The core of my dissertation involves assessing genome-wide patterns of gene flow during anole speciation. For a variety of reasons, I ultimately want to acquire DNA sequences from throughout the genome. As a first pass, however, I’ve been using Amplified Fragment Length Polumorphisms (AFLPs). As a molecular technique, AFLPs are experiencing a comeback of sorts [1] [2]. Popular in the early 2000’s, AFLPs went out of favor as Sanger sequencing became cheaper and easier. The resurgence AFLPs has largely been due to the realization that evolutionary patterns often vary throughout genomes and therefore methods that survey as much of the genome as possible are preferable to those that look at one or a few regions.
Among genome-wide analyses, AFLPs stand out: they are cheap, can be collected quickly, do not require taxon specific development (as in microsatellites), and can be used in phylogenetic, population genetic and quantitative genetic studies. Perhaps most importantly, they serve as a starting point for restriction associated DNA sequencing, a popular and useful way to use Next-Gen sequencing technologies to randomly sample homologous regions from multiple individuals.
I first used AFLPs to assess population structure in Diplodactylid geckos and I have adapted that protocol (that can be downloaded here) to our current work on anoles. So far, we have performed analyses on the Webster and Burns reinforcement transect and across all distichus clade populations. We’ve had great results so far. For instance, by comparing the results of an AFLP analysis of distichoids to an in silico AFLP analysis of the carolinensis genome we found that some of the unique characteristics to the A. carolinensis genome may be conserved in distichoids, despite a relatively old common ancestor.
AFLPs have inherent drawbacks: they are anonymous, dominant (you can’t identify heterozygous loci), and homology can’t be directly assessed. Eventually, as genome sequencing becomes cheaper this technique will again fade into obscurity, but for now I find them to be a useful method to gain insight into genomic processes without having access to genome sequences.
- John David Curlis - March 1, 2022
- Joe Macedonia - January 31, 2022
- Lindsey Swierk - January 27, 2022
Thomas Sanger
I have never looked at AFLP data and its been years since I even thought about them. Can you please explain what we are looking at in the figure? I see colors, lanes, and bands but don’t know what any of them represent.
geneva
AFLP fragments are generated by digestion of genomic DNA using two restriction enzymes. Adaptors are then annealed to the resulting fragments. The total number of fragments is large (too many to analyze via capillary electrophoresis) and depends on the size of the organism’s genome, it’s GC content and the recognition sites of the restriction enzymes. A subset of these fragments are amplified using primers (one of which is fluorescently labelled) that match the adaptor and restriction site sequences as well as one or more bases into the fragments. This reduced set is then scored via capillary electrophoresis. By comparing the electropherograms of multiple individuals you can create a binary matrix of fragment presence and absence.
Regarding the figure in the post… its there for its visual appeal, not for analytical purposes. Its a pseudogel, where the size and intensity of peaks scored via capillary electrophoresis are translated to a visual “gel” of the data. Each column represents the fragments generated by a single individual. The two colors represent different primer combinations scored via different fluorescent dyes.