Molecular Techniques for Isolate Identification and Development of Diagnostic Markers
There are a number of molecular approaches available for identification of isolates to a species level, the selection of which one to use is dependent on the type of analysis that is needed. If the isolate has been cultured the most accurate method for identification is DNA sequence analysis, but if it is not feasible to use this approach there are several gel based techniques that should work as well. For diagnosis from infected plant tissue there are PCR markers that are specific for detection of a Phytophthora species at a genus-specific level as well as those capable of identification at a species-specific level. Several approaches also are available for following subpopulations of a particular species. While not fully developed at this point in time, work is in progress on the development of macroarrays for identification of Phytophthora to a species level.
The most accurate method for identification of isolates to a species level is DNA sequence analysis. A range of loci have been used for this purpose, with perhaps the most extensive database available for the nuclear encoded ITS region of the rDNA. However, 7 nuclear and 4 mitochondrial genes have been sequenced for a large number of isolates representing all described species in the genus and the data deposited in this database. Click the links below to search the database to identify unknown isolates by BLAST analysis. For additional details on loci that have been sequenced click here.
Identify your species by BLAST using reference strains - CLICK HERE to link to the BLAST page of the website
Gel based identification of species
There are several gel-based techniques that are useful for identification of isolates to a species level. While they require more data interpretation than DNA sequence analysis, they can provide an alternative when cost or accessibility to an automated sequencer is a limiting factor. RFLP techniques include digestion of the ITS region of the rDNA as well as the mitochondrially encoded cox1 and cox2 gene cluster. In addition, single stranded conformation polymorphism analysis has been found to be useful for differentiation of species. For an overview of RFLP and SSCP techniques click here.
Genus specific diagnostic markers
There have been several Phytophthora genus-specific primer pairs that have been reported in the literature, however not all of them have been confirmed to be specific to the point where false positives are not obtained with the closely related genus Pythium. Loci that have been reported on include the ITS region, the Ras-related protein Ypt1, and the spacer region between the mitochondrially encoded cox1 and 2 gene. For additional details click here.
Species-specific diagnostic markers
When the objective is to determine if a particular species is present in a plant sample, species-specific diagnostic markers are needed. These primers should not amplify plants or other organisms that may be present, including closely related Phytophthora species. A variety of sequences have been used for development of these diagnostic markers for a number of species in the genus, including random clones of genomic DNA and SCARs as well as individual genes. Species-specific diagnostic marker systems for detection of multiple species have been designed from particular regions of the genome. For additional details click here.
Identification of subpopulations
There are a number of different approaches that can be used to identify subpopulations within a single Phytophthora spp., including sequence analysis of specific loci, single nucleotide polymorphisms (SNPs), restriction fragment length polymorphisms (RFLP) analysis, random amplified polymorphic DNAs (RAPDs), amplified fragment length polymorphism (AFLP), microsatellites, and mitochondrial haplotype. For additional details click here.
DNA arrays are more technically challenging to perform than RT PCR or DNA sequencing but they have an almost unlimited multiplexing capability to detect many different species from environmental samples. For overview of array based methods click here.
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