Data from: 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).本研究报道了一套优化后的扩增片段长度多态性（amplified fragment length polymorphism, AFLP）实验方案，该方案采用识别8碱基对序列的限制性内切酶AscI与SbfI，可为基因组大小超过70 Gb的物种提供适配性优化。该经济型优化方案在选择性扩增阶段仅使用带有3个选择性碱基的引物，大幅缩减了扩增片段的总数量。本研究验证了片段数量与基因组大小对非同源共迁移片段（长度同态性，size homoplasy）出现概率的影响，此类片段会对AFLP基因型的信息价值产生负面影响。本研究还探究了多种反应条件对槲寄生（Viscum album）超大基因组样本的重现性及条带强度的影响。本八切点酶切方案的重现性通过多个物种得到验证，其涵盖的基因组大小范围为1 Gb（细叶苔草Carex panicea）至76 Gb（槲寄生Viscum album）。该优化方案还成功在大基因组物种（槲寄生Viscum album、雪花莲Galanthus nivalis与矮松Pinus pumila）中检测到显著的种内变异。