Molecular markers

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…

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..

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 .

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

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

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.

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

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.

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

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 .

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.

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

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.

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

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

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

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

Molecular markers

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Molecular markers