Molecular markers

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Molecular markers

• PCR based
• A-priori sequence
• knowledge
• not required

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PCR markers a prior sequence knowledge

• RAPD
• AFLP
• SCARS
• CAPS
• AP-PCR
• RAMPO

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RAPD

• Randomly Amplified Polymorphic DNA
• Using a short primer (8-12 nucleotides)
• No prior sequence knowledge is required
• Require intact genome
• Dominant marker
• A major short fall Lack of reproducibility
• Welsh, J. and McCelland M. Nucleic Acid Res 1990
  187213-7218
• Williams et al. Nucleic Acid Res. 1990
  186531-6535

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3 individuals
Oligos (8 to 12 nucleotides)
Will produce products when primers are close
together to produce fragment sizes that can be
visualized
Oligos will anneal on both strands search for
palidome sequences on both strands
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PCR products formed for all individuals
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PCR products for only two individuals
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• RAPD marker has a major problem with dominance
• Previous example of individuals are shown as 2N
• In the next slide we will use chromatids (4 per
  individual) to demonstrate dominance

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RAPD Gels
size ladder
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Example of RAPD gel
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Scoring RAPD gel
Sample name (120bp) (130bp) (180bp) (220bp)
1101 1 1 0 1
1102 1 0 0 0
1103 0 1 1 1
1104 0 0 0 1
Sample name Locus A (Allele type) Locus B Locus C Locus D
1 2 2 1 2
2 1 2 1 2
3 2 1 2 1
4 1 2 1 1
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A B C D E F G H I J
2000bp   800bp 600bp   300bp   100 bp  
Score the following RAPD gel for these 10 samples
(A-J). Indicate the loci you are scoring with an
arrow on the right side of the image
  Ladder (bp) A B C D E F G H I J
1                      
2                      
3                      
4                      
5                      
6                      
7                      
8                      
9                      
10                      

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Issues to consider

• Some reproducibility problems, needs to use same
  lot for all chemicals eg. buffer, Taq, dNTP etc.
• Same band on gel same DNA fragment?
• One band on gel one DNA fragment?
• (Allele homoplasy)
• Selecting the band or lack of them to score

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More issues.

• Anonymous markers - but can be converted to SCARs
  or CAPs
• Dominant markers - homozygotes cannot be
  distinguished from heterozygotes
• Fast, easy and cheap - commercial primer sets
  available
• Scoring is subjective and individual dependent

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Applications

• Genetic Maps
• Fingerprinting isolates and cultivars
• Limited use today

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AFLP

• Amplified Fragment Length Polymorphism
• Very sensitive
• Good reproducibility but can be technically
  demanding
• Combining RFLP and RAPD technique
• Dominant marker
• Generate fingerprint
• Can use DNA and cDNA
• Vos et al. Theor. Appl. Genet. 1995 234407-4414

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AFLP flowchart
Courtesy of Ritland and Ritland
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AFLP flowchart
Courtesy of Ritland and Ritland
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AFLP flowchart
Courtesy of Ritland and Ritland 2000 Molecular
Methods in Ecology
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Simple
Complex
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A
B
C
D
E
Samples 1 2 3 4 5 27 28
Linanthus Courtesy of Carol Goodwille
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Sample Locus A (bp) Locus B Locus C Locus D Locus E
1 1 1 0 1 0
2 0 1 0 1 1
3 1 0 1 1 1
4 1 0 1? 1 1
5 1 1 0 1 0
Example of sampling from Linanthus data (see
previous slide for image)
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Scoring AFLP exercise
Samples 16 (A-P)
Indicate the loci you are scoring with an arrow
on the right side of the image
  2550bp   2300bp   2004bp 2000bp   1750 bp  
  Size (bp) A B C D E F G H I J K L M N O P
1                                  
2                                  
3                                  
4                                  
5                                  
6                                  
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Issues to consider

• Discriminating homozygotes from heterozygotes
• requires band quantification (possible gel
  scanner)
• Bands are anonymous - interpretation of patterns
• can be challenging
• Same position equal only one band?
• Dominant marker (difficult to test
  Hardy-Weinberg)
• Can try to look for co-dominant bands
• More expensive than RAPD markers
• Much more repeatable than RAPD markers
• Need to be technically consistent
• Need clean intact genomes and min. amount (500ng)
• May not be useful for population assignment
  pending on population structure
• Subjective scoring problem much like RAPD markers
• Non independence bands are difficult to detect,
  problem for phylogeny construction

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Applications for AFLP

• Physical mapping
• Genome mapping
• Population structure (clone detection)
• Genetic diversity
• DNA fingerprinting isolates or cultivars
• Detection of somatic clone contaminations
• Can convert segregating bands to co-dominant
  markers
• Forensic sciences
• QTL analysis
• Locating possible genes related to complex traits
  (cDNA template)

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Clonality study

• Wilson A.S.G., van der Kamp, B.J. and Ritland C.
  (2005) Can J. Bot 831126-1132

• Clone identification
• Clone diversity
• Spatial structure

Maianthemum dilatatum
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Comparing RAPD to AFLP

• Kjolner, S., Sastad, S.M.,Taberlet, P. and
  Brochmann, C. (2004) Molecular Ecology 1381-86
• 4 populations (13-15 individuals per population)

Saxifraga cernua
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Comparing AFLP and RAPD

• Both markers produced similar results in
  estimating clone identity, clone relationships,
  gene diversity, linkage disequilibrium
• AFLP is superior in terms of efficiency but RAPD
  may still be used as a cheaper method
• Major caution with repeatability