Zhang XiujieJin Wujun3Xu WentaoLi XiayingShang YingLi ShaOuyang Hongsheng

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Comparison of Five Endogenous Reference Genes for Specific PCR Detection and Quantification of Rice

Zhang Xiujie1, 2,#, Jin Wujun3, #, Xu Wentao4, Li Xiaying2, Shang Ying4, Li Sha4, Ouyang Hongsheng1

(College of Animal Sciences, Jilin University; , China; Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, China;College of Food Science and Nutritional Engineering, ; These authors contributed equally to this work)

Endogenous reference genes (ERGs) provide vital information regarding genetically modified organisms (GMOs). The successful detection of ERGs can identity GMOs and the source of genes, verify stability and reliability of the detection system, and calculate the level of genetically modified (GM) ingredients in mixtures. The reported ERGs in rice include(),(),and rice root-specificgenes. Based on the characteristics of ERGs, a new ERG gene,(), was selected, and further compared with the four existing genes. A total of 18 rice varieties and 29 non-rice crops were used to verify the interspecies specificity, intraspecies consistency, sensitivity, stability and reliability of these five ERGs using qualitative and quantitative PCR. Qualitative detection indicated thatanddisplayed sufficient specificity, and the detection sensitivity was 0.05% and 0.005%, respectively. Although the specificity of bothandwere adequate, the amplicons were small and easily confused with primer dimers. Non-specific amplification of thegene was present in maize and potato. Real-time quantitative PCR detection indicated that,anddisplayed good specificity, with2of the standard curve greater than 0.98, while the amplification efficiency ranged between 90% and 110%. Both the detection sensitivities ofandwere five copies and that ofwas ten copies.showed typical amplification in maize, beet and, whilewas found in maize, tobacco and oats.exhibited excellent detection sensitivity and species specificity, which made it a potentially useful application in GM-rice supervision and administration. Additionally,andare also suitable for GM-rice detection. This study effectively established a foundation for GMO detection, which not only provides vital technical support for GMO identification, but also is of great significance for enhancing the comparability of detection results, and the standardization of ERG testing in GM-rice.

endogenous reference gene; rice; genetically modified crop;gene;gene;gene;gene; rice root-specificgene

Increasing safety concerns regarding genetically modified (GM) plants and the products derived from them have prompted more than 50 countries to adopt and implement a genetically modified organisms (GMOs) labeling system (Bawa and Anilakumar, 2013; Cheng et al, 2017). The Chinese government attaches great importance to the safety management of agricultural GMOs and has formulated a specific set of regulations for the administration of these products (Li et al, 2014). Furthermore, various regulatory processes and documentation regarding GMOs have been implemented in China, including safety evaluations, appropriate labeling, as well as permits for processes involving the production, retail, importation and processing of GMOs (Kou et al, 2015). The standardization of detection technology ensures the proper implementation of these regulations (Wang et al, 2011).

In recent years, considerable progress has been made in China regarding transgenic rice research. Particular GM rice varieties are resistant to insect or disease, and have satisfied environmental and production requirements. Some lines have already obtained safety certification or are in the process of doing so (Pray et al, 2018). The industrialization and safety issues of GM rice have attracted unprecedented domestic and global attention with the occasional report of incidents involving safety. Therefore, detection methods that are scientific and accurate are vital to standardize the safety management of GM rice and its production process. The establishment of such a system not only protects human health and promotes environmental safety, but also eliminates public doubt regarding GM research and industrialization (Wang et al, 2017).

Since GM ingredient detection is vital for the safety evaluation and supervision of agricultural transgenic organisms, polymerase chain reaction (PCR) has been widely utilized for this purpose, due to its high sensitivity and good reproducibility (Gryson, 2010; Shang et al, 2013; Xu et al, 2013). The endogenous reference gene (ERG) is significant in forming the basis for determining the suitability of the isolated and purified DNA for further PCR amplification detection by evaluating the presence of components denoting specific species (Yang et al, 2005; Xu et al, 2008; Wu et al, 2010; Chaouachi et al, 2013). The established detection methods for various GM rice lines involving ERGs are significant in their diversity and affected the comparability of the detection results.

This study examined the rice related ERGs that were reported in published works and standards both domestically and abroad. These genes include() gene (Ding et al, 2004),() gene (JRC, 2006),() gene (Jeong et al, 2007) and rice root-specificgene (Hernández et al, 2005). Moreover, in this study, a novel ERG of rice was developed namely phosphoenolpyruvate carboxylase() gene. All the primers and probes were synthesized according to existing information regarding the sequences of these genes. The species specificity sensitivity and consistent copy numbers of these rice ERGs were verified. Furthermore, to establish the standardized qualitative and real-time quantitative PCR detection standard of GM rice based on the ERGs, the reaction program and the system of the selected primer sets were optimized.

