Selection of suitable reference genes for quantitative real-time PCR gene expression analysis in Mulberry (Morus alba L.) under different abiotic stresses

Shukla, Pawan; Reddy, Ramesha A.; Ponnuvel, Kangayam M.; Rohela, Gulab Khan; Shabnam, Aftab A.; Ghosh, M. K.; Mishra, Rakesh

doi:10.1007/s11033-019-04631-y

Cited by 41 publications

(23 citation statements)

References 32 publications

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“…GAPDH was the best choice in Chinese cabbage (Qi et al 2010) under drought stress, in Eleusine coracana (Jatav et al 2018) ( Wang et al 2019). UBI showed peak stability under ABA stress in Hordeum brevisubulatum (Zhang et al 2018), under cold stress in Morus alba (Shukla et al 2019), under drought stress in wheat (Kiarash et al 2018;Dudziak et al 2020), and across different tissues in Miscanthus lutarioriparia (Cheng et al 2019). The most proper reference gene for cold stress in Hordeum brevisubulatum (Zhang et al 2018), salinity stress in Panicum virgatum (Huang et al 2014) and drought stress in Miscanthus sinensis (Zhong et al 2020) was 18S.…”

Section: Discussionmentioning

confidence: 99%

“…Quantitative real-time PCR (qRT-PCR) analysis, with many benefits of simplicity, accuracy, specificity, short turn-around time and high-throughput characteristics, has been used in a variety of fields about study relative expression level of target genes (Bustin et al 2005;Huggett et al 2005;Shukla et al 2019). There are many rules that must be followed to ensure reproducible and accurate results using qRT-PCR (Udvardi et al 2008;Derveaux et al 2010).…”

Section: Introductionmentioning

confidence: 99%

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Selection of Reference Genes for qRT-PCR in Bletilla striata under Heat Stress

Liang¹

2021

IJAB

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Bletilla striata (Thunb.) Reihb.f., a traditional Chinese herbal medicine, has attracted increasing attention because of its wide range of applicability to the medical field and chemical industry. B. striata has been identified to be particularly sensitive to high temperatures. Thus, the study of temperature stress on gene transcription is of interest in the field. Use of reliable reference genes is essential for qRT-PCR analysis of genes. However, little information regarding suitable reference genes for the genus Bletilla has been published. In this study, the sequences of seven potential reference genes in B. striata were obtained via a homology cloning strategy. These genes were as follows: glyceraldehydes-3-phosphate dehydrogenase (GAPDH), 18S ribosomal RNA (18S), elongation factor 1 alpha (EF1α), α-tubulin (TUA), β-tubulin (TUB), ubiquitin (UBI), and NAC domain protein (NAC). We then evaluated the stability levels of these transcripts in different tissues (root, tuber, and leaf) exposed to high temperature using three conventional software and comprehensive RefFinder algorithms. The results indicated that 18S and TUB were the best internal control genes among different periods of heat treatment and that a combination of 18S and UBI was the best in different tissues. Altogether, 18S and UBI were identified to be the best reference genes for all the samples, while NAC and TUA were the least stable reference genes. The results will be useful for studies on target gene expression in plants of the Bletilla genus. © 2021 Friends Science Publishers© 2021 Friends Science Publishers© 2021 Friends Science Publishers© 2021 Friends Science Publishers© 2021 Friends Science Publishers© 2021 Friends Science Publishers© 2021 Friends Science Publishers© 2021 Friends Science Publishers© 2021 Friends Science Publishers© 2021 Friends Science Publishers© 2021 Friends Science Publishers© 2021 Friends Science Publishers© 2021 Friends Science Publishers© 2021 Friends Science Publishers© 2021 Friends Science Publishers© 2021 Friends Science Publishers© 2021 Friends Science Publishers© 2021 Friends Science Publishers© 2021 Friends Science Publishers© 2021 Friends Science Publishers© 2021 Friends Science Publishers© 2021 Friends Science Publishers© 2021 Friends Science Publishers© 2021 Friends Science Publishers © 2021 Friends Science Publishers© 2021 Friends Science Publishers© 2021 Friends Science Publishers© 2021 Friends Science Publishers© 2021 Friends Science Publishers© 2021 Friends Science Publishers© 2021 Friends Science Publishers©

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Selection of Reference Genes for qRT-PCR in Bletilla striata under Heat Stress

