Generation and Analysis of AFLP Data

ESPM 150/290: Biology, Ecology, and Genetics of Forest Diseases

Laboratory ExerciseApril 1, 2010

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

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

1. Double strand denaturation

2. Annealing of the primers

3. Elongation

5’5’

5’

3’3’

A review of PCR amplificationRequirements: DNA template 2 oligonucleotides - Primers Nucleotides dATP, dCTP, dGTP, dTTP Taq polymerase

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

http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/R/RestrictionEnzymes.html

Tobin & Dusheck, Asking About Life, 2nd ed. Copyright 2001, Harcourt, Inc.

Random Genomic Markers

DNA sequence of suitable SNPs is not availableRelatively inexpensiveScan the entire genome producing information

on several variations in the same reaction

RAPD Random Amplification of Polymorphic DNA

AFLP Amplified Fragment Length Polymorphism

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

GAATTCCTTAAG

TTAAAATT

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)

NN N N

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

NNNNNN NNN NNN

AFLP OVERVIEW(VOS ET AL., 1995)

Sample AFLP Gel

AFLP Electropherogram

Source: Wikimedia Commons

Peak Height

Fragment Size (bp)

AFLPFluorescent electrophoresis

AFLP Data Map from Urbanelli et al. (2007)

Rows: individuals

Columns: alleles

AFLP genotyping

PCR amplification using primers corresponding to the new sequenceIf there are 2 new priming sites within 400 – 1600 bp there is amplification

The result is: Presence or absence of amplification1 or 0Dominant marker: does not distinguish between heterozygote and homozygote

Due mostly to SNPs but also to deletions/insertions

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

Image Source: http://media.wiley.com/CurrentProtocols/BI/bi0603/bi0603-fig-0002-1-full.gif

Example of a sequence distance matrix

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

Image Source: http://media.wiley.com/CurrentProtocols/BI/bi0603/bi0603-fig-0002-1-full.gif

Example of a sequence distance matrix

AFLP Clustering Analysis

Source: Wikimedia Commons

Clustering Dendrogram Fragment Visualization

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) http://steinpilz.up.seesaa.net

Example Structure Output

Rosenberg et al. (2002). Science 298: 2381-2385.

“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: http://rosenberglab.bioinformatics.med.umich.edu/clumppExample.html)