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RAPD markers

RAPD markers

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RAPD markers

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  1. RAPD markers Larisa Gustavsson (Garkava) Balsgård-Department of Crop Sciences Swedish University of Agricultural Sciences

  2. What is RAPD? RAPD is a PCR-based method which employs single primers of arbitrary nucleotide sequence with 10 nucleotides to amplify anonymous PCR fragments from genomic template DNA

  3. RAPD technology A B C A + + + Arbitrary primers Taq polymerase Nucleotides + Genomic DNA PCR (under relaxed conditions) Buffer

  4. A B C 520bp 360bp 260 bp PCR 360 bp Electrophoresis 260 bp 520 bp A B C

  5. PCR product occurs when: • The primers anneal in a particular orientation (such that they point towards each other) • The primers anneal within a reasonable distance of one another (150 -3000 bp)

  6. 1 3 2 4 5 6 PCR reaction DNA template Product 1 Product 2 The number of amplification products is related to the number and orientation of the genome sequences which are complementary to the primer

  7. The nature of RAPD polymorphism

  8. 2 3 1 4 5 6 DNA template PCR reaction No product Product 2 nucleotide substitution within target sites may affect the annealing process - either no fragment is detected

  9. 2 1 3 4 5 6 DNA template PCR reaction Product 1 Product 2 or detected fragment is of increased size

  10. Small fragment DNA Insertion 3 1 2 Deletion 4 5 6 DNA template PCR reaction Product 1 Product 2 b)insertion or deletion of a small fragment of DNA - the amplified fragments are changed in size

  11. The insertion of large fragment 3 2 5 6 DNA template PCR reaction No product Product 2 c) insertion of alargepiece of DNA between the primer -binding sites may exceed the capacity of PCR - no fragment is detected

  12. A schematic picture of an agarose gel - Marker Plant A Plant B Plant C Monomorphic bands Polymorphic bands + Presens of a band, ”1” Absence of a band, ”0”

  13. And a real picture of a gel…

  14. … and one more

  15. Data analysis

  16. RAPD bands are treated as independent loci: 1 2 3 4 5 6 7 8 9 10 11 12 13 14 Locus A Locus B Locus C Locus D

  17. RAPD bands are scored for presens ”1” and absens ”0”. Only clear, consistent and polymorphic bands are usually used to create a binary matrix for future statistical analyses

  18. A binary matrix:

  19. Statistical analyses(some examples) Measurements of genetic diversity by means of different genetic diversity indexes (i.e. Nei’s diversity index, modified by Lynch and Milligan (1994) for dominant markers, Shannon’s index etc)

  20. Evaluation of genetic diversity in Lingonberry populations

  21. Cluster analysis,Multidimensional Scaling and Principal co-ordinate analyses are used mainly for evaluation of genetic relatedness among individual organizms or among groups of organizms (i.e. populations)

  22. Genetic relatedness among populations of lingonberry (A) and indidual plants of Japanese quince (B) revealed by cluster analyses B A Fig.1. Dendrogram based on UPGMA analysis of genetic similarity estimates among 15 populations of lingonberry

  23. Genetic relationships among lingonberry popula-tions (A) and individual plants of Japanese quince (B) revealed by MDS analysis A B Fig.2 An MDS analysis of genetic relationships Among ligonberry populations

  24. A three-dimentional representation of phenetical relationships between populations of Japanese quince revealed by PCA

  25. Genetic relationships among 23 cultivars from Gene bank at Balsgård revealed by RAPD markers Similarity % Fig.1. Dendrogram based on UPGMA analysis (Jaccard’s coefficient) for RAPD data, showing relationships among apple cultivars

  26. Advantages, limitations and applications of RAPD markers

  27. Advantages: • No prior knowledge of DNA sequences is required • Random distribution throughout the genome • The requirement for small amount of DNA (5-20 ng) • Easy and quick to assay • The efficiency to generate a large number of markers

  28. Commercially available 10mer primers are applicable to any species • The potential automation of the technique • RAPD bands can often be cloned and sequenced to make SCAR (sequence-characterized amplified region) markers • Cost-effectiveness!

  29. Limitations: • Dominant nature (heterozygous individuals can not be separated from dominant homozygous) • Sensitivity to changes in reaction conditions, which affects the reproducibility of banding patterns • Co-migratingbands can represent non-homologous loci

  30. The scoring of RAPD bands is open to interpretation • The results are not easily reproducible between laboratories

  31. Applications: • Measurements of genetic diversity • Genetic structure of populations • Germplasm characterisation • Verification of genetic identity • Genetic mapping

  32. Development of markers linked to a trait of interest • Cultivar identification • Identification of clones (in case of soma- clonal variation) • Interspecific hybridization

  33. Verification of cultivar and hybrid purity • Clarification of parentage

  34. RAPD is probably the cheapest and easiest DNA method for laboratories just beginning to use molecular markers

  35. Thank you