Abstract
Kentucky bluegrass (Poa pratensis L.) belong to Gramineae and is widely used in lawns, golf courses, landscapes, and sport fields as a prominent cool-season grass. Gene expression patterns during different stages of plant development can provide clues toward the understanding of its biological functions. The selection and validation of reference genes are the first steps in any real-time quantitative PCR gene expression study. Therefore, suitable reference genes are necessary for obtaining reliable results in real-time quantitative PCR analyses of Kentucky bluegrass. In the present study, 9 candidate reference genes were chosen, and their expression stability in the leaves and roots of Kentucky bluegrass under different stresses (drought, salt, heat, and cold) were evaluated using the GeNorm, NormFinder, BestKeeper, and RefFinder programs. The results showed that the expression stability of the candidate reference genes was dependent on the experimental conditions. The combination of SAM with GAPDH was the most stable in leaves under salt stress and cold stress, while TUB combined with ACT or GAPDH was stable in roots under salt or cold stress, respectively. ACT and SAM maintained stable expression in drought-treated leaves, and GAPDH combined with ACT was stable in drought-treated roots. SAM and TUB exhibited stable expression in heat-treated leaves. ACT and RPL were stable in heat-treated roots. In addition, the expression patterns of PpFEH in response to drought and cold stress were used to confirm the reliability of the selected reference genes, indicating that the use of an inappropriate reference gene as the internal control will cause erroneous results. This work is the first study on the expression stability of reference genes in Kentucky bluegrass and will be particularly useful in the selection of stress-tolerance genes and the identification of the molecular mechanisms conferring stress tolerance in this species.
Highlights
The study of gene expression is fundamental to understanding signal transduction, metabolic pathways, and development
Studies aimed at identifying suitable reference genes as internal controls for RTqPCR analysis have been reported in many turfgrass species, such as Lolium perenne (Lee et al, 2010), bermudagrass (Chen et al, 2014), buffalo grass (Li et al, 2014), tall fescue (Yang et al, 2015), and creeping bentgrass (Chen et al, 2015)
Experiments aimed at selecting stable reference genes for Kentucky bluegrass have not yet been conducted, which might be due to the fact that nothing is present to be searched in the Kentucky bluegrass
Summary
The study of gene expression is fundamental to understanding signal transduction, metabolic pathways, and development. The application of RT-qPCR is often affected by methodological errors, including variations in the quality of RNA, variations in the efficiency of cDNA synthesis, and variations in the efficiency of amplification (Delporte et al, 2015). These errors will cause the quantitation of target gene transcripts to be unreliable. It has been widely reported that the most effective way to normalize the data is to use appropriate reference genes (Hong et al, 2008; Delporte et al, 2015)
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