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 Genomic DNA

Taq

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

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

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 1 2 3 DNA template 4 5 PCR reaction 6 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 2 1 3

DNA template

4 5 PCR reaction No product Product 2 6

or detected fragment is of increased size 2 1 3

DNA template

4 5 PCR reaction Product 1 6 Product 2

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

c) insertion of a large piece of DNA between the primer -binding sites may exceed the capacity of PCR - no fragment is detected 2

The insertion of large fragment

3 DNA template No product 5 PCR reaction Product 2 6

A schematic picture of an agarose gel

Marker Plant A Plant B Plant C Presens of a band, ”1” + Absence of a band, ”0” Monomorphic bands Polymorphic bands

And a real picture of a gel…

… and one more

Data analysis

Locus A Locus B Locus C Locus D

RAPD bands are treated as independent loci:

1 2 3 4 5 6 7 8 9 10 11 12 13 14

1

AA/ Aa

bb

CC/ Cc

dd 2 aa 3 aa 4 5 aa aa 6 aa 7 aa 8 aa 9 10 11 12 13 14 aa aa

BB/ Bb

cc

BB/ Bb

cc

BB/ Bb

cc

BB/ Bb

cc

DD/ Dd DD/ Dd DD/ Dd DD/ Dd BB/ Bb

cc

BB/ Bb

cc

DD/ Dd DD/ Dd BB/ Bb

cc

BB/ Bb

cc

DD/ Dd DD/ Dd

bb

CC/ Cc DD/ Dd

aa bb aa bb

CC/ Cc DD/ Dd CC/ Cc DD/ Dd

aa bb aa bb

CC/ Cc

dd cc DD/ Dd

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

Plant A Plant B Plant C Plant D Plant E Plant F Plant G

A

binary

matrix:

Band 1 1 1 1 0 1 1 0 Band 2 0 0 0 1 1 1 1 Band 3 0 1 0 0 1 0 0 Band 4 1 0 1 1 0 1 1

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)

Evaluation of genetic diversity in Lingonberry populations

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Sampling site no.

Table 1. Analysed populations o f lingonb erry, number of plants sampled, location o f population , within-population gen e diversity (including s tandard error) estim ated by the Lynch and Mil ligan index (H

pop

) and Shannon’ s index (H’

pop

) Sampling site code No. of plants Country Location Latitud e (N) Longi tude (E) H

pop

H’

pop

SK SÖ SV SN SG SH FT FS NS EV EP RM RK JF CM 15 15 15 10 13 11 15 15 15 15 10 15 14 14 15 Sweden Sweden Sweden Sweden Sweden Sweden Finland Finland Norway Estonia Estonia Russia Russia Japan Canada Kristianstad Örebro Västerbotten Norrbotten Gävleborg Halland Toijala Simo Sogneda l Vöru Pärnu Murmansk Kirov Fuji San Montreal 56°13´ 59°24´ 63°37´ 66°42´ 60°18´ 57°05´ 60°13´ 65°41´ 61°12´ 57°55´ 58°25´ 68°55´ 58°53´ 35°20´ 45°50´ 14°12´ 14°39´ 19°51´ 19°33´ 16°46´ 13°20´ 24°10´ 25°01´ 7°05 ´ 27°03´ 24°40´ 33°05´ 49°30´ 138°45´ 73°50´ 0.227 (0.026) 0.483 (0.049) 0.214 (0.025) 0.517 (0.051) 0.245 (0.027) 0.513 (0.050) 0.197 (0.026) 0.400 (0.051) 0.248 (0.028) 0.523 (0.048) 0.219 (0.026) 0.500 (0.051) 0.187 (0.026) 0.375 (0.049) 0.178 (0.026) 0.374 (0.050) 0.225 (0.029) 0.434 (0.055) 0.241 (0.029) 0.456 (0.055) 0.209 (0.027) 0.434 (0.055) 0.110 (0.024) 0.190 (0.043) 0.135 (0.024) 0.265 (0.047) 0.180 (0.028) 0.349 (0.056) 0.274 (0.025) 0.654 (0.048) x = 0.206 x = 0.431

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 Similarity (%) 100 90 80 70 60 50 40 SH SN FT SV FS

A

SK SÖ RM RK SG EV EP NS CM JF Fig.1. Dendrogram based on UPGMA analysis of genetic similarity estimates among 15 populations of lingonberry

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

Genetic relationships among 23 cultivars from Gene bank at Balsgård revealed by RAPD markers Gravensteiner Röd Gravensteiner Ingrid Marie Guldborg James Grieve Maglemer Cox Orange Alice Grågylling från Skokloster Vit astrakan Stor klar astrakan Arvidsäpple Oranie Spässerud Särsö Hanaskog Åkerö Åkerö från Gripsholm Fagerö Flädie Kavlås John-Georg Golden Delicious 100 80 70 60 50 40 30 Fig.1. Dendrogram based on UPGMA analysis (Jaccard’s coefficient) for RAPD data, showing relationships among apple cultivars

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