Liang¹

2021

IJAB

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“…Studies on the evaluation and validation of reference genes have been conducted in many plant species, especially in herbaceous plants, such as Arabidopsis thaliana [17], Glycine max [18]. Similar studies have been carried out in certain woody plants, such as Morus alba L. [19], Carex rigescens [20]. However, the systematic evaluation and validation of appropriate reference genes in birch ( Betula platyphylla ) under different abiotic stress conditions has not been reported.…”

Section: Introductionmentioning

confidence: 99%

Selection of appropriate reference genes for quantitative real-time reverse transcription PCR in Betula platyphylla under salt and osmotic stress conditions

et al. 2019

PLoS ONE

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Selecting appropriate reference genes is vital to normalize gene expression analysis in birch (Betula platyphylla) under different abiotic stress conditions using quantitative realtime reverse transcription PCR (qRT-PCR). In this study, 11 candidate birch reference genes (ACT, TUA, TUB, TEF, 18S rRNA, EF1α, GAPDH, UBC, YLS8, SAND, and CDPK) were selected to evaluate the stability of their expression in different tissues and under different abiotic stress conditions. Three statistical algorithms (GeNorm, NormFinder, and BestKeeper) were used to analyze the stability of the 11 candidate reference genes to identify the most appropriate one. The results indicated that EF-1α was the most stable reference gene in different birch tissues, ACT was the most stable reference gene for normal conditions, ACT and TEF were the most stable reference genes for salt stress treatment, TUB was the most stable reference gene for osmotic stress treatment, and ACT was the most appropriate choice in all samples of birch. In conclusion, the most appropriate reference genes varied among different experimental conditions. However, in this study, ACT was the optimum reference gene in all experimental groups, except in the different tissues group. GAPDH was the least stable candidate reference gene in all experimental conditions. In addition, three stress-induced genes (BpGRAS1, BpGRAS16, and BpGRAS19) were chosen to verify the stability of the selected reference genes in different tissues and under salt stress. This study laid the foundation for the selection of appropriate reference gene(s) for future gene expression pattern studies in birch.

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“…Genes such as ACT [ 17 ], GAPDH [ 18 ], TUA [ 19 ], TUB [ 20 ], and EF1α [ 21 ] are normally used as reference genes. However, some of these reference genes may have different expression among plant tissues, species, and growth conditions [ 22 ]. As no single reference gene is universally suitable for all experiments, the selection of reference genes under various experimental conditions is crucial for exactly quantifying the expression levels of genes induced by all kinds of abiotic stress [ 23 ].…”

Section: Introductionmentioning

confidence: 99%

Selection and Validation of Reference Genes for Quantitative Real-Time PCR in White Clover (Trifolium repens L.) Involved in Five Abiotic Stresses

Qin

Zhou

Nie

et al. 2020

Plants

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White clover (Trifolium repens L.) is a widely cultivated cool-season perennial forage legume in temperate grassland systems. Many studies have analyzed the gene expression in this grass species using quantitative real-time reverse transcription PCR (qRT-PCR). The selection of stable reference genes for qRT-PCR is crucial. However, there was no detailed study on reference genes in different tissues of white clover under various abiotic stress conditions. Herein, 14 candidate reference genes (ACT7, ACT101, TUA1109, TUB, CYP, 60SrRNA, UBQ, E3, GAPDH1, GAPDH2, PP2A, BAM3, SAMDC, and ABC) were selected and analyzed by four programs (GeNorm, NormFinder, BestKeeper, and RefFinder). Samples were taken from two tissues (leaves and roots) under five different abiotic stresses (drought, salt, heat, cold, and heavy metal stress). Our results showed that 60SrRNA and ACT101 were the two top-ranked genes for all samples. Under various experimental conditions, the most stable gene was different; however, SAMDC, UBQ, 60SrRNA, and ACT101 were always top ranked. The most suitable reference genes should be selected according to different plant tissues and growth conditions. Validation of these reference genes by expression analysis of Cyt-Cu/Zn SOD and CAT confirmed their reliability. Our study will benefit the subsequent research of gene function in this species.

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Selection of suitable reference genes for quantitative real-time PCR gene expression analysis in Mulberry (Morus alba L.) under different abiotic stresses

Cited by 41 publications

References 32 publications

Selection of Reference Genes for qRT-PCR in Bletilla striata under Heat Stress

Selection of Reference Genes for qRT-PCR in Bletilla striata under Heat Stress

Selection of appropriate reference genes for quantitative real-time reverse transcription PCR in Betula platyphylla under salt and osmotic stress conditions

Selection and Validation of Reference Genes for Quantitative Real-Time PCR in White Clover (Trifolium repens L.) Involved in Five Abiotic Stresses

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