title: Optimization of AFLP for extremely large genomes over 70 Gb.
abstract: 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).
authors: Department of Forest Botany Dendrology and Geobiocenology, Faculty of Forestry and Wood Technology, Mendel University in Brno, Zemědělská 3, 613 00, Brno, Czech Republic.
authors: Petra
authors: P
authors: Veselá
authors: Department of Forest Botany Dendrology and Geobiocenology, Faculty of Forestry and Wood Technology, Mendel University in Brno, Zemědělská 3, 613 00, Brno, Czech Republic.
authors: Daniel
authors: D
authors: Volařík
authors: Department of Forest Botany Dendrology and Geobiocenology, Faculty of Forestry and Wood Technology, Mendel University in Brno, Zemědělská 3, 613 00, Brno, Czech Republic.
authors: Jaroslav
authors: http://orcid.org/0000-0002-9561-0412
authors: J
authors: Mráček
mesh_terms: D054458:Amplified Fragment Length Polymorphism Analysis / Q000379:methods*; D004262:DNA Restriction Enzymes / Q000378:metabolism; D018744:DNA, Plant / Q000235:genetics* / Q000378:metabolism; D018745:Genome, Plant; D060005:Genotyping Techniques / Q000379:methods*; D010944:Plants / Q000145:classification* / Q000235:genetics*; D015203:Reproducibility of Results
chemical_list: D018744:DNA, Plant; D004262:DNA Restriction Enzymes
keywords: amplified fragment length polymorphism; in silico AFLP; large genome; octo-cutter restriction enzyme; reproducibility; size homoplasy