Comprehensive evaluation of candidate reference genes for quantitative real-time PCR-based analysis in Caucasian clover

AbstractThe forage species Caucasian clover (Trifolium ambiguum M. Bieb.), a groundcover plant, is resistant to both cold and drought. However, reference genes for qRT-PCR-based analysis of Caucasian clover are lacking. In this study, 12 reference genes were selected on the basis of transcriptomic data. These genes were used to determine the most stably expressed genes in various organs of Caucasian clover under cold, salt and drought stress for qRT-PCR-based analysis. Reference gene stability was analyzed by geNorm, NormFinder, BestKeeper, the ∆Ct method and RefFinder. Under salt stress, RCD1 and PPIL3 were the most stable reference genes in the leaves, and NLI1 and RCD1 were the most stable references genes in the roots. Under low-temperature stress, APA and EFTu-GTP were the most stable reference genes in the leaves, and the RCD1 and NLI2 genes were highly stable in the roots. Under 10% PEG-6000 stress, NLI1 and NLI2 were highly stable in the leaves, and RCD1 and PPIL3 were the most stable in the roots. Overall, RCD1 and NLI2 were the most stable reference genes in organs under normal conditions and across all samples. The most and least stable reference genes were validated by assessing their appropriateness for normalization via WRKY genes.

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Identification and validation of reference genes for quantitative real-time PCR under salt stress in a halophyte, Sesuvium portulacastrum

Plant Gene ◽

10.1016/j.plgene.2017.11.003 ◽

2018 ◽

Vol 13 ◽

pp. 18-24 ◽

Cited By ~ 5

Author(s):

Ganesh C. Nikalje ◽

Ashish K. Srivastava ◽

Gaurav Sablok ◽

Girdhar K. Pandey ◽

Tukaram D. Nikam ◽

...

Keyword(s):

Salt Stress ◽

Real Time ◽

Real Time Pcr ◽

Reference Genes ◽

Quantitative Real Time Pcr ◽

Sesuvium Portulacastrum

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

Biologia Plantarum ◽

10.1007/s10535-016-0670-y ◽

2017 ◽

Vol 61 (3) ◽

pp. 463-472 ◽

Cited By ~ 3

Author(s):

C. Fan ◽

Z. Qiu ◽

B. Zeng ◽

Y. Liu ◽

X. Li ◽

...

Keyword(s):

Salt Stress ◽

Real Time ◽

Real Time Pcr ◽

Reference Genes ◽

Casuarina Equisetifolia ◽

Quantitative Real Time Pcr ◽

Selection Of

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Germin Like Protein Genes Exhibit Modular Expression During Salt and Drought Stress in Elite Rice Cultivars

10.21203/rs.3.rs-713600/v1 ◽

2021 ◽

Author(s):

Jazba Anum ◽

Charlotte O’Shea ◽

M Zeeshan Hyder ◽

Sumaira Farrukh ◽

Karen Skriver ◽

...

Keyword(s):

Salt Stress ◽

Drought Stress ◽

Expression Profiles ◽

Rice Variety ◽

Chromosome 8 ◽

Tissue Type ◽

Rice Cultivars ◽

Relative Expression ◽

Salt And Drought Stress ◽

Leaves And Roots

Abstract Germin-like proteins (GLPs) are ubiquitous plant proteins, which play significant role in plant responses against various abiotic stresses. However, the potential functions of GLPs in rice (Oryza Sativa) against salt and drought stress are still unclear. In this study, transcriptional variation of 8 OsGLP genes (OsGLP3-6, OsGLP4-1, OsGLP8-4, OsGLP8-7, OsGLP8-10, OsGLP8-11 and OsGLP8-12) was analyzed in leaves and roots of two economically important Indica rice cultivars, KS282 and Super Basmati under salt and drought stress at early seedling stage. The relative expression analysis from qRT-PCR indicated the highest increase in expression of OsGLP3-6 in leaves and roots of both rice varieties with a significantly higher expression in KS282. Moreover, relative change in expression of OsGLP8-7, OsGLP8-10 and OsGLP8-11 under salt stress and OsGLP8-7 under drought stress was also commonly higher in leaves and roots of KS282 as compared to Super Basmati. Whereas, OsGLP3-7 and OsGLP8-12 after salt stress and OsGLP8-4 and OsGLP8-12 after drought stress were observed with higher relative expression in roots of Super Basmati than KS282. Importantly, the OsGLP3-6 and OsGLP4-1 from chromosome 3 and 4 respectively showed higher expression in leaves whereas most of the OsGLP genes from chromosome 8 exhibited higher expression in roots. Overall, as a result of this comparative analysis, OsGLP genes showed both general and specific expression profiles depending upon a specific rice variety, stress condition as well as tissue type. These results will increase our understanding of role of OsGLP genes in rice crop and provide useful information for the further in-depth research on their regulatory mechanisms in response to these stress conditions.

