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

Qin, Pu; Zhou, Li; Nie, Gang; Zhou, Jiqiong; Liu, Lin; Yan, Peng

doi:10.3390/plants9080996

Cited by 14 publications

(13 citation statements)

References 41 publications

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“…Ideally, the stability of the reference gene should not be influenced by the experimental conditions. Nevertheless, several studies point out that the mRNA transcript levels could be affected by tissue type [ 19 , 20 ], developmental stage [ 26 ], treatment type [ 27 , 28 ], and genotype [ 26 ] as well. Furthermore, the expression stability of the same reference gene may also depend on the investigated species [ 17 ].…”

Section: Discussionmentioning

confidence: 99%

“…The majority of candidate reference genes were chosen based on existing literature that utilised them as suitable stable internal control genes for qRT-PCR analysis in different plant species [ 18 , 21 , 28 , 41 ]. The applicability of ADPR (Unigene cluster: Ta30797) and PGD (Unigene cluster: Ta2291) (candidate reference genes proposed by Paolacci et al [ 27 ]) were confirmed previously by our research group in wheat ( Triticum aestivum L.) under different stress conditions [ 40 , 42 , 43 , 44 ].…”

Section: Methodsmentioning

confidence: 99%

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Validation of Reference Genes for Studying Different Abiotic Stresses in Oat (Avena sativa L.) by RT-qPCR

Tajti

Pál

Janda

2021

Plants

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Oat (Avena sativa L.) is a widely cultivated cereal with high nutritional value and it is grown mainly in temperate regions. The number of studies dealing with gene expression changes in oat continues to increase, and to obtain reliable RT-qPCR results it is essential to establish and use reference genes with the least possible influence caused by experimental conditions. However, no detailed study has been conducted on reference genes in different tissues of oat under diverse abiotic stress conditions. In our work, nine candidate reference genes (ACT, TUB, CYP, GAPD, UBC, EF1, TBP, ADPR, PGD) were chosen and analysed by four statistical methods (GeNorm, Normfinder, BestKeeper, RefFinder). Samples were taken from two tissues (leaves and roots) of 13-day-old oat plants exposed to five abiotic stresses (drought, salt, heavy metal, low and high temperatures). ADPR was the top-rated reference gene for all samples, while different genes proved to be the most stable depending on tissue type and treatment combinations. TUB and EF1 were most affected by the treatments in general. Validation of reference genes was carried out by PAL expression analysis, which further confirmed their reliability. These results can contribute to reliable gene expression studies for future research in cultivated oat.

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Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Validation of Reference Genes for Studying Different Abiotic Stresses in Oat (Avena sativa L.) by RT-qPCR

Tajti

Pál

Janda

2021

Plants

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“…Ideally, the stability of the reference gene should not be in uenced by the experimental conditions. Nevertheless, several studies point out, that the mRNA transcript levels could be affected by tissue type [11,12], developmental stage [18] treatment type [19,20] and genotype [18] as well. Furthermore, the expression stability of the same reference gene may also depend on the investigated species [21].…”

Section: Discussionmentioning

confidence: 99%

“…The majority of candidate reference genes were chosen based on their description being stable internal control genes for qRT-PCR analysis in different plant species ( [10,13,19,34]. The applicability of ADPR (Unigene cluster: Ta30797) and PGD (Unigene cluster: Ta2291) (candidate reference genes proposed by [20]) were con rmed previously by our research group in wheat (Triticum aestivum L.) under different stress conditions [33,[35][36][37].…”

Section: Reference Gene Selection and Pcr Primer Designmentioning

confidence: 99%

Validation of Reference Genes for Studying Different Abiotic Stresses by RT-qPCR in Oat (Avena Sativa L.)

Tajti

Pál

Janda

2021

Preprint

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Background: Oat (Avena sativa L.) is a widely cultivated cereal with high nutritional value growing mainly in temperate regions. The number of studies dealing with gene expression changes in oat increases and to obtain reliable RT-qPCR results it is essential to use references genes that are least influenced by experimental conditions. However, no detailed study was conducted on reference genes in different tissues of oat under diverse abiotic stress conditions. Results: In our work 9 candidate reference genes (ACT, TUB, CYP, GAPD, UBC, EF1, TBP, ADPR, PGD) were chosen and analysed by 4 statistical methods (GeNorm, Normfinder, BestKeeper, RefFinder). Samples were taken from two tissues (leaves and roots) of 13-day-old oat plants exposed to 5 abiotic stresses (drought, salt, heavy metal, low and high temperatures). ADPR was the top-rated reference gene for all samples, while different genes proved to be the most stable depending on tissue type and treatment combinations. TUB and EF1 were most affected by the treatments in general. Validation of reference genes was carried out by PAL expression analysis which further confirmed their reliability. Conclusions: Our main goal was to identify reference genes with stable expression in oat under different abiotic stress conditions in different tissues. These results can contribute to reliable gene expression studies for future researches in cultivated oat.

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“…For accurate normalization purpose, the expression of reference genes should have minimal variation independent of tissue types, developmental stages, and experimental treatments (Wong and Medrano, 2005;Huang et al, 2016Huang et al, , 2019. Indeed, such ideal universal reference genes do not exist at all (Cubero-Leon et al, 2012;Pu et al, 2020). Therefore, it is crucial to select and validate reliable reference genes for target gene expressions under specific experimental conditions in the species studied.…”

Section: Transcriptional Responses Under Ammonia Challenge and Implications Of Ammonia Stress Coping Strategiesmentioning

confidence: 99%

Ammonia Stress Coping Strategy in a Highly Invasive Ascidian

et al. 2021

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The outbreak of invasive ascidian Molgula manhattensis has negatively affected marine and coastal ecosystems and caused huge economic loss in various industries such as aquaculture. In mariculture systems usually characterized by high ammonia nitrogen, the capacity of M. manhattensis to defend against drastic ammonia elevation plays a crucial role in its survival and subsequent invasions. However, ammonia coping strategies and associated genes/proteins remain largely unknown. Here we investigated rhesus glycoproteins (Rh)-mediated ammonia transport by identifying all Rh proteins and exploring their mRNA expression regulations under ammonia stress. Three types of primitive Rh proteins were identified, and all contained conserved amino acid residues and functional domains. Ammonia stress largely suppressed the expression of immune-related genes, but rapidly induced the increased expression of Rh genes. Ammonia was converted into glutamine as indicated by the increased expression of glutamine synthetase gene, rather than urea as illustrated by the stable expression of arginase gene. Collectively, M. manhattensis mitigates ammonia challenge by enhancing ammonia excretion through Rh channels and detoxifying ammonia into glutamine. Our results provide insights into the molecular mechanisms underlying high tolerance and invasion success to high ammonia environments by invasive ascidians.

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

Cited by 14 publications

References 41 publications

Validation of Reference Genes for Studying Different Abiotic Stresses in Oat (Avena sativa L.) by RT-qPCR

Validation of Reference Genes for Studying Different Abiotic Stresses in Oat (Avena sativa L.) by RT-qPCR

Validation of Reference Genes for Studying Different Abiotic Stresses by RT-qPCR in Oat (Avena Sativa L.)

Ammonia Stress Coping Strategy in a Highly Invasive Ascidian

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