Stability determination of candidate reference genes in cucumber plants subjected to stresses from Phytophthora melonis

Stable reference genes are indispensable for ensuring the fi delity of determined gene expression levels. However, the expression levels of reference genes are unable to remain constant under all possible experimental conditions. Therefore, the stability determination of reference genes is necessary in an experimental system set. In the preset study, the stability of nine cucumber candidate reference genes (CsACT, CsUBQ, CsEF1α, CsCYP, CsαTU, CsCACS, CsTIP41, CsYSL8 and CsHEL) subjected to stresses from Phytophthora melonis (P. melonis) were determined using four different analysis methods, including Delta Ct, BestKeeper, NormFinder and GeNorm. The study results revealed that CsUBQ and CsCYP were the most stable genes suitable as internal control in cucumber plants under attack by P. melonis condition. Research Article Stability determination of candidate reference genes in cucumber plants subjected to stresses from Phytophthora melonis Rui Wang, Hu Du, Qingmin Jin, Yu’e Lin, Yujuan Zhong, Chunpeng Yao, Xiaomei Xu and Tingquan Wu* Vegetable Research Institute, Guangdong Academy of Agricultural Sciences/ Guangdong Key Laboratory for New Technology Research of Vegetables, Guangzhou, 510640, China *Address for Correspondence: Tingquan Wu, Vegetable Research Institute, Guangdong Academy of Agricultural Sciences/ Guangdong Key Laboratory for New Technology Research of Vegetables, Guangzhou, 510640, China, Tel: +86 20 38469301; Email: tingquanwu@sina.com Submitted: 29 March 2019 Approved: 12 April 2019 Published: 15 April 2019 Copyright: © 2019 Wang R, et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited


Introduction
Gene expression analysis is very important for the study of gene function. Quantitative RT-PCR (qRT-PCR) with reliable speci icity and high sensitivity has been extensively used for analyzing the gene expression pro iles [1,2]. However, qRT-PCR is dependable only when experimental data are normalized. Furthermore, the reliability of the reference gene is the most important for the normalization of data [3][4][5]. Some housekeeping genes involved in basic biological activities, such as ubiquitin (UBQ), actin (ACT) and elongation factor 1-alpha (EF1α), have been commonly used as an internal control, because these have been considered to express at constant levels, regardless of the experimental condition [6,7]. Since these genes are necessary for basic cellular lives, their expression levels are considered to be constant in various conditions. However, increasing studies have shown that the expression levels of these genes are not independent of experimental conditions [8,9], and the stability of reference genes differs when plants were in different stages of growth and development, or in different environmental conditions [2,[9][10][11][12][13]. The most suitable reference genes should be constantly expressed in a plant, and should not be in luenced by the internal factors of the plant and the external environment [4,14]. Therefore, it is very necessary to select a stable reference gene for the ef icient quanti ication of target genes by qRT-PCR in given samples collected under different conditions. Delta-Ct [15], BestKeeper [16], NormFinder [17] and GeNorm [18], are the four presently available major statistical algorithms widely used for evaluating the stability of candidate reference genes.
Cucumber, which is one of the most important members of the Cucurbitaceae family, is widely cultivated worldwide. Studies on cucumber molecular biology has made signi icant progress [19][20][21][22][23], after cucumber genome sequencing data were released [24]. Cucumber blight caused by Phytophthora. melonis (P. melonis) is a destructive disease that leads to severe loss in yield and quality and yield losses of up to 80% in portions of affected ields. However, researches on the mechanism of the cucumber-P. melonis interaction have been rarely reported and there are only several papers published. CsLecRK6.1 was found that it was especially induced by P. melonis and P. capsici among the 25 CsLecRK genes of JSH cucumber plants [25] and Based on results of RNA-seq and qPCR, cucumber CsWRKY2, 20, 26, 35, 44 and 52 may be involved in disease resistance of JSH against Phytophthora melonis by SA and (or) JA signaling pathway(s) [26]. Therefore, it is of great value and signi icance to ind one or more of the stable reference genes for studying the function of disease resistance genes under the infection conditions.

Plant materials, growth conditions and treatments
Cucumber plants from the B80 cultivar used throughout the study were cultivated in a growth chamber at 26-28°C with 85% relative humidity and a 10-hour light and 14-hour dark cycle. Ten-day old seedlings were used for the experiments. The underground parts of the plants were treated by zoospore suspension of P. melonis (5×10 3 zoospores/ml), while mixtures of roots and stems, and leaves of the seedlings were respectively collected for RNA extraction at 0, 6, 12, 24 and 48 hours after treatment, and there are Obvious symptoms appeared at 24 -48 hours after inoculation: the constriction of stem base, and the plants begin to wilt. The phenotypes of cucumber plants CK (no inoculation) and 48h post-inoculation with P. melonis (5×10 3 zoospores/ml) were shown in Supplemental Figure 1. P. melonis were grown on V8 solid culture medium and zoospores were induced in V8 liquid culture medium. V8 juice is purchased from Campbell Soup Company. V8 liquid culture medium of 1 liter contains 200 ml of clari ied V8 juice, 3 g of CaCO3, and about 800 ml of dH2O, Ph=5.8. V8 Solid culture medium of 1 liter Contains all the components of the liquid medium plus 16 g of agar, Ph=5.8.

Experimental design
The nine cucumber genes (CsACT, CsUBQ, CsEF1α, CsCYP, CsαTU, CsCACS, CsTIP41, CsYSL8 and CsHEL) were usually chosen as the reference genes under some experimental conditions, so we predict that one or some of them may be as reference genes under the experimental condition of cucumber plants infected by P. melonis. The expression of every candidate reference gene was tested at the ive time points (0, 6, 12, 24 and 48 hours) post-inoculation with P. melonis. The stability of candidate reference genes was determined using four different analysis methods, including Delta Ct, BestKeeper, NormFinder and GeNorm. Finally, we combined these results to screen out the most stable reference gene(s) in our experimental system.

