There are several types of DNA markers:

1. Restriction fragment length polymorphism-RFLPs

2. Amplified fragment length polymorphism- AFLP

3. Random Amplified polymorphic DNA- RAPD

4. Cleaved Amplified polymorphic Sequence-CAPS

5. Simple sequence repeats- SSRs

6. Single strand conformational polymorphisms- SSCPs

7. Single Nucleotide Polymorphism (SNP)

8. Heteroduplex Analysis (HA)

9. Expressed Sequence Tags-ESTs

10. Sequence Tagged Sites-STSs

1. RFLP

   This is the polymorphism which is detected based on the differential hybridization of cloned DNA to DNA fragments in a sample of restriction enzyme digested DNAs and can be defined by a specific enzyme-probe combination.

   Strength:

   -It has good repeatability and particularly useful in comparative genome mapping. It is co-dominant nature and hence can be effectively used to differentiate homozygotes from the heterozygotes.

   Weakness:

   - The assay is tedious and time consuming.

   - Requires large quantities of DNA

   - Extremely limited utility in marker-assisted selection due to very low assay efficiency.

    

    

2. AFLP

   These polymorphism are generated using a procedure that combines restriction digestion and PCR amplification. The basic procedure is as follows:

   -Digestion of genomic DNA with a combination of two restriction enzyme- a rare and a frequent cutter, e.g. EcoRI & MSeI; EcoRI&PStI

   -Ligation of double stranded adapters ot cut ends of DNA fragments

   -Pre-selective amplification

   -Selective amplification

   -Separation of amplified fragments

   -Visualization using autoradiography

   Strength:

   - Stable amplification and high repeatability

   -Can generate fingerprints of any DNA regardless of their origin

   -Can act as bridge between genomic and physical maps

   -Hypervariability

   Weakness:

   -Time consuming procedure

   -Required significant technical skills and financial resources

   -Dominant in nature

    

    

3. SSR

   The microsatellite with variable tandem repeats range from 1-10 nucleotides dispersed throughout the genomes of most eukaryotic organisms.

   Strength:

   -Abundant and uniformly distributed in the genome

   -Hypervariable (large number of allele per locus)

   -Co-dominant markers

   -Highly reliable and reproducible assay

   -Powerful tool for genotypic differentiation, seed purity evaluation, MAS, etc.

   Weakness:

   -Expensive and time consuming and primers are usually species specific.

   -Needs prior information of the sequence to design primers

    

4. RAPD

   The RAPD markers are based on the differential PCR amplification of a sample of DNAs from oligonucleotide sequence and genetically dominant in nature. The random primers are usually of 10 nucleotides long and can anneal at multitude of genomic locations.

   Strength:

   -Requires less quantities of DNA

   -Needs limited investment in time and training

   -Commercially available

   Weakness:

   -Lack of reproducibility in markers patterns across lab and experiments

           - Inability to discern differences in sequence homology among similarly sized fragments

    

5. SCAR

   SCAR is a PCR-based marker that represents single, genetically defined loci that are identified by PCR amplification of genomic DNA with a pair of specific oligonucletide primers. The SCAR primers are designed based on the end sequence of the polymorphic RAPD fragment to amplify the corresponding locus. In conversion of RAPD to SCAR markers, polymorphism is retained either as present or absence of amplification products or appeared as length polymorphism. SCAR are advantageous over RAPD markers as they detect a single locus, their amplification is less sensitive to reaction condition and they can potentially be converted in co-dominant markers. While SCAR share the advantages of STSs, their distinct from the STSs in two aspects. SCARs are primarily defined genetically and therefore they can be used not only as physical landmarks in the genome but also as genetic markers. In addition, SCARs can contain repetitive DNA sequences within the amplified fragments as they are analyzed by PCR only and their uniqueness are determined by sequence and spacing of the primer sequence rather than by hybridization.

    

6. DAF

   DNA Amplification Fingerprinting (DAF): DNA amplification fingerprinting resembles RAPD since the amplification of the genomic DNA sequences is achieved by single short oligonucleotide primers of arbitrary sequence of 5-8 bases. DAF produces a spectrum of short DNA fragments of varying length that are resolved on polyacrylamide gel electrophoresis (PAGE) following silver staining. They can hardly be converted to locus specific SCAR or STS markers which limit their use in gene tagging and MAS.

    

7. ISSR

   Inter simple sequence repeat (ISSR): In this technique, radiolabelled di or tri-nucleotide repeats anchored through 2-4 nucleotides at one of the two ends are used as single primer, so that if two inversely oriented microsatellites are present within an amplified distance from each other, the inter-repeat sequence is amplified.

   Strength:

   -Useful for detecting genetic polymorphism

   -High polymorphism.

   Weakness:

   -However, appearance of multiple band pattern and high background noise makes this technique less favourable for MAS.

    

8. STS

    Sequence tagged sites: STS is a short, unique sequence that identifies a specific locus and can be amplified by polymerase chain reaction. Each STS is characterized by a pair of PCR primers, which are designed by a sequencing an RFLP probe representing a mapped low- copy number sequence. Polymorphism in STS is retained either as the presence or absence of amplification product or appeared as length polymorphism that converts STS into co dominant marker. STS can also be developed from any cloned sequence such as RAPD and AFLP.

    

9. CAPS

    Cleaved amplified polymorphic sequence: CAPS are a form of gentic variation in the length of DNA fragments generated by the restriction digestion of PCR products. The source for the primers can come from a gene-bank, genomic or cDNA clones and cloned RAPD bands. This marker class is codominant in nature.

    

10. ESTs

    Expressed sequence tags: An EST is a DNA fragment representing the sequence from Cdna clone that corresponds to a mRNA molecule or part of it. Thus, ESTs serve as markers for genetic and physical mapping and as clones for expression analysis. ESTs can be matched with sequences available in nucleotide sequence databases or Genebank and protein sequence databases and then assigned to specific genes with the help of computer programs and software. EST databases have proven to be a tremendous resource for finding genes and for interspecific sequence comparison. ESTs have also been utilized for developing the SSR markers.

     

11. RGAs

    Resistance gene analogs: In recent years, a large number of disease resistance genes conferring resistance to diverse spectrum of pathogens have been isolated from wide range of plant species. Most of these disease resistance genes have conserved amino acid motifs. The most notable being the presence of nucleotide binding sites (NBS) and leucine rich repeat regions(LRR). The candidate resistance gene approach, employing primers derived from conserved motifs of resistance genes, has been used successfully to develop RGA markers.

     

12. SNPs

-Useful for gene mapping

-Useful in positional cloning of a mutant locus

-Useful in detection of mutations at molecular level

-most SNPs are biallelic and thus less informative than SSRs

-multiplexing is not possible for all loci

-SNP assay is costly

-In preparing genetic map, three times more SNP markers are needed than SSR markers