Larisa Gustavsson (Garkava)
Balsgård-Department of Crop Sciences
Swedish University of Agricultural Sciences
RAPD markers
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
What is RAPD?
RAPD technologyA B C
Genomic DNA
+
Taq polymerase
+
Arbitrary primers
A
+
Nucleotides
+
Buffer
PCR
(under relaxed conditions)
Electrophoresis
PCR
360 bp
260 bp
520 bp
520bp
260 bp
360 bp
A B C
A B C
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)
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
The nature of RAPD polymorphism
a) 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
or detected fragment is of increased size
2
PCR reaction
1 3
4 6
Product 2
DNA template
5
Product 1
b) insertion or deletion of a small fragment of DNA - the amplified fragments are changed in size
2 3
5DNA template
PCR reaction
6
Product 1 Product 2
Small fragment DNA
Insertion
Deletion1
4
2 3
5DNA template
PCR reaction
6
Product 2No product
The insertion of large fragment
c) insertion of a large piece of DNA between the primer -binding sites may exceed the capacity of PCR - no fragment is detected
A schematic picture of an agarose gel
Plant AMarker Plant B -
+
Plant C
Monomorphic bands
Polymorphic bands
Presens of a band, ”1” Absence of a band, ”0”
And a real picture of a gel…
… and one more
Data analysis
RAPD bands are treated as independent loci:
AA/Aa
aa aa aa aa aa aa aa aa aa aa aa aa aa
bb BB/Bb
BB/Bb
BB/Bb
BB/Bb
BB/Bb
BB/Bb
BB/Bb
BB/Bb
bb bb bb bb bb
CC/Cc
cc cc cc cc cc cc cc cc CC/Cc
CC/Cc
CC/Cc
CC/Cc
cc
dd DD/Dd
DD/Dd
DD/Dd
DD/Dd
DD/Dd
DD/Dd
DD/Dd
DD/Dd
DD/Dd
DD/Dd
DD/Dd
dd DD/Dd
1 2 3 4 5 6 7 8 9 10 11 12 13 14
Locus ALocus BLocus CLocus D
1 2 3 4 5 6 7 8 9 10 11 12 13 14
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
Band 1 Band 2 Band 3 Band 4
Plant A 1 0 0 1
Plant B 0 1 0 1
Plant C 1 1 1 0
Plant D 1 1 0 1
Plant E 0 1 0 1
Plant F 1 0 0 1
Plant G 1 0 1 0
A binary matrix:
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)
Table 1. Analysed populations of lingonberry, number of plants sampled, location of population, within-population gene diversity(including standard error) estimated by the Lynch and Milligan index (Hpop) and Shannon’s index (H’pop)
Samplingsite no.
Samplingsite code
No. ofplants
Country Location Latitude(N)
Longitude(E)
Hpop H’pop
1 SK 15 Sweden Kristianstad 56°13´ 14°12´ 0.227 (0.026) 0.483 (0.049) 2 SÖ 15 Sweden Örebro 59°24´ 14°39´ 0.214 (0.025) 0.517 (0.051) 3 SV 15 Sweden Västerbotten 63°37´ 19°51´ 0.245 (0.027) 0.513 (0.050) 4 SN 10 Sweden Norrbotten 66°42´ 19°33´ 0.197 (0.026) 0.400 (0.051) 5 SG 13 Sweden Gävleborg 60°18´ 16°46´ 0.248 (0.028) 0.523 (0.048) 6 SH 11 Sweden Halland 57°05´ 13°20´ 0.219 (0.026) 0.500 (0.051) 7 FT 15 Finland Toijala 60°13´ 24°10´ 0.187 (0.026) 0.375 (0.049) 8 FS 15 Finland Simo 65°41´ 25°01´ 0.178 (0.026) 0.374 (0.050) 9 NS 15 Norway Sognedal 61°12´ 7°05´ 0.225 (0.029) 0.434 (0.055)10 EV 15 Estonia Vöru 57°55´ 27°03´ 0.241 (0.029) 0.456 (0.055)11 EP 10 Estonia Pärnu 58°25´ 24°40´ 0.209 (0.027) 0.434 (0.055)12 RM 15 Russia Murmansk 68°55´ 33°05´ 0.110 (0.024) 0.190 (0.043)
13 RK 14 Russia Kirov 58°53´ 49°30´ 0.135 (0.024) 0.265 (0.047)14 JF 14 Japan Fuji San 35°20´ 138°45´ 0.180 (0.028) 0.349 (0.056)15 CM 15 Canada Montreal 45°50´ 73°50´ 0.274 (0.025) 0.654 (0.048)
x = 0.206 x = 0.431
Evaluation of genetic diversity in Lingonberry populations
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)
Genetic relatedness among populations of lingonberry (A) and indidual plants of Japanese quince (B)
revealed by cluster analyses
100 90 80 70 60 50 40
SH
SN
FT
SV
SK
SÖ
RM
RK
SG
EV
EP
NS
CM
JF
Similarity (%)
FS
Fig.1. Dendrogram based on UPGMA analysis of genetic similarity estimates among 15 populations of lingonberry
A B
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
A three-dimentional representation of phenetical relationships between populations of Japanese quince revealed by PCA
GravensteinerRöd GravensteinerIngrid MarieGuldborgJames GrieveMaglemer
Cox OrangeAliceGrågylling från SkoklosterVit astrakanStor klar astrakanArvidsäpple
OranieSpässerud
SärsöHanaskog
ÅkeröÅkerö från Gripsholm
FageröFlädie
KavlåsJohn-GeorgGolden Delicious
100 80 60 5070 40 30
Likhet %
Fig.1. Dendrogram based on UPGMA analysis (Jaccard’s coefficient) for RAPD data, showing relationships among apple cultivars
Similarity %
Genetic relationships among 23 cultivars from Gene bank at Balsgård revealed by RAPD markers
Advantages, limitations and applications of RAPD markers
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
• 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!
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
• The scoring of RAPD bands is open to interpretation
• The results are not easily reproducible between laboratories
Applications:
• Measurements of genetic diversity
• Genetic structure of populations
• Germplasm characterisation
• Verification of genetic identity
• Genetic mapping
• Development of markers linked to a trait
of interest
• Cultivar identification
• Identification of clones (in case of soma-
clonal variation)
• Interspecific hybridization
• Verification of cultivar and hybrid purity
• Clarification of parentage
RAPD is probably the cheapest and easiest DNA method for laboratories just beginning to use molecular markers
Thank you