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Molecular markers

Molecular markers. PCR based A-priori sequence knowledge not required. PCR markers a prior sequence knowledge. RAPD AFLP SCARS CAPS AP-PCR RAMPO. RAPD. R andomly A mplified P olymorphic D NA Using a short primer (8-12 nucleotides) No prior sequence knowledge is required

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Molecular markers

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  1. Molecular markers PCR based A-priori sequence knowledge not required courtesy of Carol Ritland

  2. PCR markers a prior sequence knowledge • RAPD • AFLP • SCARS • CAPS • AP-PCR • RAMPO

  3. 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 18:7213-7218 • Williams et al. Nucleic Acid Res. 1990 18:6531-6535

  4. 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

  5. PCR products formed for all individuals

  6. PCR products for only two individuals

  7. 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

  8. RAPD Gels = size ladder

  9. Example of RAPD gel

  10. Scoring RAPD gel

  11. 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

  12. 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

  13. 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

  14. Applications • Genetic Maps • Fingerprinting isolates and cultivars • Limited use today

  15. 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 23:4407-4414

  16. AFLP flowchart: Courtesy of Ritland and Ritland

  17. AFLP flowchart: Courtesy of Ritland and Ritland

  18. AFLP flowchart: Courtesy of Ritland and Ritland 2000 Molecular Methods in Ecology

  19. Simple Complex

  20. A B C D E Samples 1 2 3 4 5 27 28 Linanthus Courtesy of Carol Goodwille

  21. Example of sampling from Linanthus data (see previous slide for image)

  22. 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

  23. 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

  24. 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)

  25. Clonality study • Wilson A.S.G., van der Kamp, B.J. and Ritland C. (2005) Can J. Bot 83:1126-1132 • Clone identification • Clone diversity • Spatial structure Maianthemum dilatatum

  26. Comparing RAPD to AFLP • Kjolner, S., Sastad, S.M.,Taberlet, P. and Brochmann, C. (2004) Molecular Ecology 13:81-86 • 4 populations (13-15 individuals per population) Saxifraga cernua

  27. 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

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