Orthogonal design in the optimization of a start codon targeted (SCoT) PCR system in Roegneria kamoji Ohwi
Abstract
Roegneria kamoji Ohwi is an excellent forage grass due to its high feeding value and high resistance to some biotic and abiotic stresses. However, the start codon targeted (SCoT) polymorphism has not been conducted on R. kamoji. In this study, an orthogonal L16 (45) design was employed to investigate the effects of five factors (Mg2+, dNTPs, Taq DNA polymerase, primer, and template DNA) on the polymerase chain reaction (PCR) to determine the optimal SCoT-PCR system for R. kamoji. The results showed that the most suitable conditions for SCoT-PCR in R. kamoji included 1.5 mM Mg2+, 0.15 mM dNTPs, 1.0 U Taq DNA polymerase, 0.4 pM primer, and 40 ng template DNA. SCoT primers 39 and 41 were used to verify the stability of the optimal reaction system, and amplification bands obtained from diverse samples were found to be clear, rich, and stable in polymorphisms, indicating that this reaction system can be used for SCoT-PCR analysis of R. kamoji. We have developed a simple and rapid way to study the mutual effects of factors and to obtain positive results through the use of an orthogonal design L16 (45) to optimize the SCoT-PCR system. This method may provide basic information for molecular marker-assisted breeding and analyses of genetic diversity in R. kamoji.
INTRODUCTION
Roegneria kamoji, which is classified under the largest genus in the wheat family, is mainly located in the warm and cold zones of the Northern hemisphere. There are about 120 R. kamoji species worldwide (Baum et al., 1991), of which 70 are located in the northwest, southwest, and north of China. R. kamoji is a fine forage grass because of its high feeding value, and high resistance to some biotic and abiotic stresses such as diseases caused by gibberella (Fusarium graminearum) and low-lying wet stress (Jiang and Liu, 1990; Yang et al., 2001). A number of different molecular marker systems have been developed to study the genetics of R. kamoji, including simple sequence repeats (Peng et al., 2012; Yang et al., 2016), gliadin (Xiao et al., 2008), RAMP (Zhang et al., 2005), and polymerase chain reaction (PCR)-RFLP (Marson et al., 2005; Zhang et al., 2006) markers. However, start codon targeted (SCoT) polymorphisms have not been applied to R. kamoji.
The SCoT polymorphism is a new method to sign target gene, which was proposed by Collard and Mackill (2009) in rice based on the single primer amplification reaction. In single primer PCR, SCoT uses single 18-mer primers to amplify the genomic region based on flanking sequences and conservation of the ATG translation start site in plant genes (Joshi et al., 1997; Collard and Mackill, 2009Bhattacharyya et al., 2013). There are many advantages of this method, including its high polymorphism, simple operation, low cost, good universality, good reproducibility, and simple primer design (Hu et al., 2009). SCoT markers have been confirmed to be highly effective for the evaluation of genetic variation and population structure in rice (Collard and Mackill, 2009) and some other species including, mango (Mangifera indica) (Luo et al., 2011), grape (Vitis vinifera) (Guo et al., 2012), peanut (Arachis hypogaea) (Xiong et al., 2011), Lonicera Flos (Lonicera macranthoides) (Chen et al., 2015), and dendrobe (Dendrobium nobile) (Bhattacharyya et al., 2013). In addition, SCoT markers that are developed from transcribed regions may be related to gene function. For example, the oligo-dT anchored cDNA-SCoT technique was used to exploit differentially expressed genes in mango (M. indica L.) under several stress conditions (Luo et al., 2014).
However, SCoT has some limitations. For example, 1) SCoT is based on the PCR method, but many factors including primer, dNTPs, Mg2+, and Taq DNA polymerase concentrations influence the stability of PCR; 2) the low stringency PCR conditions may limit polymorphism detection; 3) PCR conditions may diverge between species (Collard and Mackill, 2009). Therefore, it is critical to establish a stable and optimized reaction system for use with SCoT molecular markers (Zeng et al., 2015).
In this study, we employed an orthogonal design referred to as L16 (45) (four levels of five factors: Mg2+, DNA template, Taq DNA polymerase, dNTPs, and primer) to optimize the SCoT-PCR system for R. kamoji and to provide a basis for the future study of genetic relationships, genetic diversity, construction of molecular linkage genetic maps, gene localization, variety identification, QTL analysis, and molecular marker-assisted breeding in R. kamoji.
MATERIAL AND METHODS
Materials
The samples used in this study were obtained from the Teaching and Research Center at Southwest University (Rongchang), Chongqing in China in May 2013 (Table 1). Fresh leaves were collected from the field and placed in an ultralow temperature refrigerator at -80°C.
