Establishment of the amplified fragment length polymorphism (AFLP) technique for genotyping of pollen beetle (Meligethes aeneus)--a noxious insect pest on oilseed rape (Brassica napus).
In order to conduct studies concerning genetic variability of pollen beetles (Meligethes aeneus), a genotyping protocol was established. No genome information is available for pollen beetles so the amplified fragment length polymorphism (AFLP) technique was chosen since it does not depend on any prior sequence information of the samples and also is a sensitive and robust technique. However, several modifications were needed in order to adapt the method for analysis of pollen beetles. Basic modifications included (i) alterations of DNA purification, (ii) use of two six-cutter restriction enzymes, (iii) and modified PCR conditions. This protocol resulted in a favourable number of fragments of an appropriate size range for standard gel analysis by a DNA sequencer applicable to a single insect and even body parts enabling different assays to be conducted on a single specimen. Pollen beetles from different areas of Sweden were analysed to verify the reproducibility and efficacy of the protocol as well as for phenetic analysis. The high reproducibility of the modified AFLP protocol allows it to be used as a reliable tool for genotype analysis of pollen beetles.
Kazachkova
Nadiya
N
Department of Plant Biology and Forest Genetics, Genetics Center, PO Box 7080, Swedish University of Agricultural Sciences, SE-750 07, Uppsala, Sweden. nadiya.kazachkova@vbsg.slu.se
Fahleson
Jan
J
Meijer
Johan
J
D000818:Animals; D029688:Brassica napus / Q000469:parasitology*; D001517:Coleoptera / Q000235:genetics*; D004247:DNA / Q000302:isolation & purification; D014644:Genetic Variation; D019105:Random Amplified Polymorphic DNA Technique / Q000379:methods*; D015203:Reproducibility of Results; D013548:Sweden
D004247:DNA