Selection of reference genes for quantitative real-time PCR in Casuarina equisetifolia under salt stress

Fan, Chunjie; Zhenfei, Qiu; Zeng, Bo; Liu, Y.; Li, X.; Guangsheng, Guo

doi:10.1007/s10535-016-0670-y

Cited by 14 publications

(12 citation statements)

References 38 publications

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“…For example, TUB was identified as the most stable reference gene by GeNorm, NormFinder and ΔCt in the sample sets, but H3 was considered to be the most stable reference gene by BestKeeper. Similar results were observed in several studies (Zhang et al, 2015 ; Fan et al, 2016 ; Yue et al, 2016 ). Different statistical approaches, which are based on different principles, may yield contradictory results from the same data (Hu et al, 2009 ; Wan et al, 2010 ).…”

Section: Discussionsupporting

confidence: 91%

Candidate Reference Genes Selection and Application for RT-qPCR Analysis in Kenaf with Cytoplasmic Male Sterility Background

Zhou¹,

Chen²,

Khan³

et al. 2017

Front. Plant Sci.

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Cytoplasmic male sterility (CMS) is a maternally inherited trait that results in the production of dysfunctional pollen. Based on reliable reference gene-normalized real-time quantitative PCR (RT-qPCR) data, examining gene expression profile can provide valuable information on the molecular mechanism of kenaf CMS. However, studies have not been conducted regarding selection of reference genes for normalizing RT-qPCR data in the CMS and maintainer lines of kenaf crop. Therefore, we studied 10 candidate reference genes (ACT3, ELF1A, G6PD, PEPKR1, TUB, TUA, CYP, GAPDH, H3, and 18S) to assess their expression stability at three stages of pollen development in CMS line 722A and maintainer line 722B of kenaf. Five computational statistical approaches (GeNorm, NormFinder, ΔCt, BestKeeper, and RefFinder) were used to evaluate the expression stability levels of these genes. According to RefFinder and GeNorm, the combination of TUB, CYP, and PEPKR1 was identified as an internal control for the accurate normalization across all sample set, which was further confirmed by validating the expression of HcPDIL5-2a. Furthermore, the combination of TUB, CYP, and PEPKR1 was used to differentiate the expression pattern of five mitochondria F1F0-ATPase subunit genes (atp1, atp4, atp6, atp8, and atp9) by RT-qPCR during pollen development in CMS line 722A and maintainer line 722B. We found that atp1, atp6, and atp9 exhibited significantly different expression patterns during pollen development in line 722A compared with line 722B. This is the first systematic study of reference genes selection for CMS and will provide useful information for future research on the gene expressions and molecular mechanisms underlying CMS in kenaf.

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Section: Discussionsupporting

confidence: 91%

Candidate Reference Genes Selection and Application for RT-qPCR Analysis in Kenaf with Cytoplasmic Male Sterility Background

Zhou¹,

Chen²,

Khan³

et al. 2017

Front. Plant Sci.

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“…Here, it was recommended as a suitable reference gene in the total sample pools under salt stress by geNorm and NormFinder. EF-1α was recognized as the most appropriate internal controls in Casuarina equisetifolia under salt stress (Fan et al, 2017). By contrast, this gene was one of the most variable genes, and should be rejected as reference genes.…”

Section: Discussionmentioning

confidence: 99%

Selection of reliable reference genes for quantitative RT-PCR in garlic under salt stress

