Optimization of AFLP for extremely large genomes over 70 Gb
Language English Country England, Great Britain Media print-electronic
Document type Evaluation Study, Journal Article
- Keywords
- amplified fragment length polymorphism, in silico AFLP, large genome, octo-cutter restriction enzyme, reproducibility, size homoplasy,
- MeSH
- Amplified Fragment Length Polymorphism Analysis methods MeSH
- DNA, Plant genetics metabolism MeSH
- Genome, Plant * MeSH
- Genotyping Techniques methods MeSH
- Reproducibility of Results MeSH
- DNA Restriction Enzymes metabolism MeSH
- Plants classification genetics MeSH
- Publication type
- Journal Article MeSH
- Evaluation Study MeSH
- Names of Substances
- DNA, Plant MeSH
- DNA Restriction Enzymes MeSH
Here, we present an improved amplified fragment length polymorphism (AFLP) protocol using restriction enzymes (AscI and SbfI) that recognize 8-base pair sequences to provide alternative optimization suitable for species with a genome size over 70 Gb. This cost-effective optimization massively reduces the number of amplified fragments 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 nonidentical comigrating 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 (V. album). The improved protocol also succeeded in detecting high intraspecific variability in species with large genomes (V. album, Galanthus nivalis and Pinus pumila).
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