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