Internal amplification control of PCR for the Glu1-Dx5 allele in wheat
Accepted: November 30, -0001
Published: August 17, 2017
Genet.Mol.Res. 16(3): gmr16039718
One of the limiting factors in using dominant markers is the unique amplification of the target fragment. Therefore, failures in polymerase chain reaction (PCR) or non-amplifications can be interpreted as an absence of the allele. The possibility of false negatives implies in reduced efficiency in the selection process in genetic breeding programs besides the loss of valuable genetic material. Thus, this study aimed to evaluate the viability of a microsatellite marker as an internal amplification control with a dominant marker for the wheat Glu1-Dx5 gene. A population of 77 wheat cultivars/breeding lines was analyzed. Fourteen microsatellite markers were analyzed in silico regarding the formation of dimers and clamps. The biplex reaction conditions were optimized, and the Xbarc117 marker was selected as the internal amplification control with a Glu1-Dx5 marker in wheat. It was concluded that the Xbarc117 microsatellite marker was effective in the simultaneous amplification with a dominant Glu1-Dx5 marker, making biplex PCR viable in wheat for the studied markers.
The utilization of molecular markers in the target genes in marker-assisted selection (MAS) in genetic breeding programs can be more efficient than the utilization of linked markers. The advantage of these specific markers is that they are not loosed during recombination, besides being possible to use them practically in all relative populations as a way of general screening in elite breeding lines or germplasm banks (Mackill and McNally, 2006).
Many times the conversion of restriction fragment length polymorphism probes or single nucleotide polymorphism markers in specific PCR markers results in dominant markers. One of the limiting factors of the utilization of dominant markers is the single amplification of the fragment corresponding to the allele under study, and the absence of alternative allele amplification, generating a common result of presence and absence of a band in gel analysis. Therefore, failures in PCR or the non-amplification can be interpreted as an allele absence. This interpretation of false negative implies in reduced efficiency in the MAS in genetic breeding programs and loss of valuable genetic material. An alternative to avoid these problems is the utilization of an internal amplification control.
Biotechnological advances have increased the selection efficiency and productivity gains (Langridge and Fleury, 2011; Walsh et al., 2017; De Ron et al., 2017). The noncompetitive internal amplification control is a DNA sequence that is amplified in all samples and co-amplified in the same PCR of the target sample. Adding an internal amplification control, the control sign will be produced even if the target sequence is not present. This implies result interpretation safety, reduction of reaction problems by secondary structures, and elimination of false negatives (Hoorfar et al., 2004; Ma Z and Michaeliedes, 2006). However, PCRs with internal amplification control are still a challenge because several parameters need to be optimized such as the choice of an internal amplification control that can be amplified in all samples. Therefore, this DNA sequence needs to be preserved in the target species and cannot be polymorphic within the studied population. It is also necessary that amplicons have a different size to make fragment visualization easy. Besides that, the relationship between the target DNA concentrations and primers needs to be optimized to avoid competition by nucleotides in PCR, the formation of dimers and clamps between primers that will be co-amplified should be avoided, and reagents and conditions for PCR need to be optimized (Abdulmawjood et al., 2002; Hoorfar et al., 2004). Several research groups use internal amplification control to ensure reliability of their results (Henegariu et al., 1997; Narvel, 2000; Abdulmawjood et al., 2002; Loridon et al., 2005; Kanchana-Udomkan, 2013), but each internal amplification control needs to be optimized regarding the specific gene and th e population of interest.
Glu-D1 locus in wheat is associated with gluten strengthening and breadmaking quality (Payne, 1987; Lukow et al., 1989; Shewry et al., 2003) and, therefore, it is interesting to incorporate this allele to new cultivars developed by genetic breeding programs. The Glu1-Dx5 marker is dominant, and the optimization of a biplex reaction using an internal amplification control can increase the efficiency of this marker in MAS breeding programs. In a comparison between singleplex and biplex PCR using internal amplification control, Masi et al. (2003) reported an economy of 50% in PCR reagent utilization and 85% of electrophoresis costs. Therefore, this study aimed to evaluate the viability of a microsatellite marker as an internal amplification control with a dominant marker for the wheat Glu1-Dx5 gene to avoid false-negative results.
