Differential effect of sample preservation methods on plant and arbuscular mycorrhizal fungal DNA.
A wide range of methods are commonly used for preserving environmental samples prior to molecular analyses. However, the effect of these preservation methods on fungal DNA is not understood. The objective of this study was to test the effect of eight different preservation methods on the quality and yield of DNA extracted from Bromus inermis and Daucus carota roots colonized by the arbuscular mycorrhizal (AM) fungus, Glomus intraradices. The total DNA concentration in sample extracts was quantified using spectrophotometry. Samples that were frozen (-80 masculineC and -20 masculineC), stored in 95% ethanol, or silica gel dried yielded total (plant and fungal) DNA concentrations that were not significantly different from fresh samples. In contrast, samples stored in CTAB solution or freeze-dried resulted in significantly reduced DNA concentrations compared with fresh samples. The preservation methods had no effect on the purity of the sample extracts for both plant species. However, the DNA of the dried samples (silica gel dried, freeze-dried, heat dried) appeared to be slightly more degraded compared with samples that remained hydrated (frozen, stored in ethanol or CTAB solutions) during storage when visualized on a gel. The concentration of AM fungal DNA in sample extracts was quantified using TaqMan real time PCR. Methods that preserved samples in hydrated form had similar AM fungal DNA concentrations as fresh samples, except D. carota samples stored in ethanol. In contrast, preservation methods that involved drying the samples had very low concentrations of AM fungal DNA for B. inermis, and nearly undetectable for D. carota samples. The drying process appears to be a major factor in the degradation of AM fungal DNA while having less of an impact on plant DNA. Based on these results, samples that need to be preserved prior to molecular analysis of AM fungi should be kept frozen to minimize the degradation of plant and AM fungal DNA.
Bainard
L D
LD
Department of Integrative Biology, University of Guelph, Guelph, ON, Canada. lbainard@uoguelph.ca
Klironomos
J N
JN
Hart
M M
MM
D031724:Bromus / Q000382:microbiology; D004249:DNA Damage; D004271:DNA, Fungal / Q000235:genetics* / Q000378:metabolism; D018744:DNA, Plant / Q000235:genetics* / Q000378:metabolism; D018552:Daucus carota / Q000382:microbiology; D038821:Mycorrhizae / Q000302:isolation & purification; D018517:Plant Roots / Q000382:microbiology; D011309:Preservation, Biological / Q000379:methods*
D004271:DNA, Fungal; D018744:DNA, Plant