Journal
FUNGAL GENETICS AND BIOLOGY
Volume 45, Issue 5, Pages 581-596Publisher
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.fgb.2007.09.007
Keywords
Glomus; Glomeromycota; hyphae; multi- and low copy genes; population size; quantification; root colonization; Scutellospora; specific primers; spores
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Funding
- Biotechnology and Biological Sciences Research Council [BB/C506205/1] Funding Source: researchfish
- Biotechnology and Biological Sciences Research Council [BB/C506205/1] Funding Source: Medline
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To enable quantification of mycelial abundance in mixed-species environments, eight new TaqMan (R) real-time PCR assays were developed for five arbuscular mycorrhizal fungal (AMF, Glomeromycota) taxa. The assays targeted genes encoding 18S rRNA or actin, and were tested on DNA from cloned gene fragments, from spores, mycelia, and from root-free soil, and on reverse-transcribed rRNA templates from entire mycelia and from colonized roots. The assays showed high specificity, sensitivity, and reproducibility, enabling reliable quantitation over broad ranges of template molecules. From cultured mycelia, DNA and RNA measures both correlated with spore number rather than extraradical hyphal length, and epifluorescence microscopy identified pronounced heterogeneity in vitality and nuclear distribution in hyphae. Root colonization was also spatially heterogeneous, as shown by a mixing experiment with root fragments of different length. Therefore, although real-time PCR can reproducibly and accurately quantify AMF nucleic acids, these are poorly correlated with visual measures because of spatial heterogeneity. (c) 2007 Elsevier Inc. All rights reserved.
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