Materials and Methods
Vegetal material, DNA extraction, and quantification
The experiments were done in seeds of 77 wheat cultivars/breeding lines of the genetic breeding program of the Central Cooperative of Agricultural Research (Coodetec) (Table 1).
|Genotype||Germplasm type||Genotype||Germplasm type||Genotype||Germplasm type|
|CD0529||Breeding line||CDI0602||Breeding line||CD0711||Breeding line|
|CD0532||Breeding line||CD0610||Breeding line||CD0714||Breeding line|
|CD0568||Breeding line||CD0614||Breeding line||CD0715||Breeding line|
|CD0572||Breeding line||CD0677||Breeding line||CD0718||Breeding line|
|CD0574||Breeding line||CD0712||Breeding line||CD0721||Breeding line|
|CD0578||Breeding line||CD0716||Breeding line||CDI0408||Breeding line|
|CD0579||Breeding line||CD0529||Breeding line||CD0511||Breeding line|
|CD0622||Breeding line||CD0544||Breeding line||CD0513||Breeding line|
|CD0664||Breeding line||CD0651||Breeding line||CD0515||Breeding line|
|CD0665||Breeding line||CD0654||Breeding line||SAFIRA||Cultivar|
|CD0666||Breeding line||CD0658||Breeding line||BRS208||Cultivar|
|CD0671||Breeding line||CD0660||Breeding line||IPR85||Cultivar|
|CD0674||Breeding line||CD0661||Breeding line||CD104||Cultivar|
|CD0680||Breeding line||CD0667||Breeding line||CD105||Cultivar|
|CD0684||Breeding line||CD0669||Breeding line||CD106||Cultivar|
|CDF2002116||Breeding line||CD0678||Breeding line||CD108||Cultivar|
|CD0619||Breeding line||CD0683||Breeding line||CD110||Cultivar|
|CD0620||Breeding line||CD0627||Breeding line||CD111||Cultivar|
|CD0644||Breeding line||CD0631||Breeding line||CD112||Cultivar|
|CD0646||Breeding line||CD0632||Breeding line||CD113||Cultivar|
|CD0647||Breeding line||CD0545||Breeding line||CD114||Cultivar|
|CD0649||Breeding line||CD0548||Breeding line||CD116||Cultivar|
|CD0542||Breeding line||CD0672||Breeding line||CD117||Cultivar|
|CD0558||Breeding line||CD0683||Breeding line||ONIX||Cultivar|
|CD0559||Breeding line||CD0704||Breeding line||Fundacep Nova Era||Cultivar|
|CD0706||Breeding line||CD0705||Breeding line|
Table 1: List of wheat cultivars/breeding lines used in evaluating microsatellite as an internal control for the Glu1-Dx5 marker.
A sample of 50 seeds of each cultivar/breeding line was ground in an MA 630 grinder (Marconi®) and submitted to DNA extraction following the protocol described by McDonald et al. (1994), with modifications (Schuster et al., 2004). In 1.5-mL microtubes containing approximately 50 mg ground seeds, 500 μL of an extraction buffer solution containing 0.1 M Tris-HCl (pH 7.5), 5 M NaCl, 5 M EDTA, and 10% SDS was added. A 3-mm diameter glass ball was placed in each tube followed by maceration in a grinder (ACS®), the addition of 500 μL extraction buffer, homogenization by Vortex agitator, and centrifugation at 18,506 g for 10 min. After centrifugation, the supernatant was transferred to a new tube with 10 μL proteinase K (10 mg/mL) and immersed in water at 37°C for 30 min. Next, 500 μL cold isopropanol was added, and the tubes were maintained at rest for 2 min and centrifuged for 15 min at 18,506 g. The supernatant was discarded, the precipitate was dried at ambient temperature for 15 min, resuspended in 300 μL TE buffer (0.1 M Tris-HCl, pH 7.5 and 5 M EDTA) with RNase A (40 μg/μL) and immersed in water at 37°C for 30 min. DNA was again precipitated by adding 500 μL cold isopropanol, maintained at rest for 2 min and centrifuged for 15 min at 18,506 g. The supernatant was discarded, and the precipitate was dried at ambient temperature and resuspended in 300 μL TE buffer.
The DNA concentration of each sample was estimated by absorbance at 260 nm in a Nanodrop1000 spectrophotometer. Every absorbance unit corresponded to 50 μg/mL doublestranded DNA (Sambrook et al., 1989).
In silico selection for internal amplification control
To verify the best internal control, 14 microsatellite markers of wheat (Table 2) with amplicons varying from 180 up to 240 bp were evaluated regarding the formation of clamps and dimers with primers of the Glu1-Dx5 marker (450 bp) by aligning its sequences utilizing the Netprimer program <http://www.netprimer.com>. The primer sequences are available in the link <http://wheat.pw.usda.gov/CGI-bin/graingenes/SSRsize>. The primers that presented clamps and/or dimers after the analysis were discarded due to a greater chance of nonamplification of the target DNA.
|SSR marker||Amplicon (bp)||In silico analysis|
Table 2: Microsatellite markers (SSR) utilized in in silico analysis to verify the formation of secondary structures with a dominant marker for the Glu1-Dx5 gene.
Amplification and electrophoresis
The wheat cultivars Frontana and Ocepar22 were used as negative and positive controls, respectively, for PCR amplification and allele identification. The primer pair of Glu1-Dx5 markers was developed by Anderson et al. (1998) and has the following sequences: 5'-GCCTAGCAACCTTCACAATC-3' and 5'-GAAACCTGCTGCGGACAAG-3'.
