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Molecular markers

Genetic markers

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Molecular markers

  2. 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. 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. 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. 5. Dis advantage  Require large quantities of high molecular weight DNA.  Expensive process  Time consuming  Labor intensive
  6. 6. Application  Used in phylogenetic studies  Gene mapping  DNA finger printing  Studies of gene flow
  7. 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. 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. 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. 10. APPLICATION  Gene mapping  DNA amplification finger printing  Study of closely related species  RAPD technique include Arbitrarily Primed Polymerase Chain Reaction (AP-PCR).
  11. 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. 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. 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. 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. 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. 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. 17. Disadvantage  main drawbacks of microsatellites is that high development costs  errors in genotype scoring  Difficulty in interpretation  PCR error
  18. 18. Application  Poppulation genetics  Gene mapping  Analysis of germplasm collection  Useful in determining functional diversity