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Application of qRT-PCR and RNA-Seq analysis for the identification of housekeeping genes useful for normalization of gene expression values during Striga hermonthica development

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Abstract

Striga is a root parasitic weed that attacks many of the staple crops in Africa, India and Southeast Asia, inflicting tremendous losses in yield and for which there are few effective control measures. Studies of parasitic plant virulence and host resistance will be greatly facilitated by the recent emergence of genomic resources that include extensive transcriptome sequence datasets spanning all life stages of S. hermonthica. Functional characterization of Striga genes will require detailed analyses of gene expression patterns. Quantitative real-time PCR is a powerful tool for quantifying gene expression, but correct normalization of expression levels requires identification of control genes that have stable expression across tissues and life stages. Since no S. hermonthica housekeeping genes have been established for this purpose, we evaluated the suitability of six candidate housekeeping genes across key life stages of S. hermonthica from seed conditioning to flower initiation using qRT-PCR and high-throughput cDNA sequencing. Based on gene expression analysis by qRT-PCR and RNA-Seq across heterogeneous Striga life stages, we determined that using the combination of three genes, UBQ1, PP2A and TUB1 provides the best normalization for gene expression throughout the parasitic life cycle. The housekeeping genes characterized here provide robust standards that will facilitate powerful descriptions of parasite gene expression patterns.

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Abbreviations

CV:

Coefficient of variation

DMBQ:

2,6-dimethoxy-p-benzoquinone

ESTs:

Expressed sequence tags

GAPC-2:

Glyceraldehyde-3-phosphate dehydrogenase-2

GR24:

Growth regulator 24: strigol analogue

MFC:

Maximum fold change of expression

PP2A:

Phosphoprotein phosphatase 2A subunit A3

PPGP:

Parasitic plant genome project

qRT-PCR:

Quantitative real-time PCR

RLI:

RNase L inhibitor protein

RNA-Seq:

High-throughput sequencing of RNA

RPKM:

Reads per kilobase per million mapped reads

TUB1:

Beta tubulin 1

TUB5:

Beta tubulin 5

UBQ1:

Ubiquitin 1

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Acknowledgments

This research was supported by an International Outgoing European Marie Curie postdoctoral fellowship (PIOF-GA-2009-252538) to M Fernández-Aparicio, a NSF Plant Genome award (DBI-0701748) to JH Westwood, CW dePamphilis, MP Timko and JI Yoder, and a grant from the U.S. Department of Agriculture (Hatch Project no. 135798) to JH Westwood.

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Authors and Affiliations

  1. Department of Plant Breeding, IAS-CSIC, Institute for Sustainable Agriculture, 14080, Córdoba, Spain

    M. Fernández-Aparicio

  2. Department of Plant Pathology, Physiology and Weed Science, Virginia Tech, Blacksburg, VA, 24061, USA

    M. Fernández-Aparicio & J H. Westwood

  3. Department of Biology, University of Virginia, Charlottesville, VA, 22904, USA

    K. Huang & M. P. Timko

  4. Department of Biology, Institute for Molecular Evolutionary Genetics, Penn State University, State College, PA, 16801, USA

    E. K. Wafula, L. A. Honaas, N. J. Wickett & C. W. dePamphilis

  5. Department of Plant Sciences, University of California-Davis, Davis, CA, 95616, USA

    J. I. Yoder

  6. Chicago Botanic Garden, Glencoe, IL, 60022, USA

    N. J. Wickett

Authors
  1. M. Fernández-Aparicio
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  2. K. Huang
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  3. E. K. Wafula
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  4. L. A. Honaas
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  7. C. W. dePamphilis
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  8. J. I. Yoder
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  9. J H. Westwood
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Correspondence to M. Fernández-Aparicio.

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Fernández-Aparicio, M., Huang, K., Wafula, E.K. et al. Application of qRT-PCR and RNA-Seq analysis for the identification of housekeeping genes useful for normalization of gene expression values during Striga hermonthica development. Mol Biol Rep 40, 3395–3407 (2013). https://doi.org/10.1007/s11033-012-2417-y

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