Materials and Methods

Plant materials

To identify the intraspecies consistency, 18 rice varieties were employed, including 6 GM and 12 non-GM rice (Table 1). They were kindly supplied by the Research Testing Center and Biotechnology Research Institute, Chinese Academy of Agricultural Science (CAAS), Beijing, China.

In order to test the interspecies specificity, 29 non-rice species were adopted. The non-rice crops, including GM and non-GM crops were supplied by the Research Testing Center of CAAS. The tobacco leaf was supplied by Wang Zhixing, Biotechnology Research Institute, CAAS.leaf was supplied by Researcher Zhang Chunyi, Biotechnology Research Institute, CAAS. The rest of the crops were purchased at local market. All the material information is shown in Table 1.

DNA extraction and purification

All genomic DNAs were extracted using DNeasy Plant Mini Kit (Qiagen, Valencia, CA, USA), except Kefeng 6, for which Plant Genomic DNA Extraction Kit DP305 were used (Tiangen, Beijing, China). The concentration and purity of DNA samples were determined by measuring the absorbance at 260 nm (260), and calculating the260/280absorbance ratio with a NANODROP2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). All DNA concentrations were finally diluted to 50 ng/μL.

Table 1. Information of the plant materials.

GM, Genetically modified.

Table 2. The detailed sequences of the PCR primers and probes for quanlitative PCR.

Novel and reported rice ERGs

Four rice ERGs were ascertained by consulting existing literature and standards, including,,andgenes. By analyzing species specificity, this study revealed a novel ERG of rice namely(GenBank No. AP003409.4). The detection specificity and sensitivity in each of the five genes were compared using quantitative and qualitative PCR to verify their applicability in supervision, administration, and research of GM rice.

Oligonucleotide sequences and qualitative PCR conditions

PCR oligonucleotide primers and TaqMan fluorescent dye-labeled probes were designed using Primer Premier 5.0 (PREMIER Biosoft International, USA) according to the sequences of the five ERGs (Table 2). The reported primers and probes were employed as guidelines to conduct the PCR amplification (Jiang et al, 2009; Chen et al, 2010). All the sequences were synthesized by the Shanghai Sangon Co. Ltd. (Shanghai, China).

Conventional qualitative PCR assays were conducted in a 25 μL final volume, containing 5× buffer (containing 1.5 mmol/L Mg2+), dNTP (0.2 mmol/L), primer (0.2 μmol/L each), GotaqDNA polymerase (0.652 U, Promega, Madison, WI, USA), and DNA template (50 ng) using an ABI SimpliAmp Thermal Cycler (Applied Biosystems, Foster City, CA, USA). Forthe amplification, the following PCR programs were used: 5 min at 95 oC; 35 cycles of 30 s at 94 oC, 30 s at (54–60) oC (the detailed annealing temperatures are listed in Table 2), and 1 min at 72 oC; and a final 5 min at 72 oC. All the products were analyzed on 2% agarose gel electrophoresis in 1× TAE, stained with ethidium bromide.

The primers, displaying excellent intraspecies specificity and interspecies consistency, were selected and further subjected to optimization of reaction conditions that included annealing temperatures and final primer concentrations.

Real-time quantitative PCR conditions

The real-time quantitative PCR reactions were performed on aCFX96TMReal-Time System instrument (BIO-RAD, USA). The amplification specificity and sensitivity were evaluated in reaction volumes of 20 μL containing 1×FastStart Universal Probe Master (Roche), and the final concentration of each reagent, as well as the corresponding programs are listed in Supplemental Table 1. Each sample was quantified in triplicate for each biological replicate, while three biological replicates were performed.

Detectionsensitivities of qualitative and quantitative PCR

The genomic DNA of Huahui 1 was used to conduct the sensitivity verification. The initial DNA concentration of was 50 ng/μL, and it was diluted with the DNA from non-rice species. The final concentration of the rice DNA were 10% (5 ng/μL), 5% (2.5 ng/μL), 1% (0.5 ng/μL), 0.5% (0.25 ng/μL), 0.1% (0.05 ng/μL), 0.05% (0.025 ng/μL), 0.01% (0.005 ng/μL), 0.005% (0.0025 ng/μL) and 0. The programs of the qualitative and quantitative PCR were following the optimized PCR systems.

Copy number analysis of PEPC gene by digital PCR

Simplex digital PCR was performed on a BioMark System (Fluidigm, South San Francisco) using the 12.765 Digital Arrays (Fluidigm). The instrument software generated PCR amplification curves and real-time cycle threshold () values for each of the 9180 chambers (765 × 12). Following the amplification process, digital raw data were processed using BioMark Digital PCR Analysis software with a manually set threshold of 0.65 and a targetranged from 20 to 40. The sequences of the primers and probes are shown in Table 2.

