Abstract
Amplified fragment length polymorphism (AFLP) is a powerful fingerprinting technique that is widely applied in ecological and population genetic studies. However, its routine use has been limited by high costs associated with the optimization of fluorescently labelled markers, especially for individual study systems. Here we develop a low-cost AFLP protocol that can be easily transferred between distantly related plant taxa. Three fluorescently labelled EcoRI-primers with anchors that target interspecifically conserved genomic regions were used in combination with a single non-labelled primer in our AFLP protocol. The protocol was used to genotype one gymnosperm, two monocot and three eudicot plant genera representing four invasive and four native angiosperm species (Pinus pinaster (Pinaceae), Pennisetum setaceum and Poa annua (Poaceae), Lantana camara (Verbenaceae), Bassia diffusa (Chenopodiaceae), Salvia lanceolata, Salvia africana-lutea, and Salvia africana-caerulea (Lamiaceae)). Highly polymorphic and reproducible genotypic fingerprints (between 37–144 polymorphic loci per species tested) were obtained for all taxa tested. Our single protocol was easily transferred between distantly related taxa. Measures of expected heterozygosity ranged from 0.139 to 0.196 for P. annua and from 0.168 to 0.272 for L. camara which compared well with previously published reports. In addition to ease of transferability of a single AFLP protocol, our protocol reduces costs associated with commercial kits by almost half. The use of highly conserved but abundant anchor sequences reduces the need for laborious screening for usable primers that result in polymorphic fingerprints, and appears to be the main reason for ease of transferability of our protocol between distantly related taxa.
Highlights
Amplified Fragment Length Polymorphism (AFLP [1]) is a versatile technique for genome-wide screening of genetic diversity and can be applied to almost any organism [2,3,4]
Our Amplified fragment length polymorphism (AFLP) protocol yielded highly polymorphic loci and was successfully transferred between the eight species included here and generated a minimum of 5.26% polymorphic loci for P. setaceum, and a maximum of 100% polymorphic loci for B. diffusa
The Salvia hybrids yielded 99 loci of which 48.86% were polymorphic. This is the first report of AFLP fingerprints for B. diffusa, P. setaceum and the three Salvia species
Summary
Amplified Fragment Length Polymorphism (AFLP [1]) is a versatile technique for genome-wide screening of genetic diversity and can be applied to almost any organism [2,3,4]. The technique relies on detecting genetic polymorphisms through differential endonuclease restriction digestion of genomic DNA. One of the biggest advantages of AFLP technology is that, unlike many other genotyping techniques, genome wide screening of genetic diversity is possible without a priori knowledge of genome sequences [4,6]. The technique was originally used for constructing high density linkage maps [1,7], but is widely applied today to estimate genetic diversity, assign parentage, determine population structure and reconstruct shallow phylogenies (reviewed by [2]). AFLP analyses have been adapted to assess epigenetic variation [8,9] and differential gene expression [4,10]
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