has increased
sensitivity and throughput of these markers tremendously (Yasodha et al., 2004).
Fragments detected are most commonly scored as biallelic dominant markers
(presence/absence) (Gupta et al., 1994; Tsumura et al., 1996) but co-dominant
segregation has been shown in some cases (Wang et al., 1998b; Sankar and Moore,
2001).
ISSR markers have been used to address a variety of issues related to genomic
fingerprinting (Charters and Wilkinson, 2000), assessment of genetic diversity and
phylogeny analysis (Tsumura et al., 1996; Assefa et al., 2003; Bornet et al., 2004),
detection of somaclonal variation (Albani and Wilkinson, 1998), genome mapping
(Arcade et al., 2000), determining frequency of microsatellites in the genome
(Yasodha et al., 2004), gene tagging and marker-assisted selection (Akagi et al.,
1996) and evolutionary biology (Wolfe et al., 1998). The ISSR-based fingerprinting
technique has been used successfully to determine genetic variability within various
fungal species (Hantula et al., 1996; Zhou et al., 2001; Grunig et al., 2001, 2002;
Rodrigues et al., 2004).
Using ISSR analysis, considerable variability was observed among isolates from
different geographical locations and hosts in different fungal populations (Han et al.,
2002; Mishra et al., 2003). These markers will also enable a greater understanding of
the movement of economically important pathogens around the world. ISSR
fingerprinting was used to differentiate Botryosphaeria species and related anamorphic
fungi indicating its powerfulness in examining species aggregates or complexes,
justifying newly identified species, and differentiating closely related fungi with
very similar morphology and ITS sequences (Zhou et al., 2001). Successful
discrimination between Pisolithus albus and P. microcarpus was achieved by
deploying ISSR markers (Hitchcock et al., 2003).
DNA sequencing gained its importance in pathogen diagnostics by the
revolutionary discovery of the DNA structure by Watson and Crick (1953). Since
then the DNA-based techniques paved their way into the discipline of fungal,
bacterial and viral systematics and proved capable of resolving the problems related
to classification, detection and discrimination between and within the closely related
genera and species. DNA sequence techniques have an added advantage of
sensitivity. This sometimes cannot be obtained and is far beyond the range of
detection by traditional methods (Mills et al., 1992; O'Donnell, 1992; Chen et al.,
1992; Levy et al., 1991). A better understanding of disease diagnosis, host-pathogen
interaction and disease management can only be achieved with precise definition of
the pathogen. The first stage in development of a diagnostic assay is to select the
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