Towards a transferable and cost-effective plant AFLP protocol

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.

Figure 1. Typical fingerprint profiles generated with the primer pairs: EcoRI-ATG- Hex™+Mse-CTT for A) two Poa annua samples and D) 2 Pinus pinaster samples; B) EcoRI-AAT-Ned+Mse-CTT for two Pennisetum setaceum and E) two Bassia diffusa samples samples; and EcoRI-CAT-FAM+Mse-CTT for C) two Lantana camara samples, and F) two Salvia spp. samples.

Figure 2. A comparison of mean fluorescence intensity (±standard error, SE) between the species sampled.

No significant differences were found between the invasive species (dark grey). The four native species and Salvia hybrids (shown in light grey) differed significantly from all of the invasive species (with exceptions), but not from each other (Kruskal-Wallis Chi squared = 125.9, df = 8, P<0.0001). Samples with the same letters do not differ significantly, whilst different letters indicate significant differences between the species and were determined with Dunn’s post hoc tests.