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Entomophthorales on cereal aphids

Appendix B

Random Amplified Polymorphic DNA- PCR

Isolation of DNA

Before isolation of DNA all fungi were grown for one to three weeks in Grace’s insect culture media added 5% Fetal Bovine Serum (Gibco BRL) at 20^oC in constant dark. The mycelium was harvest after centrifugation under sterile conditions and DNA was isolated following the protocol of Hodge et al. (1995) but modified by a RNase treatment. After precipitation of nucleic acid with isopropanol RNA was degraded by resuspending the nucleic acid in 250 m l sterile TE buffer (10 mM Tris-HCL; 0.1 nM EDTA). 1:125 RNase PLUS was added (5 Prime ® 3 prime, Inc.) and samples were incubated for 2 hours at 37^oC. Samples were then centrifuged at 13000 rpm at 5^oC for 20 minutes and ammonium acetate was added to a concentration of 5.0 M. DNA was precipitated overnight at –20^oC with 2.5 vol cold 95% ethanol, centrifuged at 13000 rpm and washed twice with 70% cold ethanol. DNA was resuspended in filter sterilised distillated water and quantified by UV spectrophotometry.

RAPD

Amplification reactions were performed in 1.5 ml centrifuge tubes with a total volume of 30 ? l. Each reaction contained 1x supplied PCR Buffer (10mM Tris-HCl, 10mM KCl, Gibco BRL), 3 mM MgCl₂, 200 ? M each of dATP, dCTP,dGTP and dTTP (Boeringer Mannheim), 0.5 ? M primer, 5 ng genomic DNA and 3 units Taq polymerase (Gibco BRL). Reactions were overlaid with 30? l mineral oil and amplifications were performed in a MJ Research PTC-100 thermocycler with the following cycle parameters: an initial denaturisation of 2 min at 93^oC; 40 cycles of 1 min at 93^oC, 1 min at 36^oC; 2 min at 72^oC; a final extension of 7 min at 72^oC. Amplification products were separated by electrophoresis in 1.4% agarose gels with 1x TBE buffer at 44 v for 18 hours and detected by staining with ethidium bromide. For each tested primer two replicates were carried out.

Lengths of DNA fragments were measured and scored from photographs of the gels. Differences in intensity were not taken into account and products not appearing in both replicates were disregarded. Data was recorded as a binary matrix and phenetic similarity was calculated (UPGMA using Jaccard’s coefficient) by use of the statistical software NTSYSpc (V.2.01e).

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