Abstract
DNA extraction methods were evaluated for the yield and purity of DNA recovered from mycorrhized roots and whether the recovered DNA is suitable for amplification of arbuscular mycorrhizal (AM) fungal SSU rDNA. The DNeasy Plant Mini Kit and three extraction buffers were used alone or in combination with either polyvinylpyrrolidone (PVP), polyvinylpolypyrrolidone (PVPP) and/or activated charcoal (AC). Among the extraction methods tested, those based on the CTAB buffers yielded more DNA than those based on the TE buffer and the DNeasy Plant Mini Kit. Moreover, the use of AC alone or in combination with PVPP reduced DNA yield, while it significantly improved the purity of recovered DNA, whatever the extraction buffer. On the other hand, the success of nested-PCR amplification was negatively correlated with the amount of template DNA and positively correlated with the purity of recovered DNA. Three methods based on the TE buffer, two on the CTAB-βM buffer and one on the DNeasy Plant Mini Kit produced high-quality DNA in terms of purity and PCR performance. However, the TE buffer-based methods are less time consuming than the CTAB-βM buffer-based methods, and cheaper than the method based on the DNeasy Plant Mini Kit.
Highlights
Arbuscular mycorrhizal (AM) fungi belonging to the Glomeromycota (SCHÜSSLER et al, 2001) form obligate symbiosis with the majority of plants, resulting in the establishment of mixed organs called endomycorrhizae, which include fungal arbuscules, hyphae and vesicles within root cortex cells
The purpose of this study is to evaluate different DNA extraction methods based on cetyl trimethylammonium bromide (CTAB) and TE buffers and the DNeasy Plant Mini Kit for the yield and purity of DNA recovered from mycorrhized roots, and whether the recovered DNA is suitable for amplification of AM fungal SSU rDNA
Obtained by the methods based on the CTAB-βM buffer was comparable to that obtained by the methods based on the CTAB-DTT buffer, and was higher than that obtained by the methods based on the TE buffer and the DNeasy Plant Mini Kit
Summary
Arbuscular mycorrhizal (AM) fungi belonging to the Glomeromycota (SCHÜSSLER et al, 2001) form obligate symbiosis with the majority of plants, resulting in the establishment of mixed organs called endomycorrhizae, which include fungal arbuscules, hyphae and vesicles (in 80% of AM fungi) within root cortex cells. The endomycorrhizae provide mineral nutrients and water to the plant which, as a reward, provides carbohydrates to its fungal partner. AM fungi are considered key to plant diversity and productivity (VAN DER HEIJDEN et al, 1998). Staining and microscopy procedures have been used to monitor AM fungi in plant roots, according to morphological characters of their intraradical mycelia (SANDERS, 2004), many of those characters are unstable during root colonization (JEFFRIES et al, 2003). To overcome the disadvantages of these procedures, molecular
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