Utilization Of DNA Markers For Identification Of Plant Species

Identification of plant species is more difficult than identification of animals because there are more problems to find right DNA marker for plants. For animal identification mitochondrial DNA (mtDNA) is used more, especially cytochrome c oxidase 1 gene sequence. Plant mtDNA is not very useful for identification of species because genetic information there is conserved and even can be different within plants of the same species. In plant identification chloroplast DNA (cpDNA) is used more often. There are guidelines to create a molecular marker, for example, target region should be polymorphic, it should be frequent and distributed evenly in the genome, easy to access, co-dominant inheritance is preferred also marker development should not be expensive. But molecular markers usually do not fulfil all these criteria, because there is no such thing as perfect marker and properties of markers can be adjusted depending on what kind of experiment would be performed.

There are several DNA markers that can be used for identification of plant species. The first molecular marker that was used to find differences between DNA sequences were restriction fragment length polymorphisms (RFLPs). The principal of this method is that DNA is cut into short, specific sequences by using restriction enzymes and then visualized using radioactive probes or gel electrophoresis. This method is not very popular because it requires more DNA and it should be good quality, also it is time – consuming method and radioactive probes are used. Randomly amplified polymorphic DNA (RAPD) it is PCR – based multi – locus method. The principal of generating RAPDs is by using short random primers in PCR reaction. These primers attach to different regions of sample DNA. If the region, where primer attached, had mutation amplification products will have different profiles. Results can be visualized using gel electrophoresis, there we can see different length bands which vary between individuals and depends on where the primer binding site was. The main advantage of this method is that it can be used even if a sample DNA sequence is unknown also small quantities of sample DNA is enough. However, there are some problems whit using RAPD markers, these markers usually are dominant, in that case, we cannot tell if DNA segment that was amplified is heterozygous or homozygous. Also, band profiles can vary between different laboratories because any adjustments made in experimental conditions can change the final product.

Amplified fragment length polymorphism (AFLP) is another marker that can be used for plant identification. The first step for generating AFLP marker is to cut DNA using two distinct restriction enzymes because we need to get DNA that has overlapping ends. On the second step two linkers are ligated to prepared DNA fragments, so all sample DNA now has the same DNA sequence on the end. Then PCR amplification can be performed using primers that anneal to linker DNA sequence. This method is useful because a high number of polymorphic DNA bands can be produced only in one experiment. However, this marker is also dominant, and for this method, high quality DNA is required.

Another marker is inter-simple sequence repeats (ISSR). It is a technique where two microsatellites are used as primers and as a result, different length DNA sequences are generated. DNA fragments can be detected by using agarose or polyacrylamide electrophoresis. ISSR marker is highly polymorphic and can be used in different studies, for example, gene diversity, evolutionary genetics. The main drawback of this marker is that it is dominant.

One more group of markers are microsatellites, or also called short tandem repeats (STRs). Microsatellites have repeated motives of 1-10 bp and these motives can be repeated up to 100 times. STRs are widely used because they are co-dominant molecular markers also very informative and highly polymorphic. PCR reaction primers used for microsatellites analysis can be tagged with a fluorophore or radioactive element and can be visualized with gel electrophoresis. Using this marker can be not useful if there is a mutation in the primer binding region in that case when visualized there could be null alleles. Sequence characterization of amplified region (SCAR) is a co-dominant marker in which primers are designed using cloned RAPD or AFLP fragment linked to feature of interest. The main advantages of this method are that it is not time consuming, not very sensitive to changes in experimental conditions and it is a co-dominant marker. The main drawback of this marker is that for DNA sequencing need to be performed to design PCR primers. Sometimes using PCR – based multi – locus methods polymorphisms cannot be found from different plants even if they have differences in morphology. For example, in Göktürk Baydar et al. (2004) research of Rosa damascene, they used AFLP and microsatellite markers. Results showed that different plants have the same marker patterns even though they had some morphological differences. They made an assumption that these differences can be caused by point mutations, which cannot be identified by scanning small regions of the plant genome. In this case is better to use single nucleotide polymorphisms (SNPs). SNPs are single base alteration which can vary in different organisms. SNPs are evenly distributed trough genome, it has low mutation rates and is the most common polymorphism in a genome. SNPs can be identified by using specific or universal primers and after PCR, amplified DNA sequencing should be performed.

To sum up there is a wide range of different markers that can be used for plant identification. They all have some disadvantages because it is impossible to have a perfect molecular marker.