Getting the most out of fluorescent amplified fragment length polymorphism

Amplified fragment length polymorphism (AFLP (TM)) is one of the most widely applied molecular marker detection systems used today. Among the reasons for its popularity are its reproducibility, capacity to generate large numbers of data points in a single assay, and "off-the-shelf" universal applicability. The original AFLP protocol was developed using radioactive detection. The transfer of this technique to fluorescent detection on automated DNA fragment analysers not only removed the undesirable requirement for radioactivity but also provided the possibility for increased effectiveness and detection throughput. Unfortunately, a number of problems are frequently encountered with fluorescent AFLPs, particularly failure to amplify high molecular-weight fragments and generation of nonuniform peak distributions. Here, we describe an improved generic protocol for fluorescent AFLPs achieved mainly thorough optimization of the multiplexed selective amplification reaction. This improved protocol gives increased production of valuable high molecular-weight markers and uniform peak intensities, facilitating unambiguous scoring. The protocol has been successfully applied, without further optimization, to species of Salix and Populus (Salicaceae), Melampsora (Melampsoraceae, rust fungi) and Heracleum (Apiaceae), as well as sugar beet (Beta vulgaris L. subsp. vulgaris, Amaranthaceae), the endangered species Ranunculus kadzunensis Makino (Ranunculaceae), and to Aphidius ervi Haliday (Braconidae), a parasitoid wasp.