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RAPD markers. Larisa Gustavsson (Garkava) Balsgård-Department of Crop Sciences Swedish University of Agricultural Sciences. What is RAPD?.
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RAPD markers Larisa Gustavsson (Garkava) Balsgård-Department of Crop Sciences Swedish University of Agricultural Sciences
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
RAPD technology A B C A + + + Arbitrary primers Taq polymerase Nucleotides + Genomic DNA PCR (under relaxed conditions) Buffer
A B C 520bp 360bp 260 bp PCR 360 bp Electrophoresis 260 bp 520 bp 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)
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
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
2 1 3 4 5 6 DNA template PCR reaction Product 1 Product 2 or detected fragment is of increased size
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
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
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”
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
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
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)
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 B A Fig.1. Dendrogram based on UPGMA analysis of genetic similarity estimates among 15 populations of lingonberry
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
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
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-migratingbands 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