Selection of Candidate Reference Genes for Gene Expression Analysis in Kentucky Bluegrass (Poa pratensis L.) under Abiotic Stress

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.

Physiological and Proteomic Responses of Contrasting Alfalfa (Medicago sativa L.) Varieties to PEG-Induced Osmotic Stress

“…Total RNA was extracted from non-stressed and stressed root tissues of two alfalfa varieties with three biological replicates (each containing 100 mg alfalfa roots) using the RNA simple Total RNA Kit (TIANGEN BIOTECH, Co., Ltd, Beijing, China) based on the kit instructions. cDNA was reverse transcribed from 1 μg of total RNA using a First Strand cDNA Synthesis Kit (TaKaRa, Dalian, China) ( Niu et al, 2017 ). Specific RT-qPCR primers were designed using Primer 3 4 ( Table 1 ).…”

“…Each 20 μL reaction mixture contained 2 μL template cDNA, 10 μL of 2× SYBR ® Premix Dimer Eraser, 0.8 μL of each primer (10 μM), 6.0 μL dd H 2 O, and 0.4 μL of ROX reference Dye. The amplification reaction conditions were as follows: 95°C for 3 min followed by 40 cycles of 95°C for 10 s and 60°C for 30 s ( Niu et al, 2017 ). The levels of relative gene expression were calculated by the ddCt algorithm using the alfalfa MtGAPDH gene as the housekeeping gene ( Zhang et al, 2003 ).…”

Physiological and transcriptome analysis of Poa pratensis var. anceps cv. Qinghai in response to cold stress

“…The specific primers of C-repeat binding factors (CBFs) were designed according to Zhang et al [70]. In qRT-PCR analysis of CBFs, Actin gene was used as an internal control [71], the expression data were normalized to the control conditions (set at 1), using the comparative threshold cycle method. Each experiment included three independent biological replicates.…”

“…Total RNA was extracted from the control and cold groups of the two genotypes using The relative level of gene expression was calculated using the 2 −ΔΔct formula [82]. The P.pratensis putative Actin gene was used as an endogenous control [71]. The specific primers (Additional file 6: Expression patterns of selected genes involved in cold stress in two P.pratensis cultivars.…”

Identifying optimal reference genes for the normalization of microRNA expression in cucumber under viral stress

“…Constitutively expressed housekeeping genes are transcribed at a relatively constant level, and are usually selected for this purpose [ 18 , 19 ], because their expression is generally assumed to be minimally affected by experimental conditions. However, recent studies have shown that housekeeping genes that are commonly used as reference genes may not be as consistently expressed as previously thought [ 20 – 23 ], and can vary according to plant species, tissue type, and environmental conditions [ 24 ]. The use of non-validated references genes can therefore result in inaccurate quantification of miRNA expression when comparing samples from different sources.…”