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RAPD markers Larisa Gustavsson (Garkava)

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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 + + + + PCR Taq polymerase Arbitrary primers
Nucleotides + Genomic DNA PCR (under relaxed conditions) Buffer

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

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 ( bp)

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

7 The nature of RAPD polymorphism

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

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

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

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

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 1 2 3 4 5 6 7 8 9 10 11 12 13 14 AA/Aa aa bb BB/Bb CC/Cc cc dd DD/Dd

17 RAPD bands are scored for presens ”1” and absens ”0”
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: Band 1 Band 2 Band 3 Band 4 Plant A 1 Plant B Plant C
Plant B Plant C Plant D Plant E Plant F Plant G

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-migrating bands 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

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