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CIPK11: a calcineurin B-like protein-interacting protein kinase from Nitraria tangutorum, confers tolerance to salt and drought in Arabidopsis

BMC Plant Biology ◽

10.1186/s12870-021-02878-x ◽

2021 ◽

Vol 21 (1) ◽

Author(s):

Lu Lu ◽

Xinying Chen ◽

Pengkai Wang ◽

Ye Lu ◽

Jingbo Zhang ◽

...

Keyword(s):

Salt Stress ◽

Drought Stress ◽

Transgenic Plants ◽

Drought Treatment ◽

Interacting Protein ◽

Expression Of Genes ◽

Nitraria Tangutorum ◽

Genes Encoding ◽

Salt And Drought Stress ◽

Weak Expression

Abstract Background The CIPKs are a group of plant-specific Ser/Thr protein kinases acting in response to calcium signaling, which plays an important role in the physiological and developmental adaptation of plants to adverse environments. However, the functions of halophyte-derived CIPKs are still poorly understood, that limits a potential application of CIPKs from halophytes for improving the tolerance of glycophytes to abiotic stresses. Results In this study, we characterized the NtCIPK11 gene from the halophyte Nitraria tangutorum and subsequently analyzed its role in salt and drought stress tolerance, using Arabidopsis as a transgenic model system. NtCIPK11 expression was upregulated in N. tangutorum root, stem and blade tissues after salt or drought treatment. Overexpressing NtCIPK11 in Arabidopsis improved seed germination on medium containing different levels of NaCl. Moreover, the transgenic plants grew more vigorously under salt stress and developed longer roots under salt or drought conditions than the WT plants. Furthermore, NtCIPK11 overexpression altered the transcription of genes encoding key enzymes involved in proline metabolism in Arabidopsis exposed to salinity, however, which genes showed a relatively weak expression in the transgenic Arabidopsis undergoing mannitol treatment, a situation that mimics drought stress. Besides, the proline significantly accumulated in NtCIPK11-overexpressing plants compared with WT under NaCl treatment, but that was not observed in the transgenic plants under drought stress caused by mannitol application. Conclusions We conclude that NtCIPK11 promotes plant growth and mitigates damage associated with salt stress by regulating the expression of genes controlling proline accumulation. These results extend our understanding on the function of halophyte-derived CIPK genes and suggest that NtCIPK11 can serve as a candidate gene for improving the salt and drought tolerance of glycophytes through genetic engineering.

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Evaluation of Optimal Reference Genes for qRT-PCR Analysis in Hyphantria cunea (Drury)

Insects ◽

10.3390/insects13010097 ◽

2022 ◽

Vol 13 (1) ◽

pp. 97

Author(s):

Xudong Zhao ◽

Yishu Geng ◽

Tianyi Hu ◽

Yongang Zhao ◽

Suling Yang ◽

...

Keyword(s):

Gene Expression ◽

Reference Genes ◽

Comprehensive Evaluation ◽

Larval Density ◽

Physiological Role ◽

Invasive Pest ◽

Pcr Analysis ◽

Hyphantria Cunea ◽

Experimental Conditions ◽

Qrt Pcr

The relative quantification of gene expression is mainly achieved through reverse transcription-quantitative PCR (qRT-PCR); however, its reliability and precision rely on proper data normalization using one or more optimal reference genes. Hyphantria cunea (Drury) has been an invasive pest of forest trees, ornamental plants, and fruit trees in China for many years. Currently, the molecular physiological role of reference genes in H. cunea is unclear, which hinders functional gene study. Therefore, eight common reference genes, RPS26, RPL13, UBI, AK, RPS15, EIF4A, β-actin, α-tub, were selected to evaluate levels of gene expression stability when subjected to varied experimental conditions, including developmental stage and gender, different tissues, larvae reared on different hosts and different larval density. The geNorm, BestKeeper, ΔCt method, and NormFinder statistical algorithms were used to normalize gene transcription data. Furthermore, the stability/suitability of these candidates was ranked overall by RefFinder. This study provides a comprehensive evaluation of reference genes in H. cunea and could help select reference genes for other Lepidoptera species.

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Selection and Evaluation of Potential Reference Genes for Quantitative Real-Time PCR in Agaricus blazei Based on Transcriptome Sequencing Data

BioMed Research International ◽

10.1155/2021/6661842 ◽

2021 ◽

Vol 2021 ◽

pp. 1-13

Author(s):

Yuan-Ping Lu ◽

Jian-Hua Liao ◽

Zhong-Jie Guo ◽

Hui-Qing Zheng ◽

Ling-Fang Lu ◽

...

