Identification and Validation of Reference Genes for Seashore Paspalum Response to Abiotic Stresses

Yu, Liu; Liu, Jun; Xu, Lei; Lai, Hui Ting; Chen, Yu; Yang, Ziyi; Huang, Bingru

doi:10.3390/ijms18061322

Cited by 26 publications

(30 citation statements)

References 37 publications

(59 reference statements)

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“…In this study, U2AF displayed the highest stability in Cd, Hg, Cr, and As treated leaves as well as in Pb, Hg, Cr, and As treated roots of switchgrass. U2AF has been reported in other studies to be the most stable reference gene, such as in Pinus massoniana L., at different stages post-inoculation by nematode [35] and roots/leaves of Paspalum vaginatum Sw. under Cd and cold stress [36]. In the roots of switchgrass subjected to Cd treatment, CYP5 and UBQ1 were determined to be the most appropriate internal reference genes.…”

Section: Discussionmentioning

confidence: 99%

Identification and Validation of Reference Genes for RT-qPCR Analysis in Switchgrass under Heavy Metal Stresses

Zhao

Zhou²,

Meng

2020

Genes

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Switchgrass (Panicum Virgatum L.) has been recognized as the new energy plant, which makes it ideal for the development of phytoremediation on heavy metal contamination in soils with great potential. This study aimed to screen the best internal reference genes for the real-time quantitative PCR (RT-qPCR) in leaves and roots of switchgrass for investigating its response to various heavy metals, such as cadmium (Cd), lead (Pb), mercury (Hg), chromium (Cr), and arsenic (As). The stability of fourteen candidate reference genes was evaluated by BestKeeper, GeNorm, NormFinder, and RefFinder software. Our results identified U2AF as the best reference gene in Cd, Hg, Cr, and As treated leaves as well as in Hg, Pb, As, and Cr stressed root tissues. In Pb treated leaf tissues, 18S rRNA was demonstrated to be the best reference gene. CYP5 was determined to be the optimal reference gene in Cd treated root tissues. The least stable reference gene was identified to be CYP2 in all tested samples except for root tissues stressed by Pb. To further validate the initial screening results, we used the different sets of combinatory internal reference genes to analyze the expression of two metal transport associated genes (PvZIP4 and PvPDB8) in young leaves and roots of switchgrass. Our results demonstrated that the relative expression of the target genes consistently changed during the treatment when CYP5/UBQ1, U2AF/ACT12, eEF1a/U2AF, or 18S rRNA/ACT12 were combined as the internal reference genes. However, the time-dependent change pattern of the target genes was significantly altered when CYP2 was used as the internal reference gene. Therefore, the selection of the internal reference genes appropriate for specific experimental conditions is critical to ensure the accuracy and reliability of RT-qPCR. Our findings established a solid foundation to further study the gene regulatory network of switchgrass in response to heavy metal stress.

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Section: Discussionmentioning

confidence: 99%

Identification and Validation of Reference Genes for RT-qPCR Analysis in Switchgrass under Heavy Metal Stresses

Zhao

Zhou²,

Meng

2020

Genes

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“…Not surprisingly, each algorithm gave a different ranking. According to previous research, the use of multiple reference genes is preferable to gain a more accurate and reliable result ( Vandesompele et al, 2002 ; Martins et al, 2016 ; Liu et al, 2017 ). The optimal number of reference genes to achieve normalization can be calculated using geNorm, where the lowest number of gene combinations which causes the threshold value to fall below 0.15 indicates the number of reference genes which will be sufficient for normalization ( Vandesompele et al, 2002 ).…”

Section: Discussionmentioning

confidence: 99%

“…The RefFinder software comprehensively ranks the candidate reference genes based on the geometric mean (GM) values of the results from the four different statistical algorithms described above ( Xie et al, 2012 ). With the help of these five statistical algorithms, much research on the validation of reference genes under different experimental conditions has been reported ( Liu et al, 2017 ; Sadangi et al, 2017 ; Sprang et al, 2017 ; Mughal et al, 2018 ).…”

Section: Introductionmentioning

confidence: 99%

Selection of Reliable Reference Genes for RT-qPCR Analysis of Bursaphelenchus mucronatus Gene Expression From Different Habitats and Developmental Stages

Zhou

Chen

et al. 2018

Front. Genet.

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Quantitative reverse transcription polymerase chain reaction (RT-qPCR), a sensitive technique for gene expression analysis, depends on the stability of the reference genes used for data normalization under different experimental conditions. Bursaphelenchus mucronatus, a pine-parasitic nematode varying in virulence, is widely distributed in natural pine forests throughout the northern hemisphere, but has not been investigated with respect to the identification of reference genes suitable for the normalization of RT-qPCR data. In the present study, eight candidate reference genes were analyzed in B. mucronatus under different habitat conditions and at different developmental stages. The expression stability of these genes was assessed by geNorm, NormFinder, BestKeeper, delta Cq, and RefFinder algorithms. In general, our results identified encoding beta-tubulin as the most stable gene. Moreover, pairwise analysis showed that three reference genes were sufficient to normalize the gene expression data under each set of conditions, with genes encoding beta-tubulin, 18S ribosomal RNA and ubiquitin-conjugating enzyme being the most suitable reference genes for different habitat conditions, whereas genes encoding beta-tubulin, histone, and 18S ribosomal RNA exhibited the most stable expression at different developmental stages. Validation of the selected reference genes was performed by profiling the expression of the fatty acid- and retinol-binding protein gene in different habitats, and by profiling the expression of the arginine kinase gene at different developmental stages. This first systematic analysis for the selection of suitable reference genes for RT-qPCR in B. mucronatus will facilitate future functional analyses and deep mining of genetic resources in this nematode.

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“…ACT and GAPDH exhibited variable expression in leaves of bermudagrass ( Cynodon dactylon ) under NaCl and PEG stress [ 18 ]. Likewise, both genes performed poorly in seashore paspalum ( Paspalum vaginatum ) exposed to drought and salinity [ 19 ]. Furthermore, GAPDH was reported to be least stable in annual ryegrass ( Lolium multiflorum ) under abovementioned stresses [ 16 ], while ACT was least stable in durum wheat ( Triticum durum ) subjected to drought [ 33 ].…”

Section: Discussionmentioning

confidence: 99%

Carex muskingumensis and Osmotic Stress: Identification of Reference Genes for Transcriptional Profiling by RT-qPCR

Sozoniuk

Parzymies

Kozak

et al. 2020

Genes

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Carex muskingumensis is a highly valued perennial ornamental grass cultivated worldwide. However, there is limited genetic data regarding this species. Selection of proper reference genes (RGs) for reverse transcription quantitative PCR (RT-qPCR) data normalization has become an essential step in gene expression analysis. In this study, we aimed to examine expression stability of nine candidate RGs in C. muskingumensis plants, subjected to osmotic stress, generated either by salinity or PEG treatment. The identification of genes exhibiting high expression stability was performed by four algorithms (geNorm, NormFinder, BestKeeper and deltaCt method). The results showed that the combination of two genes would be sufficient for reliable expression data normalization. ADP (ADP-ribosylation factor) and TBP (TATA-box-binding protein) were identified as the most stably expressed under salinity treatment, while eIF4A (eukaryotic initiation factor 4A) and TBP were found to show the highest stability under PEG-induced drought. A set of three genes (ADP, eIF4A and TBP) displayed the highest expression stability across all experimental samples tested in this study. To our best knowledge, this is the first report regarding RGs selection in C. muskingumensis. It will provide valuable starting point information for conducting further analyses in this and related species concerning their responses to water shortage and salinity stress.

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Identification and Validation of Reference Genes for Seashore Paspalum Response to Abiotic Stresses

Cited by 26 publications

References 37 publications

Identification and Validation of Reference Genes for RT-qPCR Analysis in Switchgrass under Heavy Metal Stresses

Identification and Validation of Reference Genes for RT-qPCR Analysis in Switchgrass under Heavy Metal Stresses

Selection of Reliable Reference Genes for RT-qPCR Analysis of Bursaphelenchus mucronatus Gene Expression From Different Habitats and Developmental Stages

Carex muskingumensis and Osmotic Stress: Identification of Reference Genes for Transcriptional Profiling by RT-qPCR

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