The effect of different DNA isolation protocols and AFLP fingerprinting optimizations on error rate estimates in the bryophyte Campylopus introflexus
Eva Mikulášková, Tomáš Fér and Veronika Ku?abováE. Mikulášková (firstname.lastname@example.org), Dept of Botany and Zoology, Faculty of Science, Masaryk Univ., CZ-61137 Brno, Czech Republic. – T. Fér and V. Ku?abová, Dept of Botany, Faculty of Science, Charles Univ. in Prague, CZ-128 01 Praha 2, Czech Republic.
Amplified fragment length polymorphism (AFLP) has become a standard method for investigating genetic variation in plants. Nevertheless, only a few applications in bryophytes have been published and there is still a need to optimize the method. We optimized DNA isolation and AFLP protocols for Campylopus introflexus (Hedw.) Brid. As DNA quality is crucial for successful AFLP analysis, three different DNA extraction protocols were compared and the Invisorb Plant Mini Kit produced the highest DNA amount (15, 38 and 47 ng μl–1 for dry, fresh and new cultivated tissues, respectively) and purity. Newly grown stems gave the purest DNA (absorbance at the wavelengths of 260 nm and 280 nm was 1.86). However, banding patterns obtained from dry herbarium specimens (up to two years old) corresponded with those from fresh material at a similarity level of 94.71%. The replicability of AFLP profiles was not dependent on the way plants were stored. We compared commercial kits and the influence of modifications of the protocols for obtaining reliable results. We tested the reproducibility of AFLP fingerprints produced by the final optimized protocol. An increased amount of restriction enzymes and prolonged restriction and ligation of up to 10 h were the most important modifications for improving results. The modified protocol was applied to 30 samples and four selective primer combinations, and gave an average genotyping error rate of 0.0451.