ISSR (Inter-Simple Sequence Repeat) and RAPD (Random Amplified Polymorphic DNA) are both PCR-based molecular marker techniques used for detecting genetic polymorphisms. While they share similarities in their principles and applications, they also exhibit differences in methodology, reproducibility, and utility. Here's a comparison of ISSR and RAPD marker systems, along with their applications and limitations:

 

ISSR Marker System:

·        Principle: ISSR markers are based on the amplification of DNA regions between two inversely oriented microsatellite repeats using primers anchored in these repeats. The primers typically consist of microsatellite motifs (e.g., (GT)_n) and are designed to anneal at multiple sites throughout the genome, resulting in the amplification of DNA fragments between adjacent microsatellite repeats.

·        Reproducibility: ISSR markers tend to be more reproducible than RAPD markers due to the specificity of primer annealing and the presence of microsatellite sequences at primer binding sites. However, ISSR reproducibility can still be influenced by factors such as PCR conditions and template DNA quality.

·        Polymorphism: ISSR markers are often highly polymorphic, as they target regions of the genome with variable numbers of tandem repeats. This high level of polymorphism makes ISSRs valuable for genetic diversity analysis, population genetics studies, and marker-assisted selection in breeding programs.

·        Applications: ISSR markers are widely used for genetic mapping, diversity analysis, population structure analysis, parentage analysis, and phylogenetic studies in various organisms, including plants, animals, and microorganisms.

·        Limitations: ISSR markers may suffer from reduced reproducibility and allele scoring difficulties compared to other marker systems, particularly when working with poorly characterized genomes or complex DNA samples. Additionally, ISSR markers may not always provide sufficient genomic coverage for certain applications, requiring the use of complementary marker systems.

 

RAPD Marker System:

·        Principle: RAPD markers rely on the amplification of random DNA segments using short, arbitrary primers of random sequence. These primers anneal at multiple sites throughout the genome, resulting in the amplification of random DNA fragments with variable sizes and sequences.

·        Reproducibility: RAPD markers are prone to variability and inconsistency due to the random nature of primer annealing and amplification. Factors such as primer quality, PCR conditions, and template DNA quality can affect the reproducibility of RAPD banding patterns.

·        Polymorphism: RAPD markers can be highly polymorphic, as they amplify regions of the genome with variable sequences and lengths. However, the level of polymorphism may vary depending on the choice of primers and the genetic diversity of the studied population.

·        Applications: RAPD markers have been used for genetic diversity analysis, linkage mapping, marker-trait association studies, population genetics studies, and phylogenetic analysis in various organisms. They are particularly useful for rapid screening of genetic variation and initial genetic characterization of populations or species.

·        Limitations: RAPD markers are less reproducible and less informative than other marker systems, such as SSRs or SNP markers. The random nature of RAPD amplification can lead to difficulties in band scoring and data interpretation, particularly for complex or low-quality DNA samples. Additionally, RAPD markers may suffer from issues such as allele dropout, non-specific amplification, and lack of transferability between studies or laboratories.

In summary, both ISSR and RAPD marker systems offer advantages and limitations for genetic analysis and molecular marker applications. ISSR markers provide higher reproducibility and specificity, making them suitable for diverse genetic studies, whereas RAPD markers offer rapid screening of genetic variation but suffer from lower reproducibility and specificity. The choice between ISSR and RAPD markers depends on the specific research objectives, sample characteristics, and analytical requirements of the study.