1. Generation and Analysis of AFLP Data
ESPM 150/290: Biology, Ecology, and Genetics of Forest Diseases
Laboratory Exercise
April 1, 2010

2. Some Considerations in Choosing a Genotyping Method
What is the level of taxonomic resolution desired? (Populations? Species? Phyla?)
Comparison of distantly related individuals requires slowly evolving markers (e.g., protein-coding DNA or Amino Acid sequences) due to saturation of changes in quickly-evolving markers
Comparison of closely related individuals requires rapidly evolving markers (e.g., microsatellites or non-coding DNA sequences)
What is the level of genotypic resolution desired?
Dominant vs. codominant markers
Fine (e.g., nucleotide-level) data vs. coarse (e.g., fragment size) genomic scale – detailed information about one or a few loci vs. less-detailed information about more loci

3. Some Considerations in Choosing a Genotyping Method
How much previous sequence knowledge is available?
DNA sequencing, microsatellite amplification, PCR-RFLP, etc. require previous sequence information so that PCR primers can be designed
AFLPs and RAPDs allow genetic fingerprinting when previous sequence knowledge is not available
What are the cost and labor constraints?
DNA sequencing is more costly than fragment analysis
Techniques requiring fluorescent labeling are generally more costly than techniques that don’t require labeling

4. A review of PCR amplification
Requirements:
DNA template
2 oligonucleotides - Primers
Nucleotides dATP, dCTP, dGTP, dTTP
Taq polymerase
Double strand denaturation
Annealing of the primers
Elongation 5’ 3’

5. Restriction Enzymes Found in bacteria
Cut DNA within the molecule (endonuclease)
Cut at sequences that are specific for each enzyme (restriction sites)
Leave either blunt or sticky ends, depending upon the specific enzyme
Tobin & Dusheck, Asking About Life, 2nd ed. Copyright 2001, Harcourt, Inc.

6. Random Genomic Markers
DNA sequence of suitable SNPs is not available
Relatively inexpensive
Scan the entire genome producing information on several variations in the same reaction
RAPD Random Amplification of Polymorphic DNA
AFLP Amplified Fragment Length Polymorphism

7. AFLP Amplified Fragment Length Polymorphisms (Vos et al., 1995)
Genomic DNA digested with 2 restriction enzymes:
EcoRI (6 bp restriction site)
cuts infrequently
MseI(4 bp restriction site)
cuts frequently
GAATTC
CTTAAG
TTAA
AATT
Technique similar to RFLP Restriction Fragment Length Polymorphisms and RAPD Random Amplification of Polymorphic DNA.
As RFLP is based on restriction digestion but, like RAPD uses random amplification of genomic DNA.
Genome is digested using 2 enzymes, 1 frequent cutter MseI (4 bp) and 1 infrequent cutter (EcoRI).

8. Fragments of DNA resulting from restriction digestion are ligated with end-specific adaptors (a different one for each enzyme) to create a new PCR priming site
Pre selective PCR amplification is done using primers complementary to the adaptor + 1 bp (chosen by the user) N N N N

9. Selective amplification using primers complementary to the adaptor (+1 bp) + 2 bp
NNN
NNN
NNN
NNN

10. AFLP OVERVIEW (VOS ET AL., 1995)

11. Sample AFLP Gel

12. AFLP Electropherogram
Peak Height
Fragment Size (bp)
Source: Wikimedia Commons

13. AFLP Fluorescent electrophoresis

14. AFLP Data Map from Urbanelli et al. (2007)
Rows: individuals
Columns: alleles

15. AFLP genotyping
PCR amplification using primers corresponding to the new sequence
If there are 2 new priming sites within 400 – 1600 bp there is amplification
The result is: Presence or absence of amplification
1 or 0
Dominant marker: does not distinguish between heterozygote and homozygote
Due mostly to SNPs but also to deletions/insertions

16. Analysis of AFLP data Similarity (cluster analysis)
NJ (Neighbor Joining)
UPGMA (Unweighted Pair Group Method with Arithmetic mean)
AMOVA (Analysis of Molecular Variance)
Model-based
Maximum likelihood
Bayesian
Example of a sequence distance matrix
Image Source:

17. Analysis of AFLP data Similarity (cluster analysis)
NJ (Neighbor Joining)
UPGMA (Unweighted Pair Group Method with Arithmetic mean)
AMOVA (Analysis of Molecular Variance)
Model-based
Maximum likelihood
Bayesian
Example of a sequence distance matrix
Image Source:

18. AFLP Clustering Analysis
Clustering Dendrogram
Fragment Visualization
Source: Wikimedia Commons

19. AFLP Data Map with UPGMA dendogram from Urbanelli et al
AFLP Data Map with UPGMA dendogram from Urbanelli et al. (2007): “Distinguishing taxa in the Pleurotus eryngii (King Oyster Mushroom) complex using AFLPs”
90 populations sampled
94 AFLP loci scored
Photos: (Top) The New York Times
(Bottom L) Wikimedia Commons
(Bottom R)

20. Example Structure Output
“Estimated population structure for 10 runs of structure using 1056 individuals from 52 human populations. Each graph represents the output of one run of structure. In each graph, each individual is represented by a vertical line, which is partitioned into 5 colors that represent its estimated membership fractions in K=5 clusters.”
(Source:
Rosenberg et al. (2002). Science 298: