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. 2020 Aug 31;11(9):1022.
doi: 10.3390/genes11091022.

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

Magdalena Sozoniuk  1 , Marzena Parzymies  1 , Danuta Kozak  1 , Bairam Solomon Ismael  1
Affiliations

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

Magdalena Sozoniuk et al. Genes (Basel). .

Abstract

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.

Keywords: RT-qPCR; drought; reference genes; salinity; sedges.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Growth of explants on the medium supplemented with PEG after 4 weeks of cultivation.
Figure 2
Figure 2
Growth of explants on the medium supplemented with NaCl after 4 weeks of cultivation.
Figure 3
Figure 3
Average expression stability M of all remaining control genes after stepwise exclusion of the least stable reference genes according to the geNorm algorithm. The lower the M value the more stable is the gene’s expression in tested samples. Analysis was performed separately for (a) NaCl treated samples (NaCl), (b) PEG treated samples (PEG) and (c) all samples combined together (Total).
Figure 3
Figure 3
Average expression stability M of all remaining control genes after stepwise exclusion of the least stable reference genes according to the geNorm algorithm. The lower the M value the more stable is the gene’s expression in tested samples. Analysis was performed separately for (a) NaCl treated samples (NaCl), (b) PEG treated samples (PEG) and (c) all samples combined together (Total).
Figure 4
Figure 4
Determination of the most stable reference genes based on their correlation coefficients (r) according to the BestKeeper algorithm. The higher the correlation coefficient, the more stable is the gene’s expression. Analysis was performed separately for (a) NaCl treated samples (NaCl), (b) PEG treated samples (PEG) and (c) all samples combined together (Total).
Figure 5
Figure 5
Determination of optimal number of reference genes calculated by geNorm. Pairwise variation (Vn/Vn+1) below 0.15 indicates no significant contribution made by inclusion of additional reference gene. Analysis was performed for NaCl treated samples (NaCl), PEG treated samples (PEG) and all samples combined together (Total).
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