DNA-Marker Technology
Phytowelt possesses a key expertise in plant biotechnology. We provide custom DNA marker analytical service and data interpretation. Our fast, low cost and reliable genetic marker methodology can be applied to nearly any biological material and species. We offer a complete service starting with defined biological material, which is then processed by DNA purification. Subsequently, DNA markers are designed, evaluated and optimized. Optionally, marker cloning and DNA sequencing can be requested. Such markers are tailored to the client´s specific application.
The application of DNA-based genetic markers has revolutionized the development of genetic maps and the efficiency of breeding programs (MAS, marker assisted selection). Nowadays, the analysis of polymorphisms completes the analysis of species and varieties as shown by the characterisation of plant genetic resources and the analysis of biodiversity within and between species (e.g. management of germplasm collections). In general, commercially available DNA-based markers assume that the DNA in question is already at hand and that its sequence is already characterised, too.
In addition, there are DNA-based markers that do not require knowledge of the sequence in advance. Each class of marker has its own set of advantages and disadvantages, especially with regards to cost, speed, and reliability. Techniques applied are:
- Amplified Fragment Length Polymorphism (AFLP),
- Restriction Fragment Length Polymorphism (RFLP),
- Randomly Amplified Polymorphic DNA (RAPD),
- Mini- and microsatellite DNAs (Variable Number Tandem Repeats, VNTRs),
- Simple Sequence Repeats (SSRs),
- Sequence-Tagged Microsatellites (STMs),
- Inverse Sequence-Tagged Repeats (ISTR).
The ISTR Methodology
The inverse sequence-tagged repeat (ISTR) analysis is a PCR-based DNA marker technology. It was developed by MPIZ researchers on the basis of gypsy/copia-like elements which are mobile genetic elements (retrotransposons) found in plant, human, animal and microbial genomes. The ISTR technique uses the ubiquitous presence of reverse transcriptase sequences of neighbouring copia-like elements, as depicted in the illustration. Copia-specific primer pairs are used to produce PCR fragments of specific length building probe-characteristic fingerprints (polymorphism analysis).
Important crop plants have been subjected to ISTR analysis including cereals (barley, maize, rye and wheat), fruits (grapevine, guava, plum, strawberry), oil crops (coconut and oil palm, rapeseed, soya) and others like cassava, potato, sugar beet, tobacco, tomato or herbs like mint or parsley. Non-crop plant material has been tested as well, like arabidopsis, trees (cedar, pine, eucalyptus) or various ornamental plants (heather, petunia, roses, snapdragon).
The principle of ISTR-based polymorphism analysis. The sequence of copia-like-elements (Copia) has been used for the design of a set of PCR-primers (ISTR-F and -B primer). These are used to amplify specific products (Fragment 1-3), which are separated by gel electrophoreses. According to specific genome positions of the copia-elements, differences in length and amount of PCR fragments come about forming a probe-specific pattern (see polymorphism A and B).
In comparison with currently available methodologies like AFLP, ISTR represents a significant breakthrough in terms of price, facility, precision, flexibility and speed. The characteristics of ISTR are summarized as follows:
- Universal primers
- Moderate infrastructure required
- Non-radioactive detection in gel possible
- Large amount of loci (>5 to 100) amplified
- High number of polymorphisms detected
- High reproducibility
- Simple DNA extraction
- Applicable for the analysis of plant, animal, human and microbial DNAs