Identification of reference genes for RT-qPCR in the Antarctic moss <i>Sanionia uncinata</i> under abiotic stress conditions

<div><p><i>Sanionia uncinata</i> is a dominant moss species in the maritime Antarctic. Due to its high adaptability to harsh environments, this extremophile plant has been considered a good target for studying the molecular adaptation mechanisms of plants to a variety of environmental stresses. Despite the importance of <i>S</i>. <i>uncinata</i> as a representative Antarctic plant species for the identification and characterization of genes associated with abiotic stress tolerance, suitable reference genes, which are critical for RT-qPCR analyses, have not yet been identified. In this report, 11 traditionally used and 6 novel candidate reference genes were selected from transcriptome data of <i>S</i>. <i>uncinata</i> and the expression stability of these genes was evaluated under various abiotic stress conditions using three statistical algorithms; geNorm, NormFinder, and BestKeeper. The stability ranking analysis selected the best reference genes depending on the stress conditions. Among the 17 candidates, the most stable references were <i>POB1</i> and <i>UFD2</i> for cold stress, <i>POB1</i> and <i>AKB</i> for drought treatment, and <i>UFD2</i> and <i>AKB</i> for the field samples from a different water contents in Antarctica. Overall, novel genes <i>POB1</i> and <i>AKB</i> were the most reliable references across all samples, irrespective of experimental conditions. In addition, 6 novel candidate genes including <i>AKB</i>, <i>POB1</i> and <i>UFD2</i>, were more stable than the housekeeping genes traditionally used for internal controls, indicating that transcriptome data can be useful for identifying novel robust normalizers. The reference genes validated in this study will be useful for improving the accuracy of RT-qPCR analysis for gene expression studies of <i>S</i>. <i>uncinata</i> in Antarctica and for further functional genomic analysis of bryophytes.</p></div

Primer sequences and amplicon characteristics of 17 candidate reference genes for RT-qPCR analysis.

<p>Primer sequences and amplicon characteristics of 17 candidate reference genes for RT-qPCR analysis.</p

Expression stability values (M) of the candidate reference genes based on the geNorm algorithm.

<p>The cutoff for reference gene stability was proposed to be an M value of 0.5. Candidate reference genes were ranked in ascending order of expression stability, from the least stable on the left to the most stable gene on the right. (A) Cold experimental set, (B) drought experimental set, (C) WGF experimental set, and (D) total experimental set.</p

Growth and abiotic stress conditions tested on Antarctic moss <i>S</i>. <i>uncinata</i>.

<p>Growth and abiotic stress conditions tested on Antarctic moss <i>S</i>. <i>uncinata</i>.</p

Pairwise variation (Vn/Vn+1) analysis of the candidate reference genes.

<p>The pairwise variation (Vn/Vn+1) was analyzed based on the geNorm algorithm to determine the optimal number of reference genes for accurate normalization. A cutoff value of 0.15 was set for the pairwise variation, as recommended by Vandesompele et al (2002) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0199356#pone.0199356.ref012" target="_blank">12</a>].</p

Stability analysis of 17 candidate reference genes using BestKeeper.

<p>Stability analysis of 17 candidate reference genes using BestKeeper.</p

Relative expression levels of the late embryogenesis abundant (<i>LEA</i>) and heat shock protein 70 (<i>HSP70</i>) genes under different stress conditions when the selected reference genes were used for normalization.

<p>(A) <i>LEA</i> and <i>HSP70</i> expression in the cold experimental set of <i>S</i>. <i>uncinata</i> was normalized using a combination of the two most stable genes <i>POB1</i> and <i>UFD2</i>, and the least stable genes <i>HIS</i> and <i>tIF</i>. Red bar indicates fold-change values of gene expression, which were calculated based on the normalized FPKM from the RNA-Seq analysis of the cold experimental set (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0199356#pone.0199356.s006" target="_blank">S2 Table</a>). (B) The expression of <i>LEA</i> and <i>HSP70</i> in the drought experimental set of <i>S</i>. <i>uncinata</i> was normalized using a combination of the two most stable genes <i>POB1</i> and <i>AKB</i>, and the least stable genes <i>UFD2</i> and <i>TUB250</i>. (C) The expression of <i>LEA</i> and <i>HSP70</i> in the WGF experimental set of <i>S</i>. <i>uncinata</i> was normalized using a combination of the two most stable genes <i>UFD2</i> and <i>AKB</i>, and the least stable genes <i>SuTub</i> and <i>ACT5</i>. Error bars represent the standard deviation of the mean of three biological replicates.</p

Distribution of the quantification cycle (Cq) values of the 17 candidate reference genes in <i>Sanionia uncinata</i> samples.

<p>The median Cq values for 11 samples are displayed in a boxplot indicating the interquartile range. Error bars represent the minimum and maximum Cq values and the middle panes show the mean values. Three biological replicates per sample and four technical replicates per biological replicate were used.</p

Expression stability values and ranking of the candidate reference genes based on the NormFinder algorithm.

<p>Candidate reference genes were ranked in ascending order of expression stability, from the least stable on the left to the most stable gene on the right. (A) Cold experimental set, (B) drought experimental set, (C) WGF experimental set, and (D) total experimental set.</p

Comprehensive stability ranking of the candidate reference genes.

<p>Comprehensive stability ranking of the candidate reference genes.</p