Keyword(s):

Gene Expression ◽

Cytochrome C ◽

Expression Pattern ◽

Real Time Pcr ◽

Reference Genes ◽

Fruit Body ◽

Pcr Analysis ◽

Agaricus Blazei ◽

Quantitative Real Time Pcr ◽

Qrt Pcr

Quantitative real-time PCR (qRT-PCR) is widely used to detect gene expression due to its high sensitivity, high throughput, and convenience. The accurate choice of reference genes is required for normalization of gene expression in qRT-PCR analysis. In order to identify the optimal candidates for gene expression analysis using qRT-PCR in Agaricus blazei, we studied the potential reference genes in this economically important edible fungus. In this study, transcriptome datasets were used as source for identification of candidate reference genes. And 27 potential reference genes including 21 newly stable genes, three classical housekeeping genes, and homologous genes of three ideal reference genes in Volvariella volvacea, were screened based on transcriptome datasets of A. blazei and previous studies. The expression stability of these genes was investigated by qRT-PCR analysis and further evaluated by four software packages, geNorm, NormFinder, BestKeeper, and RefFinder. Among these candidates, α-TUB (Tubulin alpha) and Cox5a (COX5A subunit VA of cytochrome c oxidase) were revealed as the most stable in fruit body, and suitable for 5 different developmental stages. α-TUB and ATP3 (ATP3 gamma subunit of the F1 sector of mitochondrial F1F0 ATP synthase) showed the most stable expression in stipe tissues and, Uqcrc (core subunit of the ubiquinol-cytochrome c reductase complex) and PUP3 (20S proteasome subunit beta 3) performed well in pileus tissues during the process of A. blazei development, while GAPDH (glyceraldehyde-3-phosphate dehydrogenase) was among the least stable genes in all sample sets. Finally, the Ableln3 (homology of eln3 gene of Coprinus cinereus) was adopted to validate the reliability of these stable and unstable reference genes, indicating that the use of unsuitable reference genes as internal controls could change the target gene’s expression pattern. This study can provide guidance for choosing reference genes for analyzing the expression pattern of target genes and facilitate the functional genomic investigation on fruit body formation and development, as well as stipe elongation and pileus expansion in A. blazei.

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Selection of reference genes for quantitative real-time PCR analysis in cucumber (Cucumis sativus L.), pumpkin (Cucurbita moschata Duch.) and cucumber–pumpkin grafted plants

PeerJ ◽

10.7717/peerj.6536 ◽

2019 ◽

Vol 7 ◽

pp. e6536 ◽

Cited By ~ 5

Author(s):

Li Miao ◽

Xing Qin ◽

Lihong Gao ◽

Qing Li ◽

Shuzhen Li ◽

...

Keyword(s):

Gene Expression ◽

Real Time ◽

Reference Gene ◽

Real Time Pcr ◽

Reference Genes ◽

Stable Expression ◽

Quantitative Real Time Pcr ◽

Graft Union ◽

Expression Levels ◽

Qrt Pcr

Background Quantitative real-time PCR (qRT-PCR) is a commonly used high-throughput technique to measure mRNA transcript levels. The accuracy of this evaluation of gene expression depends on the use of optimal reference genes. Cucumber–pumpkin grafted plants, made by grafting a cucumber scion onto pumpkin rootstock, are superior to either parent plant, as grafting conveys many advantages. However, although many reliable reference genes have been identified in both cucumber and pumpkin, none have been obtained for cucumber–pumpkin grafted plants. Methods In this work, 12 candidate reference genes, including eight traditional genes and four novel genes identified from our transcriptome data, were selected to assess their expression stability. Their expression levels in 25 samples, including three cucumber and three pumpkin samples from different organs, and 19 cucumber–pumpkin grafted samples from different organs, conditions, and varieties, were analyzed by qRT-PCR, and the stability of their expression was assessed by the comparative ΔCt method, geNorm, NormFinder, BestKeeper, and RefFinder. Results The results showed that the most suitable reference gene varied dependent on the organs, conditions, and varieties. CACS and 40SRPS8 were the most stable reference genes for all samples in our research. TIP41 and CACS showed the most stable expression in different cucumber organs, TIP41 and PP2A were the optimal reference genes in pumpkin organs, and CACS and 40SRPS8 were the most stable genes in all grafted cucumber samples. However, the optimal reference gene varied under different conditions. CACS and 40SRPS8 were the best combination of genes in different organs of cucumber–pumpkin grafted plants, TUA and RPL36Aa were the most stable in the graft union under cold stress, LEA26 and ARF showed the most stable expression in the graft union during the healing process, and TIP41 and PP2A were the most stable across different varieties of cucumber–pumpkin grafted plants. The use of LEA26, ARF and LEA26+ARF as reference genes were further verified by analyzing the expression levels of csaCYCD3;1, csaRUL, cmoRUL, and cmoPIN in the graft union at different time points after grafting. Discussion This work is the first report of appropriate reference genes in grafted cucumber plants and provides useful information for the study of gene expression and molecular mechanisms in cucumber–pumpkin grafted plants.