RNA isolation and qPCR analysis
In order to determine the expression levels of reference genes, qRT-PCR was performed using the BIO-RAD CFX96 real-time PCR system (Bio-Rad, USA) and SYBR ® Premix Ex Taq™ II mix kit (Takara). Total RNA was extracted using the RNAiso Plus reagent (takara), and irst-strain cDNA was synthesized using a PrimeScript™ RT reagent Kit with the gDNA Eraser (TaKaRa Perfect Real Time), according to manufacturer's protocol. A 10-fold dilution of the resulting cDNA was amplified employing SYBR ® Premix Ex Taq™ II in a 10-μL volume with the following program: 95 ° C for 30 seconds, 30 cycles of 95°C for ive seconds, 60°C for 15 seconds, and 72°C for 15 seconds. Three experimental and three biological replicates were carried out for assaying the expression of the reference genes.
Origin and culture of P. melonis and the preparation of zoospore suspension P. melonis was isolated from cucumber plants infected in the experimental ield of Baiyun of the Vegetable Research Institute, Guangdong Academy of Agricultural Sciences (Guangzhou), and these were cultured in V 8 solid culture medium. Pieces of fresh mycelia were added in 10% V8 l culture medium for 2-3 days, the mycelia were placed in water to induce sporangia production for two days, and the water was renewed once every 12 hours. The zoospore release was induced by putting sporangia in water at 4°C for 30 minutes, the number of zoospores was determined by microscopy, and zoospore suspension were adjusted to 5×10 3 zoospores/ml.

Analysis method of the stability of cucumber candidate reference genes
The stability of cucumber candidate reference genes was analyzed using presently available major computational programs, such as Delta-Ct, BestKeeper, NormFinder and geNorm, in order to compare and rank the tested candidate reference genes.

Transcription profi ling of cucumber candidate reference genes
In order to identify the most stable reference genes for studies on cucumber gene expression under inoculation with P. melonis condition, the primers of nine candidate reference genes (Table 1) in cucumber were respectively designed for the qRT-PCR assay based on SYBR green detection. The speci icity of the qRT-PCR ampli ication was con irmed through the single-peak melting curve of the PCR products ( Figure 2). The expression levels of these candidate reference genes in mixture of roots and stem, and leaves were respectively presented with threshold cycle (Ct) values (T ables 2,3).

GeNorm analysis
The criteria for choosing the reference genes by GeNorm is that the most stable gene(s) has the smallest stability value(s) and when the stability values of the determined genes are less than 1.5, the genes can be as the reference genes. F or cucumber roots and stems, t he results revealed that except CsHEL, the stability values of other eight genes were less than 1.5 and for cucumber leaves, the stability values of ive genes (CsUBQ, CsCYP, C sACT, CsCACS and CsɑTU) were less than 1.5 ( Figure 2). So, CsUBQ, CsCYP, CsACT, CsCACS and Cs ɑTU can be as reference genes in cucumber leaves or roots and stems und er the inoculation conditions of P.melonis using GeNorm analysis.

BestKeeper analysis
The criteria for choosing the reference genes by BestKeeper is that the most stable gene(s) has (or have) the smallest SD values and when SD value is less than 1, the gene can be as reference gene.
For cucumber roots and stems, BestKeeper analysis revealed that two genes (CsUBQ and CsCYP) reached the standard of the reference genes (Table 4). For cucumber leaves, BestKeeper analysis showed the same result that CsUBQ and CsCYP with SD values less than 1, reached the standard of the reference genes (Table 5). So CsU BQ and CsCYP are suitable as reference genes in cucumber roots and stems or leaves under the inoculation conditions of P.melonis using BestKeeper analysis.
Ranking order of gene stability by Bestkeeper:

Norm Finder analysis
The criteria for choosing the reference genes by NormFinder is that the most stable gene(s) has (or have) the smallest stability value(s). Using the method, we can only get the ranking order of the stability of genes and choose the most stable gene as the reference gene. Nine candidate reference genes were analyzed using the NormFinder method in three repeat experiments.
For cucumber roots and s tems or leaves the results revealed that CsUBQ and CsCYP ranked in the t op two positions and (Figure 3). Comprehensively, CsUBQ and CsCYP are the best ge nes as reference genes in cucumber under P. melonis infection condition.

Delta CT analysis
The criteria for choosing the reference genes by Delta CT is that the most stable gene(s) has (or have) the smallest STDEV value(s), as the similar to NormFinder. Using Delta CT method, we can get the ranking order of the stability of genes and determine the most stable gene(s). Delta CT analysis revealed that CsUBQ and CsCYP are the most stable genes in all experiments (Tables 6,7).

Discussion
Several approaches have been proposed to evaluate the stability of reference gene expression, such as the GeNorm, BestKeeper, NormFinder and Delta-Ct method. In the present study, four analysis methods were used to compare and rank nine    In cucumber plants subjected to growth regulators and abiotic stress, CsCACS, CsTIP41 and CsEF1α are the best reference genes, while the stability rankings of CsACT, CsUBQ, CsαTU and CsCYP were ranked lowe r [9]. However, the study results revealed that CsUBQ and CsCYP were the most stable reference genes under the condition of cucumber plants infected by P. melonis. Our research results suggest that under the condition of biotic stress, the stability of reference genes in cucumber plants are different with those under the condition of abiotic stress or growth regulators, providing a solid foundation for future studies.