rapd markers l.
Download
Skip this Video
Loading SlideShow in 5 Seconds..
RAPD markers PowerPoint Presentation
Download Presentation
RAPD markers

Loading in 2 Seconds...

play fullscreen
1 / 35

RAPD markers - PowerPoint PPT Presentation


  • 1862 Views
  • Uploaded on

RAPD markers. Larisa Gustavsson (Garkava) Balsgård-Department of Crop Sciences Swedish University of Agricultural Sciences. What is RAPD?.

loader
I am the owner, or an agent authorized to act on behalf of the owner, of the copyrighted work described.
capcha
Download Presentation

PowerPoint Slideshow about 'RAPD markers' - Ava


An Image/Link below is provided (as is) to download presentation

Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author.While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server.


- - - - - - - - - - - - - - - - - - - - - - - - - - E N D - - - - - - - - - - - - - - - - - - - - - - - - - -
Presentation Transcript
rapd markers

RAPD markers

Larisa Gustavsson (Garkava)

Balsgård-Department of Crop Sciences

Swedish University of Agricultural Sciences

slide2

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
RAPD technology

A

B

C

A

+

+

+

Arbitrary primers

Taq polymerase

Nucleotides

+

Genomic DNA

PCR

(under relaxed conditions)

Buffer

slide4

A

B

C

520bp

360bp

260 bp

PCR

360 bp

Electrophoresis

260 bp

520 bp

A

B

C

pcr product occurs when
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)
slide6

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

slide8

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

slide9

2

1

3

4

5

6

DNA template

PCR reaction

Product 1

Product 2

or detected fragment is of increased size

b insertion or deletion of a small fragment of dna the amplified fragments are changed in 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
slide11

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
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
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

slide17

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
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)

slide21

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)

slide22
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

slide23
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

slide24
A three-dimentional representation of phenetical relationships between populations of Japanese quince revealed by PCA
slide25

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
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
slide28
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
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
slide30
The scoring of RAPD bands is open to interpretation
  • The results are not easily reproducible between laboratories
applications
Applications:
  • Measurements of genetic diversity
  • Genetic structure of populations
  • Germplasm characterisation
  • Verification of genetic identity
  • Genetic mapping
slide32
Development of markers linked to a trait

of interest

  • Cultivar identification
  • Identification of clones (in case of soma-

clonal variation)

  • Interspecific hybridization
slide34
RAPD is probably the cheapest and easiest DNA method for laboratories just beginning to use molecular markers