Larisa Gustavsson Garkava

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RAPD markers

• Larisa Gustavsson (Garkava)
• Balsgård-Department of Crop Sciences
• Swedish University of Agricultural Sciences

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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
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RAPD technology
A
B
C
A



Arbitrary primers
Taq polymerase
Nucleotides

Genomic DNA
PCR
(under relaxed conditions)
Buffer
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PCR
360 bp
Electrophoresis
260 bp
520 bp
A
B
C
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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)

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The number of amplification products is related
to the number and orientation of the genome
sequences which are complementary to the primer
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The nature of RAPD polymorphism
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• nucleotide substitution within target sites may
  affect
• the annealing process - either no fragment is
  detected

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• or detected fragment is of increased size

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b) insertion or deletion of a small fragment of
DNA - the amplified fragments are changed in
size
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c) insertion of a large piece of DNA between the
primer -binding sites may exceed the capacity of
PCR - no fragment is detected
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A schematic picture of an agarose gel
-
Plant A
Marker
Plant B
Plant C
Monomorphic bands
Polymorphic bands

Presens of a band, 1
Absence of a band, 0
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And a real picture of a gel
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and one more
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Data analysis
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RAPD bands are treated as independent loci
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Locus A
Locus B
Locus C
Locus D
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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
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A binary matrix
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Statistical analyses (some examples)

• Measurements of genetic diversity by means of
  different genetic diversity indexes (i.e. Neis
  diversity index, modified by Lynch and Milligan
  (1994) for dominant markers, Shannons index etc)

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Evaluation of genetic diversity in Lingonberry
populations
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• 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)

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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
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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
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A three-dimentional representation of phenetical
relationships between populations of Japanese
quince revealed by PCA
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Genetic relationships among 23 cultivars from
Gene bank at Balsgård revealed by RAPD markers
Similarity
Fig.1. Dendrogram based on UPGMA analysis
(Jaccards coefficient) for RAPD data, showing
relationships among apple cultivars
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Advantages, limitations and applications of RAPD
markers
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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

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• 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!

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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

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

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Applications

• Measurements of genetic diversity
• Genetic structure of populations
• Germplasm characterisation
• Verification of genetic identity
• Genetic mapping

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

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• Verification of cultivar and hybrid purity
• Clarification of parentage

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

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Thank you