Identification of Four Soybean Reference Genes for Gene Expression Normalization

Libault, Marc; Thibivilliers, Sandra; Bilgin, Damla D.; Radwan, Osman; Benítez, Mariana; Clough, Steven J.; Stacey, Gary

doi:10.3835/plantgenome2008.02.0091

Cited by 300 publications

(289 citation statements)

References 64 publications

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“…LinRegPCR 7.5 software (Ramakers et al, 2003) was used to determine PCR efficiency for each reaction. GmATP synthase and GmCons6 (Libault et al, 2008) were used as housekeeping genes.…”

Section: Qrt-pcrmentioning

confidence: 99%

Systemic Regulation of Soybean Nodulation by Acidic Growth Conditions

2012

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Mechanisms inhibiting legume nodulation by low soil pH, although highly prevalent and economically significant, are poorly understood. We addressed this in soybean (Glycine max) using a combination of physiological and genetic approaches. Split-root and grafting studies using an autoregulation-of-nodulation-deficient mutant line, altered in the autoregulation-of-nodulation receptor kinase GmNARK, determined that a systemic, shoot-controlled, and GmNARK-dependent mechanism was critical for facilitating the inhibitory effect. Acid inhibition was independent of aluminum ion concentration and occurred early in nodule development, between 12 and 96 h post inoculation with Bradyrhizobium japonicum. Biological effects were confirmed by measuring transcript numbers of known early nodulation genes. Transcripts decreased on both sides of split-root systems, where only one side was subjected to low-pH conditions. Our findings enhance the present understanding of the innate mechanisms regulating legume nodulation control under acidic conditions, which could benefit future attempts in agriculture to improve nodule development and biological nitrogen fixation in acid-stressed soils.

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“…LinRegPCR 7.5 software (Ramakers et al, 2003) was used to determine PCR efficiency for each reaction. GmATP synthase and GmCons6 (Libault et al, 2008) were used as housekeeping genes.…”

Section: Qrt-pcrmentioning

confidence: 99%

Systemic Regulation of Soybean Nodulation by Acidic Growth Conditions

2012

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“…It should be noted that Rer1 and ATPase genes that were included as candidates on the basis of their stability of expression in a differential macroarray hybridization of alfalfa ESTs had not been previously tested as putative reference genes in other species. New reference genes identified by microarray analysis in Arabidopsis thaliana [33] and soybean (Glycine max) [34] were shown to outperform commonly used housekeeping genes in these species and other plant systems as well [21]. It is also noteworthy that eEF-1α consistently maintained an M value well below the 0.5 geNorm threshold across all treatments and genetic sources and is another candidate to consider in the validation of reference genes in RT-qPCR analysis of gene expression in alfalfa.…”

Section: Stability Of the Expression Of Candidate Reference Genesmentioning

confidence: 99%

Reference Genes for RT-qPCR Analysis of Environmentally and Developmentally Regulated Gene Expression in Alfalfa

Castonguay¹,

Jl²,

Dubé³

2015

AJPS

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Reverse transcription quantitative PCR (RT-qPCR) is a highly sensitive technique that has become the standard for the analysis of differences in gene expression in response to experimental treatments or among genetic sources. The accuracy of the RT-qPCR results can be significantly affected by uncontrolled sources of variation that can be accounted for normalization with so-called reference genes stably expressed under various conditions. In this study we assessed the stability of 21 reference gene candidates in crowns of two alfalfa cultivars (Apica and Evolution) exposed to various environmental conditions (cold, water stress and photoperiod) and from above ground biomass of the cultivar Orca sampled at three developmental stages (vegetative, full bloom and mature pods). Candidates were selected based on their previous identification in other plant species or their stable expression in a differential hybridization of alfalfa ESTs with cDNA from nonacclimated and cold-acclimated alfalfa. Genes encoding ubiquitin protein ligase 2a (UBL-2a), actin depolymerizing factor (ADF) and retention in endoplasmic reticulum 1 protein (Rer1) were the most stable across experimental conditions. Conversely β-actin (Act), α-tubulin (Tub) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) frequently used as "housekeeping genes" in gene expression studies showed poor stability. No more than two reference genes were required to normalize the gene expression data under each condition. Normalization of the expression of genes of interest with unstable reference genes led to observations that were conflicting with those made with validated reference genes and that were in some cases inconsistent with the current knowledge of the trait. The reference genes identified in this study are strong candidates for normalization of gene expression in cultivated alfalfa.

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“…Transcript levels of GmHGO genes were determined by qRT-PCR using an ABI17500 real-time PCR following the SYBR Green method (Applied Biosystems). Gene expression levels were normalized to the expression of the soybean housekeeping genes cons6 and cons4 (Libault et al, 2008). Primers used for RT-PCR are listed in Supplemental Table S4.…”

Section: Determination Of Tocochromanol Content and Compositionmentioning

confidence: 99%

Identification of Homogentisate Dioxygenase as a Target for Vitamin E Biofortification in Oilseeds

et al. 2016

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Soybean (Glycine max) is a major plant source of protein and oil and produces important secondary metabolites beneficial for human health. As a tool for gene function discovery and improvement of this important crop, a mutant population was generated using fast neutron irradiation. Visual screening of mutagenized seeds identified a mutant line, designated MO12, which produced brown seeds as opposed to the yellow seeds produced by the unmodified Williams 82 parental cultivar. Using forward genetic methods combined with comparative genome hybridization analysis, we were able to establish that deletion of the GmHGO1 gene is the genetic basis of the brown seeded phenotype exhibited by the MO12 mutant line. GmHGO1 encodes a homogentisate dioxygenase (HGO), which catalyzes the committed enzymatic step in homogentisate catabolism. This report describes to our knowledge the first functional characterization of a plant HGO gene, defects of which are linked to the human genetic disease alkaptonuria. We show that reduced homogentisate catabolism in a soybean HGO mutant is an effective strategy for enhancing the production of lipid-soluble antioxidants such as vitamin E, as well as tolerance to herbicides that target pathways associated with homogentisate metabolism. Furthermore, this work demonstrates the utility of fast neutron mutagenesis in identifying novel genes that contribute to soybean agronomic traits.

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Identification of Four Soybean Reference Genes for Gene Expression Normalization

Cited by 300 publications

References 64 publications

Systemic Regulation of Soybean Nodulation by Acidic Growth Conditions

Systemic Regulation of Soybean Nodulation by Acidic Growth Conditions

Reference Genes for RT-qPCR Analysis of Environmentally and Developmentally Regulated Gene Expression in Alfalfa

Identification of Homogentisate Dioxygenase as a Target for Vitamin E Biofortification in Oilseeds

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