Rationalizing molecular analysis of field-collected roots for assessing diversity of arbuscular mycorrhizal fungi: to pool, or not to pool, that is the question

For rationalizing molecular analysis of field-collected roots in diversity studies on arbuscular mycorrhiza, we compared three different approaches. After DNA extraction from 50 root samples of Plantago lanceolata grown on monoculture plots at a former arable field site, (1) DNAs were amplified sepa...

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Bibliographic Details
Published in:Mycorrhiza 2006-11, Vol.16 (8), p.525-531
Main Authors: Renker, C, Weißhuhn, K, Kellner, H, Buscot, F
Format: Article
Language:English
Subjects:
Biodiversity
Biological and medical sciences
Cloning, Molecular
DNA
DNA, Fungal - analysis
DNA, Fungal - genetics
DNA, Intergenic - analysis
DNA, Intergenic - genetics
Fundamental and applied biological sciences. Psychology
Fungi
internal transcribed spacers
Methods
Microbiology
Mycorrhizae - genetics
Mycorrhizae - isolation & purification
mycorrhizal fungi
nontarget organisms
Parasitism and symbiosis
Phylogeny
Plant physiology and development
Plant Roots - microbiology
Plantago - microbiology
Plantago lanceolata
Polymerase chain reaction
roots
Symbiosis
vesicular arbuscular mycorrhizae
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Summary:For rationalizing molecular analysis of field-collected roots in diversity studies on arbuscular mycorrhiza, we compared three different approaches. After DNA extraction from 50 root samples of Plantago lanceolata grown on monoculture plots at a former arable field site, (1) DNAs were amplified separately by nested PCR and each amplicon was cloned separately; (2) DNAs were amplified separately by nested PCR, 1 μl of each amplicon was pooled, and a single cloning was made from the resulting amplicons mix; and (3) DNAs were pooled and the single amplicon derived from the nested PCR was cloned. Based on these three different methods, 109 nuclear ribosomal internal transcribed spacer sequences were obtained. Methods 1 and 2 enabled the detection of almost similar levels of arbuscular mycorrhizal fungal diversity. However, method 1 was expensive and time-consuming as much more cloning had to be done. Method 3 was completely biased by preferential amplification of nontarget organisms, which were only detected in low frequencies by the other methods.
ISSN:0940-6360
1432-1890
DOI:10.1007/s00572-006-0067-4