Names and types of Roegneria kamoji samples used in the experiment.
| No. | Material type (name) | Source |
|---|---|---|
| 1 | Cultivar, Du Jiang Yan (ZY) | Sichuan, China |
| 2 | Cultivar, Japan (88) | Japan |
| 3 | Cultivar, Gansi One (CK) | Jiangxi, China |
| 68 | Wild material | Sichuan, China |
| 69 | Wild material | Sichuan, China |
| 70 | Wild material | Sichuan, China |
| 71 | Wild material | Sichuan, China |
| 72 | Wild material | Sichuan, China |
| 73 | Wild material | Sichuan, China |
| 74 | Wild material | Sichuan, China |
| 75 | Wild material | Sichuan, China |
| 76 | Wild material, 004 | Chongqing, China |
| 77 | Wild material, 005 | Chongqing, China |
DNA extraction and PCR program
The DNA of 13 fresh young leaf samples was extracted using a genomic DNA extraction kit (ComWin Biotechnology Co., Ltd., Beijing, China). The concentration and quality of the DNA were confirmed by electrophoresis on 0.8% agarose gels and spectrophotometric analysis with the NanoDrop 2000 nucleic acid/protein analyzer (Nanodrop Technologies, Wilmington, DE, USA). Samples were stored at -20°C and DNA was diluted to 20 μg/µL prior to conducting the experiment, and then stored at -4°C.
The SCoT primer sequences (SCoT S16: 5'-ACCATGGCTACCACCGAC-3'; SCoT S39: 5'-CAATGGCTACCACTAGCG-3', and SCoT S41: 5'-CAATGGCTACCACTGACA-3') were previously described by Luo et al. (2011), and synthesized by Shanghai Shenggong Biological Engineering Technology Services Ltd. (Shanghai, China). Taq DNA polymerase, 6X buffer, 10X buffer, dNTPs, Mg2+, and DL2000 marker were provided by Takara Biotechnology (Dalian) Co., Ltd. (Shiga, Japan). PCR amplification was performed on an Eppendorf Mastercycler (Hamburg, Germany) and included the following thermal profile: 94°C for 3 min, followed by 36 cycles of denaturing at 94°C for 30 s, annealing at 50°C for 1 min, and extension at 72°C for 2 min. The final extension was set at 72°C for 10 min, followed by storage at 4°C. The PCR amplification products were then added to 2 μL 6X buffer, and a 8-10 μL sample from each treatment was separated on 1.5% agarose gels in 1X Tris-borate EDTA buffer, and then stained with GoldView™ dye (Beijing Bioteke Biotechnology Co., Beijing, China). Finally, a gel documentation system (Bio-Rad, Hercules, CA, USA) was used to visualize the DNA fragments under UV light.
Orthogonal array design
The optimum concentrations of template DNA (Gansi One), Taq DNA polymerase, dNTPs, Mg2+, and primer (SCoT S16) were determined using an orthogonal design L16 (45). We selected four concentrations based on experience. Further details about these five factors, and detailed information on the experimental concentrations, are provided in Table 2. The L16 (45) orthogonal experimental design is shown in Table 3. The amplification conditions were as follows: 2.0 μL 10X buffer and other components, and ddH2O was added to obtain a final volume of 20 μL. A scoring system was applied to determine the variance between patterns of SCoT-PCR fingerprints obtained under different treatments. A scoring criterium was used as described by He et al. (1998), and the generated DNA amplification patterns were scored from the best (16 points) to the worst (1 point), including the number of amplified fragments and the clear degree of the PCR amplification results (Table 3).
Four different levels of factors and volume for SCoT-PCR amplification.
| No. | Mg2+ (mM) | dNTPs (mM) | Taq DNA polymerase (U) | Primers (pmol/µL) | Template DNA (ng) |
|---|---|---|---|---|---|
| 1 | 1.5 | 0.15 | 0.75 | 0.2 | 20 |
| 2 | 2.0 | 0.20 | 1.00 | 0.3 | 30 |
| 3 | 2.5 | 0.25 | 1.25 | 0.4 | 40 |
| 4 | 3.0 | 0.30 | 1.50 | 0.5 | 50 |
L16 (45) orthogonal design used for SCoT-PCR amplification.
