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Real-time PCR to quantify composition of arbuscular mycorrhizal fungal communities - marker design, verification, calibration and field validation

Thonar, C.; Erb, A. and Jansa, J. (2012) Real-time PCR to quantify composition of arbuscular mycorrhizal fungal communities - marker design, verification, calibration and field validation. Molecular Ecology Resources, 12 (2), pp. 219-232.

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Summary

Quantitative real-time PCR (qPCR) is slowly becoming established as a tool to quantify abundance of different arbuscular mycorrhizal fungal (AMF) taxa in roots and in soil. Here, we describe the development and field validation of qPCR markers (i.e. primers with associated hydrolysis probes), targeting taxon-specific motifs in the nuclear large ribosomal subunit RNA genes. Design of such markers is complicated by the multinuclear and multigenomic cellular organization of these fungi and the high DNA sequence diversity within the smallest biologically relevant units (i.e. single-spore isolates). These limitations are further compounded by inefficient biomass production of these fungi, resulting in limited availability of pure genomic DNA (gDNA) of well-defined isolates for cross-specificity testing of the markers. Here we demonstrate, using a number of AMF isolates, the possibility to establish stringent qPCR running conditions allowing quantification of phylogenetically disjunctive AMF taxa. Further, we show that these markers can more generally be used to quantify abundance (i.e. number of target gene copies or amount of gDNA) of what is usually considered the level of AMF species, regardless of the isolate identities. We also illustrate the range of variation within qPCR signal strength across different AMF taxa with respect to the detected number of gene copies per unit amount of gDNA. This information is paramount for interpretation of the qPCR analyses of field samples. Finally, the field validation of these markers confirmed their potential to assess composition of field AMF communities and monitor the changes owing to agricultural practices such as soil tillage.


EPrint Type:Journal paper
Keywords:calibration, field validation, marker design, nuclear large ribosomal subunit (nLSU, 28S), specificity, taxon-specific sequence motifs, Bodenwissenschaften, Indikatoren, arbuskuläre Mykorrhiza Pilze(AMP)
Subjects: Knowledge management > Research methodology and philosophy > Specific methods > Indicators and other value-laden measures
Soil
Research affiliation: Switzerland > FiBL - Research Institute of Organic Agriculture Switzerland > Soil Sciences
Switzerland > ETHZ - Agrarwissenschaften
DOI:10.1111/j.1755-0998.2011.03086.x
Related Links:http://www.fibl.org/en/switzerland/research/soil-sciences.html, http://onlinelibrary.wiley.com/doi/10.1111/j.1755-0998.2011.03086.x/abstract
Deposited By: Forschungsinstitut für biologischen Landbau, FiBL
ID Code:21817
Deposited On:20 Nov 2012 22:15
Last Modified:20 Nov 2012 22:18
Document Language:English
Status:Published
Refereed:Peer-reviewed and accepted

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