Abstract
BackgroundDiscriminating taxa with the nuclear marker, amplified fragment length polymorphism (AFLP) has been accomplished for various organisms in economic, ecological, and evolutionary studies. The protocol available for AFLP generation does not require prior knowledge of the genome; however, it is often extensively modified to fit the needs of the researcher. Modification of this protocol for new labs is intimidating and time-consuming, particularly for taxa in which AFLP have not been previously developed. Furthermore, determining what constitutes quality output during different stages of fragment generation is not well defined and this may further hinder the use AFLP by new researchers.FindingsWe present a step-by-step AFLP protocol, using flourophore-labeled primers for use with automated sequencers, including examples of both successful and unsuccessful results. We sufficiently normalized peak intensity and standardized allele calling across all samples for each primer combination. Repeatability was assessed with a phylogenetic tree in which replicate samples clustered together using the minimum evolution procedure. We found differences greater than 10% in allele position among replicated samples would cause replicates to no longer cluster. To minimize offset allele positions, we suggest that researchers analyze different primer combinations at the same time using multiple dyes with the automated sequencer to minimize mismatched alleles across replicates.ConclusionFor researchers wanting to use AFLP, this molecular technique is difficult and time-consuming to develop. Clarifying what constitutes quality output for each step in AFLP generation will help to reduce redundant trials in protocol development and, in turn, advance the discipline of population genetics.
Highlights
Discriminating taxa with the nuclear marker, amplified fragment length polymorphism (AFLP) has been accomplished for various organisms in economic, ecological, and evolutionary studies
Amplified fragment length polymorphism (AFLP) has been extensively used to investigate population genetics [1,2], genome mapping [3,4], and genetic structure of intra- and interspecific taxa [5,6,7], especially in plants, microbes, and fungi, but less often for animal taxa [8]. This method has many benefits over other genetic techniques for addressing questions in population genetics including: low start-up cost, high repeatability, the ability to assay a large number of polymorphic loci in many individuals in a relatively short period of time, and no prior knowledge of the genome or sequence data is necessary [8,9,10,11,12,13,14]
There is a standard protocol available for AFLP generation [9] that has been modified by researchers for their specific study taxa [8]
Summary
Discriminating taxa with the nuclear marker, amplified fragment length polymorphism (AFLP) has been accomplished for various organisms in economic, ecological, and evolutionary studies. The protocol available for AFLP generation does not require prior knowledge of the genome; it is often extensively modified to fit the needs of the researcher Modification of this protocol for new labs is intimidating and time-consuming, for taxa in which AFLP have not been previously developed. Understanding and interpretation of successful results can be challenging to researchers first using AFLP and redundant trials in protocol development can hinder advancement of research Because of these challenges, the objectives of this study were to: (1) provide guidelines for AFLP generation with automated analyses using a protocol amenable for disparate animal groups; (2) construct procedures to normalize and standardize AFLP electropherograms; and (3) test the repeatability of AFLP samples processed with an automated capillary sequencer and analyzed with applicable software
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