| Treatment No. | Mg2+ (mM) | dNTPs (mM) | Taq DNA polymerase (U) | Primers (pmol/µL) | Template DNA (ng) | Score |
|---|---|---|---|---|---|---|
| 1 | 1.5(1) | 0.15(1) | 0.75(1) | 0.2(1) | 20(1) | 11 |
| 2 | 1.5(1) | 0.20(2) | 1.00(2) | 0.3(2) | 30(2) | 12 |
| 3 | 1.5(1) | 0.25(3) | 1.25(3) | 0.4(3) | 40(3) | 13 |
| 4 | 1.5(1) | 0.30(4) | 1.50(4) | 0.5(4) | 50(4) | 7 |
| 5 | 2.0(2) | 0.15(1) | 1.00(2) | 0.4(3) | 50(4) | 16 |
| 6 | 2.0(2) | 0.20(2) | 0.75(1) | 0.5(4) | 40(3) | 10 |
| 7 | 2.0(2) | 0.25(3) | 1.50(4) | 0.2(1) | 30(2) | 3 |
| 8 | 2.0(2) | 0.30(4) | 1.25(3) | 0.3(2) | 20(1) | 2 |
| 9 | 2.5(3) | 0.15(1) | 1.25(3) | 0.5(4) | 30(2) | 8 |
| 10 | 2.5(3) | 0.20(2) | 1.50(4) | 0.4(3) | 20(1) | 5 |
| 11 | 2.5(3) | 0.25(3) | 0.75(1) | 0.3(2) | 50(4) | 6 |
| 12 | 2.5(3) | 0.30(4) | 1.00(2) | 0.2(1) | 40(3) | 14 |
| 13 | 3.0(4) | 0.15(1) | 1.50(4) | 0.3(2) | 40(3) | 15 |
| 14 | 3.0(4) | 0.20(2) | 1.25(3) | 0.2(1) | 50(4) | 1 |
| 15 | 3.0(4) | 0.25(3) | 1.00(2) | 0.5(4) | 20(1) | 9 |
| 16 | 3.0(4) | 0.30(4) | 0.75(1) | 0.4(3) | 30(2) | 4 |
Determination of an optimal reaction system
According to the score for each treatment, the optimal treatment was calculated (Table 4). The experimental Ki value represents the total score for each factor at the same level, the experimental ki value represents the average of the total score for each factor at the same level, and the experimental R value represents the range of the average total score for the same factors between various levels.
L16 (45) orthogonal design for SCoT-PCR amplification.
| Results | Mg2+ (mM) | dNTPs (mM) | Taq DNA polymerase (U) | Primers (pmol/µL) | Template DNA (ng) |
|---|---|---|---|---|---|
| K1 | 43.00 | 50.00 | 31.00 | 29.00 | 27.00 |
| K2 | 31.00 | 28.00 | 51.00 | 35.00 | 27.00 |
| K3 | 33.00 | 31.00 | 24.00 | 38.00 | 52.00 |
| K4 | 29.00 | 27.00 | 30.00 | 34.00 | 30.00 |
| k1 | 10.75 | 12.50 | 7.75 | 7.25 | 6.75 |
| k2 | 7.75 | 7.00 | 12.75 | 8.75 | 6.75 |
| k3 | 8.25 | 7.75 | 6.00 | 9.50 | 13.00 |
| k4 | 7.25 | 6.75 | 7.50 | 8.50 | 7.50 |
| R | 3.50 | 5.75 | 6.75 | 2.25 | 6.25 |
Stability analysis of two reaction systems
The DNA samples (1, 2, 68, 70, 72, 74, and 76) were used to compare the stability of the highest scored and statistically optimum reaction systems using the SCoT primer S16. The best reaction system was then selected to verify its stability using the SCoT S39 and SCoT S41 primers for 13 samples shown in Table 1.
RESULTS
Visual analysis of the PCR orthogonal design
Multiple fingerprinting patterns were observed from the PCR amplification products after the orthogonal experiment treatments. Treatment 5 showed the clearest fragments (the score was 16), while the treatment 14 displayed the vaguest fragments (the score was 1) (Figure 1). Electrophoresis of samples obtained using an orthogonal design. Lane M: marker (DL2000); lanes 1-16: treatment numbers are the same as those noted in Table 3. Bar charts showing the relationships between the following factors. 
Electrophoresis of the optimal reaction system (right) and the highest scored orthogonal reaction system (left). Lane M: marker (DL2000).
Stability of the best reaction system
SCoT primers 39 and 41 were used to verify the stability of the best reaction system. As shown in Figure 4, amplified bands from diverse samples were clear, rich, and stable in polymorphisms. The fingerprinting patterns of the R. kamoji samples revealed the genetic differences between samples, and confirmed the genetic stability of each sample, indicating that the best reaction system can be applied in SCoT-PCR analysis of R. kamoji.
Electrophoresis of start codon targeted (SCoT) S39.
DISCUSSION
The five factors studied here generate different fingerprinting patterns and Taq DNA polymerase is a key factor affecting the PCR. Whether high or low, the concentration of Taq DNA polymerase would lead to poor amplification; therefore, the most effective concentration is 1 U, which is consistent with the result of Yang et al. (2007). There are interactions between each factor in the PCR system. Taq DNA polymerase is an Mg2+-dependent enzyme, which is influenced by the concentration of Mg2+, while Mg2+ is also affected by other factors, especially dNTP (Zheng et al., 2008). In the present study, the primer concentration had the smallest influence on the PCR result, which conflicts with the results of Zeng et al., (2015) who used Dactylis glomerata as the test material, indicating that samples from different species may generate different results.
Optimizing the SCoT-PCR system for R. kamoji is essential. We have developed a simple and rapid method that can be used to study the mutual effects of different factors and obtained good results through the use of an orthogonal design L16 (45) to optimize the SCoT-PCR system. In addition, this method can be used in follow-up studies in R. kamoji, especially for molecular marker-assisted breeding and in analyses of genetic diversity.