All copy number variation (CNV) assays were coupled with areference assay. The genewas chosen as the housekeeping gene during the digital PCR, since it was identified as a single-copy gene in the rice genome.

The thermal cycling condition forwas: 10 min at 94 oC; 40 cycles of 30 s at 94 oC, and60 s at 55 oC; 10 min at 98 oC, and final maintenance at 4 oC. The thermal cycling condition forwas mostly the same as that for, with only the annealing temperature at 60 oC. The digital PCR was conducted in a 20 μL volume, containing 2× Taqman Universal Mastermix, 500 nmol/L primer each, 250 nmol/L probe and a 50 ng DNA sample.

Results

Specificity verification of the five ERGs

Specificity verification was conducted in two steps, including the intraspecies consistency and interspecies specificity. PCR reactions were performed using the 19 primer pairs (quantitative PCR primers without the probes and the qualitative PCR primers).

First, among the 18 rice varieties, the PCR results should be positive. Based on the results of agarose gel electrophoresis, except for the primers PLD-F2/R2 and GOS9-F1/R1, the remaining 17 primer pairs exhibited adequate consistency in amplifying the different rice varieties (Supplemental Fig. 1). For the primer PLD-F2/R2, the PCR amplicons displayed variation in their length, while GOS9-F1/R1 failed to amplify all the rice varieties.

When using the DNAs of the 29 non-rice species as templates, no amplicon should be obtained. Of the 17 pairs of primers, 7 pairs displayed sufficient interspecies specificity, including GOS9-F/R, GOS9-F3/R3, PEPC-F1/R1, PEPC-F3/R3, RBE4-F/R, SPS-F1/R1 and SPS-F2/R2. The remaining 10 primer pairs showed either specific or non-specific amplification bands in the non-rice species (Table 3). Although the primers GOS9-F/R (68 bp) and RBE4-F/R (106 bp) displayed good intraspecies and interspecies specificity, however, the lengths of the PCR amplicons were short, making them difficult to distinguish from the primer dimers. Therefore, they were not suitable for the qualitative PCR detection. Finally, five primer pairs were obtained, including SPS-F1/R1, SPS-F2/R2, GOS9-F3/R3, PEPC-F1/R1, and PEPC-F3/R3.

Table 3.The interspecific specificity of the five endogenous reference genes among the 29 non-rice species using qualitative PCR.

‘P’means the positive result and ‘–’means the negative result.

PCR reaction optimizations of selected specific primers

The PCR reaction was further optimized employing the five primer pairs, and the DNAs of 13 rice varieties were used as templates, including Huahui 1, II Kefeng 6, Kefeng 6, Bar 68-1, Wan 21B, Kangyou 97, 80-4B, Donglong, Songjing 6, Zhonghua 11, Zhongzuo 58, Sanjiang 1 and Liaojing 371.

The annealing temperatures during the PCR reaction were adjusted to 56 oC, 58 oC and 60 oC, respectively. Based on the optimal reaction results, the final primer concentrations were set as 0.1 μmol/L, 0.2 μmol/L and 0.4 μmol/L. The optimization results are shown in Table 4.

Detection sensitivity of qualitative PCR assays

All the qualitative PCR reactions were performed in the optimized conditions using the five qualified primers. From the results (Fig. 1), it was evident that the sensitivity of the primer pair PEPC-F3/R3 was as low as 0.005% in SPS-F2/R2 and 0.05% in GOS9-F3/R3, and 0.5% in PEPC-F1/R1 and SPS-F1/R1. The PEPC- F3/R3 was the most sensitive primer pair, and together with the SPS-F2/R2 and GOS9-F3/R3 primers satisfied the requirements necessary for qualitative PCR detection.

Table 4. Optimized PCR reaction of the five selected specific primers.

Fig. 1. Qualitative detection sensitivity of the five primers.

Species specificity of real-time quantitative PCR assays

In this section, the interspecies specificity was first discussed. The 29 non-rice species were used as templates. Allvalues that exceeding 38 were accepted, which meant the amplification was not happened. The results of the real-time PCR detection (Table 5) indicated the primer and probe set ofandgenes exhibited nonspecific amplification, while,andgenes displayed good interspecies specificity.

And then, the intraspecies consistency verification was conducted. The real-time fluorescent PCR tests were performed for the,andgenes using the DNA templates of 18 rice varieties. Ideally, all thevalues should be under 38, which meant the amplification was went well. As results shown in Table 6, all the primer and probe sets obtained satisfactory results and displayed excellent intraspecific stability.