PCRs were done in a Verit thermocycler (Applied Biosystems®) in 0.2-mL microtubes with a total volume of 20 μL containing 1 X PCR buffer (20 mM Tris-HCl, 50 mM KCl), 2 or 3 mM MgCl2, 250 mM dNTP, 75 ng DNA, and primers at the concentration of 0.2 μM. The PCRs were done in biplex with microsatellite primer and specific primer for the Glu1-Dx5 gene.
Two different concentrations of magnesium chloride at 2 and 3 mM were tested at different annealing temperatures: 51°, 53°, 55°, 57°, and 59°C utilizing different amplification conditions: initial cycle at 94°C for 5 min, 94°C of denaturation for 60 s, annealing for 30 s at the temperatures cited before, extension of 72°C for 60 s during 45 cycles, and a final cycle of 72°C for 10 min.
After the amplification, PCR fragments were separated by electrophoresis on 3% agarose gel with ethidium bromide (1 μg/mL). The fragments were visualized under ultraviolet light in a Vilber Lourmat photo-documentation device (Marne La Valle®).
The PCR program that produced results without non-specificity amplification such as faint, fuzzy, or smeared bands was selected to amplify all 77 samples.
Results and Discussion
Among the 14 in silico microsatellite markers with the Glu1-Dx5 marker analyzed in the Netprimer program, six presented dimers or clamps and were excluded from the study. Among the remaining markers, Xbarc117 was the most monomorphic within this population and, therefore, selected to continue this study. The in silico alignment for the analysis of the possible formation of secondary structures such as dimers and clamps has not been reported in previous studies (Henegariu et al., 1997; Narvel, 2000; Loridon et al., 2005) and can compromise the success of PCRs. Moreover, in silico analysis saves time and reagents, and is an important factor to quickly reach the aims of studies with great amounts of samples.
The concentration of 3 mM MgCl2 at an annealing temperature of 57°C was the most effective simultaneous amplification with neat bands without non-specific bands such as smeared or phantom bands. The bands were of the expected fragment sizes of 223 bp for Xbarc117 and 450 bp for Glu1-Dx5. The amplification in this condition makes possible to analyze the presence and absence of high molecular weight (HMW) glutenin alleles, excluding false negatives. The annealing temperatures at 51°, 53°, 55°, and 59°C were not promising. The annealing temperature of 57°C presented the best result for the amplification of the Glu1-Dx5 marker in the cultivar Ocepar 22 (Figure 1H). This temperature is closer to the optimal temperature of the Xbarc117 marker (54.4°-55°C) than Glu1-Dx5 marker (63°-65°C). The positive and negative controls, cultivars Ocepar 22 and Frontana, were properly amplified with the annealing temperature of 57°C. This PCR condition, with 3 mM MgCl2 and an annealing temperature of 57°C also works well when both markers were used in the biplex amplification (Figure 2).
Annealing temperature and MgCl2 concentration: A = 51°C and 2 mM, B = 51°C and 3 mM, C = 53°C and 2 mM, D = 53°C and 3 mM, E = 55°C and 2 mM, F = 55°C and 3 mM, G = 57°C and 2 mM, and H = 57°C and 3 mM.
Paro (2011) observed that of the 77 individuals analyzed, only 62 presented the Glu1- Dx5 allele amplified after standard PCR analysis. However, the same author, after analyzing the same samples by SDS-PAGE, verified the presence of the Glu1-Dx5 subunit in 66 samples. Thus, PCR analysis with dominant markers without the presence of an internal reaction control, despite being practical, is limited and less effective.
In our study, the same 77 samples utilized by Paro (2011) were evaluated, and the presence of the Glu1-Dx5 allele was verified in the 66 individuals that presented Glu1-Dx5 subunit of HMW glutenin. Also, the presence of the Xbarc117 marker was observed in all 77 samples of the population without false-negative results or non-specific amplification bands. This suggests that the PCR simultaneous amplification with Glu1-Dx5 and Xbarc117 markers was effective and the internal amplification control (Xbarc117) is valuable with the dominant marker for the wheat Glu1-Dx5 gene. Besides, this molecular analysis can reduce the analysis time and increase the reliability of results obtained by PCR.
The Xbarc117 microsatellite marker can be amplified and detected with or without the dominant marker for the Glu1-Dx5 gene in different wheat cultivars/breeding lines and is valuable as an internal control for PCR.
Conflicts of interest
The authors declare no conflict of interest.
The authors thank Paranaense University, the Postgraduate Program of Biotechnology applied to Agriculture and the Central Cooperative of Agricultural Research.
About the Authors
Programa de Pós-Graduação em Biotecno, Laboratório de Biologia Molecular, Universidade Paranaense, Umuarama, PR, Brasil
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