Optimisation of AFLP for extremely large genomes over 70 Gb.

Here, we present an improved amplified fragment length polymorphism (AFLP) protocol using restriction enzymes (AscI and SbfI) that recognise 8-base pair sequences to provide alternative optimisation suitable for species with a genome size over 70 Gb. This cost-effective optimisation massively reduces the number of amplified fragments by using only +3 selective bases per primer during selective amplification. We demonstrate the effects of the number of fragments and genome size on the appearance of non-identical co-migrating fragments (size homoplasy), which has a negative impact on the informative value of AFLP genotypes. We also present various reaction conditions and their effects on reproducibility and the band intensity of the extremely large genome of Viscum album. The reproducibility of this octo-cutter protocol was calculated using several species with genome sizes ranging from 1 Gb (Carex panicea) to 76 Gb (Viscum album). The improved protocol also succeeded in detecting high intraspecific variability in species with large genomes (Viscum album, Galanthus nivalis and Pinus pumila).