Molecular markers are DNA sequences that can be easily detected and whose inheritance can be monitored. They are based on natural polymorphisms and allow studying the inheritance of genes. Common types of molecular markers include RFLPs, RAPDs, AFLPs, SSRs, and SNPs. RFLPs use restriction enzymes to detect differences in fragment lengths. RAPDs use random primers to detect sequence polymorphisms. AFLPs selectively amplify restriction fragments to detect length differences. SSRs detect variability in simple sequence repeats. Molecular markers are useful for applications like gene mapping, phylogenetic studies, and analyzing genetic diversity.

1. MOLECULAR MARKERS

2. Molecular marker
 A DNA sequence that is readily detected and whose inheritance can be easily moniterd.
 The uses of molecular markers are based on the naturally occouring polymorphism.
 A marker is a gene of known function and location, that allow the studying of the inheritance of
the gene.
 A marker must be a polymorphic ie, it must exist in different forms so that chromosomes
carrying mutant gene can be distinguished from the chromosome with the normal gene by a
marker.
 NB: polymorphism involves existence of different forms of same gene in plants or population of
plants.
 Examples: RFLP,RAPD,AFLP,SSR,SNP etc…

3. RFLP(Restriction Fragment Length Polymorphism)
 Organism can be differentiated by analysis of patterns derived from cleavage of
their DNA.
 Technique is mainly based on the special enzyme called Restriction Endonucleases.
 In RFLP restriction enzyme digested DNA is resolved by Gel electrophoresis and
then blotted to a nitro cellulose membrane.
 Specific binding patterns can be visualized by hybridization with labelled probes.
 Different size or length of restriction fragments are produced,such polymorphism
are used to distinguish plant species , genotypes etc..

4. Advantages
 High reproducibility
 Show codominant alleles
 Detect coupling phase of dna
 Reliable marker in linkage and breeding analysis
 Easily determine a linked trait present in both homozygous and
heterozygous .

5. Dis advantage
 Require large quantities of high molecular weight DNA.
 Expensive process
 Time consuming
 Labor intensive

6. Application
 Used in phylogenetic studies
 Gene mapping
 DNA finger printing
 Studies of gene flow

7. RAPD(Random Amplified Polymorphic DNA)
 It is a PCR based technology.
 In 1991 Welsh and Maclelland developed this technique.
 This procedure detects nucleotide sequence polymorphism in DNA.
 It is used to analyze genetic diversity of an individual by random primers.
 In RAPD the decamer primers will or will not amplify a segment of DNA
depending on the positions that are complimentary to the primer sequence.
 If the priming sites are in the amplifiable region a discrete DNA product is formed
through cyclic amplification.
 Amlified products are separated on agarose gel in presence of ETBR and view
under UV.

8. Advantages
 Quick and easy to assay.
 Low quantities of template DNA required.
 Dominant markers.
 In expensive.
 Do not require any specific knowledge of the target

9. DISADVANTAGES
 Low reproducibility
 Highly sensitive and complicated procedure.
 PCR cycling conditions greatly influence the out come.
 Mismatches between primer and template may result in total
absence of PCR product.

10. APPLICATION
 Gene mapping
 DNA amplification finger printing
 Study of closely related species
 RAPD technique include Arbitrarily Primed Polymerase Chain
Reaction (AP-PCR).

11. AFLP(Amplified Fragment Length Polymorphism)
 AFLP is based on a selectively amplifying a subset of restriction fragments from a
complex mixture of DNA fragments obtained after digestion of genomic DNA
with restriction endonucleases.
 Polymorphisms are detected from differences in the length of the amplified
fragments by polyacrylamide gel electrophoresis (PAGE)
 The technique involves four steps: (1) restriction of DNA and
ligation of oligonucletide adapters (2) preselective amplification (3) selective
amplification (4) gel analysis of amplified fragments.
 AFLP involves the restriction of genomic DNA, followed by ligation of adaptors
complementary to the restriction sites and selective PCR amplification of a subset
of the adapted restriction fragments. These fragments are viewed on denaturing
polyacrylamide gels either through autoradiographic or fluorescence
methodologies .

12. Advantages
 High genomic abundance.
 Considerable reproducibility.
 AFLPs can be analyzed on automatic sequencers.
 The generation of many informative bands per reaction.
 Capability to amplify between 50 and 100 fragments at one time.
 Higher resolution and sensitivity.

13. Disadvantages
 Need for purified, high molecular weight DNA.
 The major disadvantage of AFLP markers is that these are
dominant markers.
 Abundance of data.

14. Application
 AFLPs can be applied in studies involving genetic identity, parentage and
identification of clones and cultivars.
 phylogenetic studies of closely related species.
 AFLP markers have successfully been used for analyzing genetic diversity in some
other plant species such as peanut.
 This technique is useful for breeders to accelerate plant improvement.
 AFLP markers are useful in genetic studies, such as biodiversity evaluation,
analysis of germplasm collections, genotyping of individuals and genetic distance
analyses.

15. SSR (Simple Sequence Repeat) or Microsatellites
 The term microsatellites was coined by Litt & Lutty (1989)and it also known as Simple
Sequence Repeats (SSRs), are sections of DNA.
 Microsatellite markers, developed from genomic libraries, can belong to either the
transcribed region or the non transcribed region of the genome.
 Microsatellite sequences are especially suited to distinguish closely related genotypes;
because of their high degree of variability, they are, therefore, favoured in population
studies .
 Microsatellite polymorphism can be detected by Southern hybridisation or PCR.
 If nucleotide sequences in the flanking regions of the microsatellite are known, specific
primers can be designed to amplify the microsatellite by PCR.
 microsatellite may be identified by screening sequence databases, poly morphism can
detected by gel electrophoresis

16. Advantage
 Because the technique is PCR-based, only low quantities of
template DNA (10–100 ng per reaction) are required.
 The strengths of microsatellites include the codominance of alleles,
their high genomic abundance
 the reproducibility of microsatellites is high and analyses do not
require high quality DNA

17. Disadvantage
 main drawbacks of microsatellites is that high development costs
 errors in genotype scoring
 Difficulty in interpretation
 PCR error

18. Application
 Poppulation genetics
 Gene mapping
 Analysis of germplasm collection
 Useful in determining functional diversity