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Screening and Evaluation of Stable Reference Genes for Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) Analysis in Chinese Fir Roots under Water, Phosphorus, and Nitrogen Stresses

Forests ◽

10.3390/f10121087 ◽

2019 ◽

Vol 10 (12) ◽

pp. 1087 ◽

Cited By ~ 2

Author(s):

Ranhong Chen ◽

Wanting Chen ◽

Mulualem Tigabu ◽

Weimin Zhong ◽

Yushan Li ◽

...

Keyword(s):

Gene Expression ◽

Drought Stress ◽

Reference Genes ◽

Chinese Fir ◽

Chain Reaction ◽

Internal Reference ◽

Qrt Pcr ◽

N Starvation ◽

Polymerase Chain ◽

The Stability

Chinese fir (Cunninghamia lanceolata) is an economical important timber species widely planted in southeastern Asia. Decline in yield and productivity during successive rotation is believed to be linked with abiotic stress, such as drought stress and nitrogen (N) and phosphorus (P) starvation. Molecular breeding could be an option to develop tolerant genotypes. For gene expression studies using quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR), stable reference genes are needed for normalization of gene expression under different experimental conditions. However, there is no internal reference genes identified for Chinese fir under abiotic stresses. Thus, nine internal reference genes based on transcriptome data were selected and analyzed in the root of Chinese fir under drought stress and N and P starvation. Data were analyzed using geNorm, NormFinder, and BestKeeper, to screen and identify the best reference genes. The results showed that the UBQ and GAPDH genes were the two most stable genes under drought stress and the Actin1 and GAPDH were the two most stable genes under P starvation. Further, it was discovered that the Actin1 and UBC were the two most stable genes under N starvation among nine candidate reference genes. The gene expression of drought stress induced expression protein 14-3-3-4, the P transporter gene ClPht1;3, and the nitrate transporter gene NRT1.1 were used to verify the stability of the selected reference genes under drought stress and P and N starvation, respectively, and the results revealed that the screened reference genes were sufficient to normalize expression of the target genes. In conclusion, the results demonstrate that the stability of reference genes was closely related to the external conditions and reference genes applied to the roots of Chinese fir under different abiotic stress treatments were different. Our data will facilitate further studies on stress ecology and gene function analysis in Chinese fir.

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Systematic assessment of reference genes for RT-qPCR across plant species under salt stress and drought stress

Acta Physiologiae Plantarum ◽

10.1007/s11738-015-1922-8 ◽

2015 ◽

Vol 37 (9) ◽

Cited By ~ 4

Author(s):

Xinlong Xiao ◽

Xiaomeng Wu ◽

Jinbiao Ma ◽

Pengbo Li ◽

Taotao Li ◽

...

Keyword(s):

Salt Stress ◽

Drought Stress ◽

Plant Species ◽

Reference Genes ◽

Systematic Assessment

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Identification and Testing of Reference Genes for qRT-PCR Analysis During Pear Fruit Development

10.21203/rs.3.rs-910473/v1 ◽

2021 ◽

Author(s):

Guoming Wang ◽

Zhihua Guo ◽

Xueping Wang ◽

Hongru Gao ◽

Kaijie Qi ◽

...

Keyword(s):

Fruit Development ◽

Reference Genes ◽

Housekeeping Genes ◽

Housekeeping Gene ◽

Pcr Analysis ◽

Quantitative Real Time Pcr ◽

Sequencing Data ◽

Pear Fruit ◽

Qrt Pcr ◽

Fruit Development And Ripening

Abstract Background: Quantitative real-time PCR (qRT-PCR) is currently one of the most reliable and improved tools for analyzing gene expression. Various studies have shown that housekeeping genes was varied with cultivars, tissues and treatment. The reliable and stable reference genes were necessarily identified and evaluated according to different experimental requirements. Result: In this study, 10 candidate reference genes were initially screened based on the transcriptome sequencing data of four pear fruit development stages of three different pear cultivars, including a candidate housekeeping gene PbrTUB. Furthermore, we ranked the expression stability of 10 candidate reference genes using algorithms GeNorm, NormFinder, BestKeeper and ReFinder. Finally, the result showed that Pbr028511, Pbr038418 and Pbr041114 were the most stable reference gene in Cuiguan, Housui and Xueqing fruit, respectively. Concludion: Thee results provide a valuable resource that serve as significant reference for gene function explorations and molecular mechanism studies in fruit development and ripening of different pear cultivars.

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