Optimization of AFLP fingerprinting of organisms with a large-sized genome: a study on Alstroemeria spp.

The recently introduced PCR-based DNA fingerprinting technique AFLP (amplif ied fragment length polymorphism) allows the selective amplification of sub sets of genomic restriction fragments. AFLP has been used for multiple purp oses such as the construction of linkage maps, marker saturation at specifi c genomic regions, analysis of genetic diversity and molecular phylogeny an d cultivar identification. AFLP can be tailored by varying the number of se lective nucleotides added to core primers and can allow accurate amplificat ion, even in complex template mixtures generated from plant species with ve ry large genomes. In this study Alstroemeria, a plant species with a very l arge genome, was tested for adapting the AFLP protocol. The results indicat ed that the estimated number of amplification products was close to the obs erved number when eight selective nucleotides were used but that seven sele ctive nucleotides did not increase the number of amplification products fou rfold. However, we found reproducibility in both +7 and +8 fingerprints. Va rious distributions of selective nucleotides over the various rounds of pre amplifications were tested. Preamplification with four selective nucleotide s followed by final amplification with eight selective nucleotides produced clear and reproducible AFLP patterns. The effects of GC content of primers and multiple preamplification steps were also discussed.