Project topics and materials
Get Complete Project Material File(s) Now! »
DNA-BASED TECHNIQUES
The use of DNA polymorphisms as a trait has added a new dimension to the recognition and classification of genetic variability in fungi. In theory, DNA-based techniques such as Random Amplified Polymorphic DNA (RAPD), Restriction Fragment Length Polymorphism (RFLP), Amplified Fragment Length Polymorphism (AFLP) and DNA sequence analyses, overcome all the limitations associated with the identification of sub-species taxa using morphology, pathogenicity, vegetative compatibility, and protein-based methods (Table 1). If correlated to pathogenicity, they could also be useful in identifying virulence groups (Woo et al., 1996). DNA-based techniques are also valuable for use in phylogenetic studies.
For the purpose of this review, the DNA-based techniques are broadly grouped into RAPD, RFLP, AFLP, electrophoretic karyotyping, Simple Sequence Repeats (SSRs) and DNA sequence analyses. A description of these tools and a review of their use in the taxonomy of F. oxysporum isolates are presented in the following sections.
Random Amplified Polymorphic DNA (RAPD)
RAPD (Williams et al., 1990) or the Arbitrarily Primed PCR (AP-PCR, Welsh and McClelland, 1990), and its variation, DNA Amplified Fingerprinting (DAF), use single primers of arbitrary sequence to generate DNA fragments. DAF differs from RAPD in that it uses shorter primers (usually, 8-12 nucleotides) with a 5’-prime mini-hairpin structure to help minimize interactions within and between the termini of amplification products during PCR (Cateno-Anollés et al., 1991). RAPD has several advantages as a means of characterizing genetic variability. These include speed, low cost, low DNA concentration, lack of radioactivity and there is no need for prior sequence information about the target DNA. It is also applicable to large numbers of isolates and enables analysis of variation at more than one locus (Bentley et al., 1995). As a result, this technique has been used extensively in the molecular characterization of F. oxysporum isolates. Several studies have revealed correspondence between virulence groups and/or VCGs on one hand, and RAPD Finger Print Groups (FPGs) on the other. For instance, Wang et al. (2001) studied 24 isolates belonging to 13 formae speciales using RAPDs. Seven RAPD primers selected from an initial set of 132, revealed RAPD fingerprints unique to each forma specialis. Probes developed from forma specialis-specific RAPD bands showed different specificity to the 13 formae speciales after Southern hybridisation (Southern, 1975) with RAPD fingerprints. Based on these findings, the authors concluded that markers based on differences in fingerprints are potentially useful for the identification of formae speciales without the need for pathogenicity tests. Similar studies showed that RAPD FPGs correspond with VCGs (Grajal-Martin et al., 1993; Bentley et al., 1995; Tantaoui et al., 1996; Nelson et al., 1997; Mes et al., 1999), formae speciales (Chiocchetti et al., 1999; Hernandez et al., 1999; Vakalounakis and Fragkiadakis, 1999; Pasquali et al., 2003), and/or races (Assigbetse et al., 1994; Manulis et al., 1994; de Haan et al., 2000). However formae speciales in these studies were represented by only one to three isolates. Unsuitability of RAPD to differentiate isolates based on pathogenicity or geographical origin has also been reported. An example is the work of Cramer et al. (2003) who studied 34 isolates including F. o. phaseoli, F. o. betae, non-pathogenic F. oxysporum and an isolate of F. solani.
Restriction Fragment Length Polymorphisms (RFLPs)
RFLPs represent differences in the size of DNA fragments produced when two or more DNA templates are digested with sequence-specific endonucleases. Commonly, these fragments are resolved by electrophoresis on agarose gels containing ethidium bromide, and subsequently visualized by UV illumination. Alternatively, the fragments can be probed using labeled nucleic acids in Southern analyses (Southern, 1975). Polymorphisms can be generated by a gain or loss in restriction sites resulting from nucleotide substitution or from the rearrangement of DNA sequences (Taylor, 1986). RFLP markers are ideally suited to genetic diversity studies because most are selectively neutral, produce more polymorphisms compared to other types of markers such as isozymes, they are reproducible and co-dominant (Koenig et al., 1997).
RFLPs can be broadly divided into two groups according to the type of template used. These are total DNA RFLPs and RFLPs of PCR-amplified products (PCR-RFLPs). Total DNA RFLPs are sub-divided into genomic DNA (gDNA) RFLPs and mitochondrial DNA (mtDNA) RFLPs depending on the type of template and/or probe used. The term ‘mtDNA RFLP’ has been used in the literature in connection with RFLPs involving mtDNA templates (e.g., Jacobson and Gordon, 1991; Kim et al., 1991a), or where mtDNA fragments are used as probes (e.g., Kistler et al., 1987; Jacobson and Gordon, 1990; Gordon and Okamoto, 1992; Kim et al., 1992; 1993b). For the sake of convenience, RFLP analyses involving mtDNA templates and/or mtDNA-based probes are treated in the mtDNA RFLPs section. RFLP analyses involving hybridisation with SSR-containing probes are discussed in section 4.6.
CHAPTER 1
Literature review: Taxonomic tools used in the classification of species in the Fusarium oxysporum complex
CHAPTER 2
A survey of Fusarium species from Ethiopia using morphology and DNA sequence information
CHAPTER 3
Characterization of Fusarium solani isolates from Ethiopia using Amplified Fragment Length Polymorphism (AFLP) and DNA sequence information
CHAPTER 4
Simple Sequence Repeat (SSR) markers for the study of species in the Fusarium oxysporum complex
CHAPTER 5
Characterization of Fusarium oxysporum isolates from Ethiopia using AFLP, SSR and DNA sequence analyses
CHAPTER 6
Species-specific primers for Fusarium redolens and a PCR-RFLP technique to distinguish among the three clades of Fusarium oxysporum
SUMMARY
