Suitable Reference Genes for Accurate Gene Expression Analysis in Parsley (Petroselinum crispum) for Abiotic Stresses and Hormone Stimuli

Abstract

Parsley, one of the most important vegetables in the Apiaceae family, is widely used in the food, medicinal, and cosmetic industries. Recent studies on parsley mainly focus on its chemical composition, and further research involving the analysis of the plant's gene functions and expressions is required. qPCR is a powerful method for detecting very low quantities of target transcript levels and is widely used to study gene expression. To ensure the accuracy of results, a suitable reference gene is necessary for expression normalization. In this study, four software, namely geNorm, NormFinder, BestKeeper, and RefFinder were used to evaluate the expression stabilities of eight candidate reference genes of parsley (GAPDH, ACTIN, eIF-4α, SAND, UBC, TIP41, EF-1α, and TUB) under various conditions, including abiotic stresses (heat, cold, salt, and drought) and hormone stimuli treatments (GA, SA, MeJA, and ABA). Results showed that EF-1α and TUB were the most stable genes for abiotic stresses, whereas EF-1α, GAPDH, and TUB were the top three choices for hormone stimuli treatments. Moreover, EF-1α and TUB were the most stable reference genes among all tested samples, and UBC was the least stable one. Expression analysis of PcDREB1 and PcDREB2 further verified that the selected stable reference genes were suitable for gene expression normalization. This study can guide the selection of suitable reference genes in gene expression in parsley.

Identification of Optimal Reference Genes for Expression Analysis in Radish (Raphanus sativus L.) and Its Relatives Based on Expression Stability

“…The four computational methods (ΔCt, BestKeeper, NormFinder and geNorm) (Niu L. et al, 2015 ; He et al, 2016 ; Li et al, 2016a ) for expression stability are based on different algorithms and analytical procedures (Vandesompele et al, 2002 ; Andersen et al, 2004 ; Pfaffl et al, 2004 ; Silver et al, 2006 ). In our research, we found that the most unstable genes identified by the four algorithms were mostly the same, but the most stable genes were not consistent.…”

“…This was possibly due to differences among the algorithms. Integrated analysis using different programs can minimize errors in the stability evaluation of candidate reference genes (Li et al, 2016a ; Shivhare and Lata, 2016 ). Here, RefFinder (Xie et al, 2012 ) was used to combine the results of the four computational methods and generate a comprehensive ranking list for the candidate reference genes.…”

Selection and Validation of Appropriate Reference Genes for Real-Time Quantitative PCR Analysis in Needles of Larix olgensis under Abiotic Stresses

“…Three different Microsoft Excel-based software programs, geNorm [3], NormFinder [15], and BestKeeper [13,34], were used to analyze the expression stability of the candidate RGs under different experimental conditions. The Cq (PCR cycle threshold) data were used directly in the BestKeeper program, but for geNorm and NormFinder, Cq values were converted into relative values and imported into geNorm and NormFinder program analyzed as described previously [13]. The output of each software program permitted us to rank the expression stability of RGs in different treatment groups.…”

“…Unlike the other two programs, BestKeeper ranks RGs based on the standard deviation (SD) and coefficient of variation (CV) of the Cq values from the qRT-PCR assay. The smaller the CV, the better the stability of the gene was [13,34]. The results of BestKeeper analysis are also listed in Table 5.…”

Evaluation of Housekeeping Gene Expression Stability in Carnation (Dianthus caryophyllus)

“…An ideal internal reference gene should have the following characteristics: (1) stable expression in different tissues and developmental stages, and not affected by experimental treatment; (2) cannot be a pseudogenes; (3) The expression level should be in a suitable range (15 < Cq < 30), not too low or too high [22,29]. ACTIN, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), elongation factor 1alpha (EF1α), and ubiquitin-conjugating enzyme (UBC) are commonly used internal reference genes in plant gene expression analysis experiments [17].…”