The Plant Pathology Journal

The Korean Society of Plant Pathology (한국식물병리학회)
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Selection of Reference Genes for Real-time Quantitative PCR Normalization in the Process of Gaeumannomyces graminis var. tritici Infecting Wheat

  • Xie, Li-hua (Institute of Plant Protection Research, Henan Academy of Agricultural Sciences, Henan Key Laboratory for Control of Crop Diseases and Insect Pests, IPM Key Laboratory in Southern Part of North China for Ministry of Agriculture) ;
  • Quan, Xin (Institute of Plant Protection Research, Henan Academy of Agricultural Sciences, Henan Key Laboratory for Control of Crop Diseases and Insect Pests, IPM Key Laboratory in Southern Part of North China for Ministry of Agriculture) ;
  • Zhang, Jie (Institute of Plant Protection Research, Henan Academy of Agricultural Sciences, Henan Key Laboratory for Control of Crop Diseases and Insect Pests, IPM Key Laboratory in Southern Part of North China for Ministry of Agriculture) ;
  • Yang, Yan-yan (Institute of Plant Protection Research, Henan Academy of Agricultural Sciences, Henan Key Laboratory for Control of Crop Diseases and Insect Pests, IPM Key Laboratory in Southern Part of North China for Ministry of Agriculture) ;
  • Sun, Run-hong (Institute of Plant Protection Research, Henan Academy of Agricultural Sciences, Henan Key Laboratory for Control of Crop Diseases and Insect Pests, IPM Key Laboratory in Southern Part of North China for Ministry of Agriculture) ;
  • Xia, Ming-cong (Institute of Plant Protection Research, Henan Academy of Agricultural Sciences, Henan Key Laboratory for Control of Crop Diseases and Insect Pests, IPM Key Laboratory in Southern Part of North China for Ministry of Agriculture) ;
  • Xue, Bao-guo (Institute of Plant Protection Research, Henan Academy of Agricultural Sciences, Henan Key Laboratory for Control of Crop Diseases and Insect Pests, IPM Key Laboratory in Southern Part of North China for Ministry of Agriculture) ;
  • Wu, Chao (Institute of Plant Protection Research, Henan Academy of Agricultural Sciences, Henan Key Laboratory for Control of Crop Diseases and Insect Pests, IPM Key Laboratory in Southern Part of North China for Ministry of Agriculture) ;
  • Han, Xiao-yun (Institute of Plant Protection Research, Henan Academy of Agricultural Sciences, Henan Key Laboratory for Control of Crop Diseases and Insect Pests, IPM Key Laboratory in Southern Part of North China for Ministry of Agriculture) ;
  • Xue, Ya-nan (Agricultural and Animal Husbandry Bureau of Mianchi) ;
  • Yang, Li-rong (Institute of Plant Protection Research, Henan Academy of Agricultural Sciences, Henan Key Laboratory for Control of Crop Diseases and Insect Pests, IPM Key Laboratory in Southern Part of North China for Ministry of Agriculture)
  • Received : 2018.03.22
  • Accepted : 2018.10.04
  • Published : 2019.02.01
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Abstract

Gaeumannomyces graminis var. tritici is a soil borne pathogenic fungus associated with wheat roots. The accurate quantification of gene expression during the process of infection might be helpful to understand the pathogenic molecular mechanism. However, this method requires suitable reference genes for transcript normalization. In this study, nine candidate reference genes were chosen, and the specificity of the primers were investigated by melting curves of PCR products. The expression stability of these nine candidates was determined with three programs-geNorm, Norm Finder, and Best Keeper. $TUB{\beta}$ was identified as the most stable reference gene. Furthermore, the exopolygalacturonase gene (ExoPG) was selected to verify the reliability of $TUB{\beta}$ expression. The expression profile of ExoPG assessed using $TUB{\beta}$ agreed with the results of digital gene expression analysis by RNA-Seq. This study is the first systematic exploration of the optimal reference genes in the infection process of Gaeumannomyces graminis var. tritici.

Keywords

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Fig. 1. Specificity of RTqPCR and amplicon size. (a) Agarose gel (1.5%) electrophoresis showing amplification of a specific PCR product of the expected size for each gene. (b) Melting curves of one target gene and nine reference genes showing single peaks. M1 and M2 represent D2000 DNA Marker (150 bp-2000 bp) and DNA Marker I (100 bp-600 bp) marker, respectively.

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Fig. 3. Average expression stability values (M) and Pairwise variation (V) calculated by geNorm to determine the optimal number of reference genes. The average pairwise variations Vn/Vn+1 was analyzed between the normalization factors NFn and NFn+1 to indicate the optimal number of reference genes required for RT-qPCR data normalization in different samples.

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Fig. 4. The expression quantification of ExoGP normalized by TUBβ and GAPDH as internal controls, respectively. Error bars represent standard error of the mean.

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Fig. 2. RT-qPCR Ct values for reference genes. Expression data displayed as Ct values for each reference gene in all Ggt and Ggtinfected wheat root samples. A line across the box is depicted as the median. The box indicates the 25th and 75th percentiles, whiskers represent the maximum and minimum values.

Table 1. Candidate reference genes, primers and different parameters derived from RT-qPCR

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Table 2. Ranking of candidate reference genes in order of their expression stability as calculated by Norm Finder

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Table 3. Statistics results by Best Keeper software for ten se-lected genes based on Ct values

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