Detection sensitivity of real-time quantitative PCR assays

According to the results, the real-time quantitative PCR detection sensitivity of theandgenes were as low as five copies, and ten copies of thegene, all of which satisfied the detection requirements. The linear coefficient,2was higher than 0.98, and the amplification efficiency was between 90% and 110%, displaying excellent repeatability and reliability (Table 7).

Confirmation of PEPC genecopynumber by digital PCR

The digital PCR was performed using thegene. Two rice varieties, Huahui 1 and Wan 21B, were used as templates, and the absolute copy number per panel was estimated depending on the number of positive partitions, as well as the total number of partitions.

Four sets of DNA samples were used to detect the copy number via digital PCR. Theratio and the confidence interval were automatically generated, and illustrated in Fig. 2 and Supplemenal Table 2. The results revealed that the copy number ratio oftowas nearly 1:1. A comparison between the copies ofanddisplayed no significant differences (= 0.95). Hence, in the genome of rice, thegene was identified as single copy.

Table 5.Interspecific specificity (Ct values) of the five endogenous reference genes among the 29 non-rice species using real-time PCR.

‘–’ means data are not available. Species with novalues for all the genes are not shown.

RSD, Relative standard deviation.

Discussion

In this study, the qualitative and quantitative PCR detection of five ERGs of rice, including four existing genes and one novel selected gene, were conducted on 18 rice varieties and 29 non-rice species. The interspecies specificity, intraspecies consistency, detection sensitivity, stability and reliability of the quantitative system in the detection of these genes were evaluated. The qualitative PCR detection results show that, the primer pairs GOS9-F/R, GOS9-F3/R3, PEPC-F1/R1, PEPC- F3/R3, RBE4-F/R, SPS-F1/R1 and SPS-F2/R2 exhibited sufficient specificity. The target fragments that were amplified by GOS9-F/R and RBE4-F/R primers were short, making them difficult to distinguish from the primer dimers, which were not suitable for the qualitative detection. The PCR reaction and sensitivity detection were used to optimize the remaining five primers. The sensitivity of the primer pair PEPC-F3/R3 was as low as 0.005%, in SPS-F2/R2 and 0.05% in GOS9-F3/R3, and all of them satisfied the qualitative PCR detection requirements.

In the real-time quantitative PCR detection system, RBE4-F/R showed typical amplification in maize, beet and, while GOS9-F/R showed typical amplification in maize, tobacco and oats. PEPC-F/R, PLD-F/R and SPS-F/R exhibited sufficient specificity among different species, as well as the consistency within the same varieties. The detection sensitivities of the PEPC-F/R and PLD-F/R systems achieved a level as low as five copies. Repeated experiments indicated that the linear coefficient2was higher than 0.98, and the amplification efficiency ranged between 90% and 110%, displaying excellent repeatability and reliability. Furthermore, the detection sensitivity for SPS-F/R was 10 copies. Thereofore,,and, as well as their corresponding primers and probes satisfied the requirements of real-time fluorescence qualitative PCR detection.

From the qualitative and quantitative detection, the nonspecific amplification was mostly occurred in maize, soybean, rapeseed, barley and mung bean materials. The homology and similarity of gene sequences among many plant species were discovered, especially in the gramineous plants, including maize, sorghum, wheat and rice. Hence, it is believed that most gramineous plants originated from the same ancestor (Jin et al, 2000).

To maintain a consistent detection result between qualitative and real-time quantitative PCR, the genesandwere ultimately selected as the ERGs for the detection of GM-rice. The five ERGs of rice were tested and verified from the full index. A total of 18 rice varieties and 29 non-rice species were used as templates to investigate the specificity and sensitivity of these genes thoroughly. This research not only evaluated the existing ERGs, but also revealed the presence of an entirely novel one. Thegene was used as a reliable ERG for the detection of GM-rice. Moreover, this study effectively established a foundation for the detection of GMOs in China, and possibly worldwide. Additionally, this research provided powerful technical support for the identification of GMOs.These findings are of great significance in enhancing the comparability of the detection results and the standardization gene testing in transgenic rice.

Table 7.Quantitative detection sensitivity of the three endogenous reference genes.

RSD, Relative standard deviation. Data in the parenthesis are the copy number of DNA.

Fig. 2. Schematic digital PCR results of the two rice samples.

SUPPlemental DATA

The following materials are available in the online version of this article at http://www.sciencedirect.com/science/ journal/16726308; http://www.ricescience.org.

Supplemental Table 1.Final real-time quantitative PCR reaction systems and programs.

Supplemental Table 2. Digital PCR results of theandgenes.

Supplemental Fig. 1.Intraspecific specificity of the five endogenous reference genes among the 18 rice varieties.

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28 January 2019;

28 April 2019

Ouyang Hongsheng (ouyh@jlu.edu.cn)

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http://dx.doi.org/10.1016/j.rsci.2019.04.005

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