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Int. J. Mol. Sci. 2015, 16(9), 23057-23075; doi:10.3390/ijms160923057

Reference Genes in the Pathosystem Phakopsora pachyrhizi/ Soybean Suitable for Normalization in Transcript Profiling

Department of Phytopathology, Institute of Phytomedicine, Faculty of Agricultural Sciences, University of Hohenheim, Otto-Sander-Straße 5, 70599 Stuttgart, Germany
These authors contributed equally to this work.
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Author to whom correspondence should be addressed.
Academic Editor: Jan Schirawski
Received: 19 August 2015 / Revised: 14 September 2015 / Accepted: 18 September 2015 / Published: 23 September 2015
(This article belongs to the Special Issue Plant Microbe Interaction)
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Abstract

Phakopsora pachyrhizi is a devastating pathogen on soybean, endangering soybean production worldwide. Use of Host Induced Gene Silencing (HIGS) and the study of effector proteins could provide novel strategies for pathogen control. For both approaches quantification of transcript abundance by RT-qPCR is essential. Suitable stable reference genes for normalization are indispensable to obtain accurate RT-qPCR results. According to the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines and using algorithms geNorm and NormFinder we tested candidate reference genes from P. pachyrhizi and Glycine max for their suitability in normalization of transcript levels throughout the infection process. For P. pachyrhizi we recommend a combination of CytB and PDK or GAPDH for in planta experiments. Gene expression during in vitro stages and over the whole infection process was found to be highly unstable. Here, RPS14 and UbcE2 are ranked best by geNorm and NormFinder. Alternatively CytB that has the smallest Cq range (Cq: quantification cycle) could be used. We recommend specification of gene expression relative to the germ tube stage rather than to the resting urediospore stage. For studies omitting the resting spore and the appressorium stages a combination of Elf3 and RPS9, or PKD and GAPDH should be used. For normalization of soybean genes during rust infection Ukn2 and cons7 are recommended. View Full-Text
Keywords: Phakopsora pachyrhizi; RT-qPCR; reference gene; HIGS/VIGS; effector Phakopsora pachyrhizi; RT-qPCR; reference gene; HIGS/VIGS; effector
This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. (CC BY 4.0).

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Hirschburger, D.; Müller, M.; Voegele, R.T.; Link, T. Reference Genes in the Pathosystem Phakopsora pachyrhizi/ Soybean Suitable for Normalization in Transcript Profiling. Int. J. Mol. Sci. 2015, 16, 23057-23075.

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