Wang

Tian

Wang

et al. 2019

PeerJ

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Quantitative real-time reverse-transcriptase PCR (qRT-PCR) has been frequently used for detecting gene expression. To obtain reliable results, selection of suitable reference genes is a fundamental and necessary step. Garlic (Allium sativum), a member from Alliaceae family, has been used both as a food flavoring and as a traditional medicine. In the present study, garlic plants were exposed to salt stress (200 mM NaCl) for 0, 1, 4 and 12 h, and garlic roots, bulbs, and leaves were harvested for subsequent analysis. The expression stability of eight candidate reference genes, eukaryotic translation initiation factor 4α (eIF-4α), actin (ACTIN), tubulin β-7 (TUB7), TAP42-interacting protein of 41 kDa (TIP41), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), SAND family protein (SAND), elongation factor 1 alpha (EF-1α), and protein phosphatase 2A (PP2A) were evaluated by geNorm, NormFinder, and BestKeeper. All genes tested displayed variable expression profiles under salt stress. In the leaf and root group, ACTIN was the best reference gene for normalizing gene expression. In garlic clove, ACTIN and SAND were the least variable, and were suitable for gene expression studies under salt stress; these two genes also performed well in all samples tested. Based on our results, we recommend that it is essential to use specific reference genes in different situations to obtain accurate results. Using a combination of multiple stable reference genes, such as ACTIN and SAND, to normalize gene expression is encouraged. The results from the study will be beneficial for accurate determination of gene expression in garlic and other plants.

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“…The relative expression level of each gene was calculated as 2 -ΔΔCT [ 60 ] compared with untreated control plants that were set as 1. Specific primers for CeqMYB genes were designed by Primer Premier 5.0 software and the EF1α was used as housekeeping gene [ 61 ]. Statistical analysis and drawing by GraphPad 8 software [ 62 ].…”

Section: Methodsmentioning

confidence: 99%

Genome-wide analysis of MYB transcription factors and their responses to salt stress in Casuarina equisetifolia

et al. 2021

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Background MYB transcription factors are a kind of DNA binding protein that can specifically interact with the promoter region. Members of MYB TFs are widely involved in plant growth and development, secondary metabolism, stress response, and hormone signal transduction. However, there is no report of comprehensive bioinformatics analysis on the MYB family of Casuarina equisetifolia. Results In this study, bioinformatics methods were used to screen out 182 MYB transcription factors from the Casuarina equisetifolia genome database, including 69 1R-MYB, 107 R2R3-MYB, 4 R1R2R3-MYB, and 2 4R-MYB. The C. equisetifolia R2R3-MYB genes were divided into 29 groups based on the phylogenetic topology and the classification of the MYB superfamily in Arabidopsis thaliana, while the remaining MYB genes (1R-MYB, R1R2R3-MYB, and 4R-MYB) was divided into 19 groups. Moreover, the conserved motif and gene structure analysis shown that the members of the CeqMYBs were divided into the same subgroups with mostly similar gene structures. In addition, many conserved amino acids in the R2 and R3 domains of CeqMYBs by WebLogo analysis, especially tryptophan residues (W), with 3 conserved W in R2 repeat and 2 conserved W in R3 repeat. Combining promoter and GO annotation analysis, speculated on the various biological functions of CeqMYBs, thus 32 MYB genes were selected to further explore its response to salt stress by using qPCR analysis technique. Most CeqMYB genes were differentially regulated following multiple salt treatments. Conclusions Seven genes (CeqMYB164, CeqMYB4, CeqMYB53, CeqMYB32, CeqMYB114, CeqMYB71 and CeqMYB177) were assigned to the “response to salt stress” by GO annotation. Among them, the expression level of CeqMYB4 was up-regulated under various salt treatments, indicating CeqMYB4 might participated in the response to salt stress. Our results provide important information for the biological function of C. equisetifolia, as well as offer candidate genes for further study of salt stress mechanism.

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Selection of reference genes for quantitative real-time PCR in Casuarina equisetifolia under salt stress

Cited by 14 publications

References 38 publications

Candidate Reference Genes Selection and Application for RT-qPCR Analysis in Kenaf with Cytoplasmic Male Sterility Background

Candidate Reference Genes Selection and Application for RT-qPCR Analysis in Kenaf with Cytoplasmic Male Sterility Background

Selection of reliable reference genes for quantitative RT-PCR in garlic under salt stress

Genome-wide analysis of MYB transcription factors and their responses to salt stress in Casuarina equisetifolia

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