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Identification of Reference Genes for Studying Herbicide Resistance Mechanisms in Japanese Foxtail (Alopecurus japonicus)

Published online by Cambridge University Press:  30 June 2017

Hongle Xu
Research Associate, College of Plant Protection, Nanjing Agricultural University, Nanjing 210095, China, and Institute of Plant Protection, Henan Academy of Agricultural Sciences, Zhengzhou450002, China
Jun Li
Associate Professor, Graduate Student, Graduate Student, and Professor, College of Plant Protection, Nanjing Agricultural University, Nanjing210095, China
Renhai Wu
Associate Research Fellow and Research Associate, Institute of Plant Protection, Henan Academy of Agricultural Sciences, Zhengzhou 450002, China
Wangcang Su
Associate Research Fellow and Research Associate, Institute of Plant Protection, Henan Academy of Agricultural Sciences, Zhengzhou 450002, China
Xibao Wu
Associate Professor, Graduate Student, Graduate Student, and Professor, College of Plant Protection, Nanjing Agricultural University, Nanjing210095, China
Lingyue Wang
Associate Professor, Graduate Student, Graduate Student, and Professor, College of Plant Protection, Nanjing Agricultural University, Nanjing210095, China
Liyao Dong*
Associate Professor, Graduate Student, Graduate Student, and Professor, College of Plant Protection, Nanjing Agricultural University, Nanjing210095, China
*Corresponding author’s E-mail:


Herbicide-resistant weeds pose a considerable threat to agriculture, but their resistance mechanisms are poorly understood. Differential gene expression analysis of a weed subjected to herbicide treatment is a key step toward more mechanistic studies. Such an analysis, often involving quantitative real-time PCR (qPCR), requires suitable reference genes as internal controls. In this study, we identified optimal reference genes in the noxious weed, Japanese foxtail. This weed has evolved resistance to acetyl-coenzyme A carboxylase (ACCase) inhibitors. We analyzed the stability of eight commonly used candidate reference genes (glyceraldehyde-3-phosphate dehydrogenase [GAPDH]; ubiquitin [UBQ]; capsine phosphatase [CAP]; beta-tubulin [TUB]; eukaryotic initiation factor 4a [EIF4A]; elongation factor-1 alpha [EF1]; 18S ribosomal RNA [18S]; 25S ribosomal RNA [25S]) from root, stem, and leaf tissue of plants that were either resistant or sensitive to ACCase inhibitors, with or without herbicide stress, using qPCR. The results were further ranked and analyzed using geNorm, NormFinder, and BestKeeper software. These analyses identified EF1 and UBQ in roots, EF1, TUB, CAP, and 18S in stems, and EF1, GAPDH, and 18S in leaves as suitable references for qPCR normalization. We have identified a set of reference genes that can be used to study herbicide resistance mechanisms in Japanese foxtail.

© Weed Science Society of America, 2017 

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Associate Editor for this paper: Patrick J. Tranel, University of Illinois.


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