Impact of Amplified Fragment Length Polymorphism Size Homoplasy on the Estimation of Population Genetic Diversity and the Detection of Selective Loci

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AFLP markers are becoming one of the most popular tools for genetic analysis in the fields of evolutionary genetics and ecology and conservation of genetic resources. The technique combines a high-information content and fidelity with the possibility of carrying out genomewide scans. However, a potential problem with this technique is the lack of homology of bands with the same electrophoretic mobility, what is known as fragment-size homoplasy. We carried out a theoretical analysis aimed at quantifying the impact of AFLP homoplasy on the estimation of within- and between-neutral population genetic diversity in a model of a structured finite population with migration among subpopulations. We also investigated the performance of a currently used method (DFDIST software) to detect selective loci from the comparison between genetic differentiation and heterozygosis of dominant molecular markers, as well as the impact of AFLP homoplasy on its effectiveness. The results indicate that the biases produced by homoplasy are: (1) an overestimation of the frequency of the allele determining the presence of the band, (2) an underestimation of the degree of differentiation between subpopulations, and (3) an overestimation or underestimation of the heterozygosis, depending on the allele frequency of the markers. The impact of homoplasy is quickly diminished by reducing the number of fragments analyzed per primer combination. However, substantial biases on the expected heterozygosity (up to 15-25%) may occur with ~50-100 fragments per primer combination. The performance of the DFDIST software to detect selective loci from dominant markers is highly dependent on the number of selective loci in the genome and their average effects, the estimate of genetic differentiation chosen to be used in the analysis, and the critical bound probability used to detect outliers. Overall, the results indicate that the software should be used with caution. AFLP homoplasy can produce a reduction of up to 15% in the power to detect selective loci.

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THE amplified fragment length polymorphism (AFLP) technique (Vos et al. 1995) is becoming one of the most popular methods in the fields of conservation and evolutionary genetics and ecology (MUELLER and WOLFENBARGER 1999; BENSCH and AKESSON 2005; BONIN et al. 2007; MEUDT and CLARKE 2007), as it combines a high reproducibility and information content with the possibility of making genomewide screenings. Because of the anonymous nature of the fragments generated by the AFLP technique, however, one major concern is the incidence of size homoplasy due to the lack ofhomology of comigrating fragments. This implies that fragments of a given size migrating in a band may involve more than one locus of the genome and, therefore, the inferences obtained from thebandcan produce misleading conclusions.

Several empirical approaches have been used to estimate levels of homoplasy in AFLP data sets. A few studies have demonstrated the presence of homoplasy in AFLP data by sequencing comigrating fragments within the same individual or from different individuals of the same or different species (ROUPPE VAN DER VOORT et al. 1997; PETERS et al. 2001; EL-RABEY et al. 2002; ROMBAUTS et al. 2003; MECHANDA et al. 2004;MENDELSON and SHAW 2005). HANSEN et al. (1999) and O'HANLON and PEAKALL (2000) developed a simplifiedmethod for detecting size homoplasy comparing the AFLP banding patterns resulting from several rounds of selective amplification using PCR primers differing in the number of selective nucleotides. This method revealed that the proportion of comigrating nonhomologous fragments within single individuals of sugar beet was 13% (Hansen et al. 1999) and as high as 100% for comparisons among pairs of individuals from distantly related taxa of Carduinae thistles (O'Hanlon and Peakall 2000). Moreover, it has been shown that in interspecific studies of Echinacea (Mechanda et al. …