Background

DNA is the precondition of molecular biology research, and the quality of DNA samples is the key to the experiment of molecular biology. Polysaccharides, pigments, tannins, phenols, and other secondary metabolites in the plant have severely affected the quality of extraction of genomic DNA, PCR amplification, restriction enzyme digestion and other downstream molecular biology experiments (Arbi et al., 2009). How to remove these interfering substances and obtain high concentration and high purity DNA is the basis of molecular biology research.

At present, there are a lot of reports about the extraction of DNA from the apple genome, young leaves are usually used as experimental materials (Zhao et al., 2002; Zhang et al., 2006; Tong et al., 2008). However, the study of molecular mechanism related to the color and texture of fruit needs to extract genomic DNA from the peel and flesh. However, there are few reports about the extraction of DNA from apple fruit.

Earlier studies had found that apple fruit contains a large number of secondary metabolites, such as polysaccharides and polyphenols (Liu and Wang, 2013). Many physical and chemical properties of polysaccharides are similar to DNA, and they can be combined with DNA to form a colloidal substance; Polyphenols are a strong oxidizing substance that are very easy to oxidize in the air and are irreversible combined with DNA, that causes browning (Dabo et al., 1993; Li et al., 2002), so the extraction quality of DNA is seriously affected. The genomic DNA extracted by traditional methods is brown and has more impurities that cannot be used in molecular biology experiments, so these methods need to be improved. There are a lot of reports about the methods of removing polysaccharides and polyphenols in the process of plant genomic DNA extraction, Zhan and Zeng (2005) took Betula platyphylla leaves as materials, and when DNA was precipitated, 1/10 volume of sodium acetate was added to achieve the purpose of polysaccharide removal by the method of dissolving polysaccharides in high salt. Mohammad et al. (2014) used the buffer solution without CTAB (0.4 mol/L glucose, 20 mmol/LEDTA (pH 8.0), 3% PVP40, 0.2% β-mercaptoethanol) to wash materials when extracting DNA from the leaves of mango, so the interference of polysaccharides and polyphenol impurities on DNA was overcomed. Wang et al. (2011) took Liriodendron chinense leaves as material, and they repeated washing materials with PEG rinse (5 mmol/L EDTA (pH 8.0), 50 mmol/L Tris-HCl (pH 8.0), 350 mmol/L Sorbitol, 10% PEG 8000, 1% β-mercaptoethanol), which effectively removed secondary metabolic substances. In this study, the fruit of 'Granny Smith' was used as the material. Drawing on the methods of removing polysaccharides and polyphenols in other plants and then improve it, so it can be applied to the extraction of DNA of the fruit genome. The study tried to find a high-quality DNA extraction method that was suitable for apple fruit, which would provide a theoretical reference for further improvement of molecular biological experiments.

1 Results and Analysis

1.1 Comparison of the content of polysaccharides and polyphenols in different tissues of apple

Polysaccharides were determined by anthrone-sulfuric acid method and polyphenols were determined by the folin-phenol method (Table 1).

The content of polysaccharides in apple leaves and fruits was very different. The content of polysaccharide in fruit was significantly higher than that in leaf. The content of polysaccharide in flesh was the highest, and the second was in the peel, which were 8.2 times and 4.1 times higher than that in leaves respectively. The content of polyphenols was the highest in the peel, and there was little difference in the leaf and flesh. The polyphenol content in the peel was 2.5 times higher than that in the leaf and 2.3 times higher than that in the flesh.

1.2 Comparison of the DNA concentration and purity of apple genomic DNA extracted by different methods

The concentration and purity of DNA were detected by Thermo NanoDrop 2000 UV spectrophotometer (Table 2).

The concentrations of genomic DNA extracted from the leaf with three methods were the highest, which were about 2 000 ng/μL, and they were significantly higher than that in the peel and flesh. The highest one in the peel was (547.6±12.3) ng/μL, while the highest one in the flesh was (257.8±8.5) ng/μL. The DNA concentrations extracted by method two and method three were lower than that by the method one, which indicated that there was a small amount of DNA loss during washing (Table 2). The OD260/OD280 value of the leaf DNA was about 1.8, and the OD260/OD230 was more than 2, which meant that they all met the requirements.

The DNA purity of fruit extracted by different methods was very different. The value of DNA OD260/OD280 extracted by the method 1 was less than 1.6, which indicated that there were residues of protein, phenol and other impurities. The value of DNA OD260/OD280 extracted by method 2 and method 3 was around 1.8, which indicated that there were no impurities such as protein and phenols (Table 2). DNA OD260/OD230 extracted by the method 1 was less than 2, which indicated that small molecular impurities containing sugar and salt were included. Method 2 and method 3 were used to wash the material before the cell was cracked. The OD260/OD230 value was close to 2, and the OD260/OD230 value of method 3 was greater than that of method 2, which indicated that the DNA impurity extracted by method 3 was less and the purity was higher (Table 2).

1.3 Detection of apple genomic DNA integrity extracted by different methods

The integrity of DNA was detected by 0.8% agarose gel electrophoresis. There was no significant difference in genomic DNA extracted by three improved methods, and DNA strip was neat and clear, with no protein, polysaccharide, polyphenols and other secondary metabolites residues (Figure 1). The genomic DNA of the peel and flesh extracted from three methods showed significantly differences. The DNA of peel and flesh was poor extracted by Method 1, the bands were dark and fuzzy, and the sample has residual impurities, which showed that the extracted DNA contained more proteins, phenols, sugar, and small molecular impurities in salt. However, Method 2 and Method 3 obtained good extraction effects with less impurity in point hole, and clear DNA strip. Among them, the integrity of method 3 was better than that of the Method 2. The DNA strip was neat and clear, and the impurities in the sample hole were few and the purity was high (Figure 1).

1.4 PCR amplification detection

ISSR primer 811 was used for PCR amplification of the DNA extracted by different methods. The PCR reaction of the genomic DNA extracted by the three methods could amplify clear strips (Figure 2). The PCR amplification products of genomic DNA of peel and flesh extracted by the three methods were quite different. The stripe amplified in PCR reaction from genomic DNA that was extracted by method one was dark, which indicated that the quality of DNA extracted by this method was poor. The strips amplified from method 2 and method 3 were clear and bright.

Among them, the stripe amplified from method 3 was clearer than that from method 2, which showed that the genomic DNA extracted by method 3 had better quality and was more suitable for further ISSR molecular biology analysis (Figure 2).

1.5 Enzyme digestion detection of EcoRI

The Leaf DNA extracted by the three methods could be cut by restriction enzyme, but with small amount of RNA pollution (Figure 3). After the EcoRI digestion, DNA residual was detected in the DNA of peel and flesh extracted by Method 1, which showed that the residual polysaccharides, polyphenols and other impurities inhibited the activity of restriction endonuclease and affected the effect of enzyme digestion. Small amount of DNA residual was detected in peel DNA extracted by Method 2 after EcoRI digestion, while flesh DNA could be completely digested by EcoRI. The genomic DNA of peel and flesh extracted by the Method 3 could be completely digested by EcoRI with no DNA residue which indicated that the DNA impurity was less, the purity was higher, and the enzyme activity was not affected (Figure 3).

2 Discussion

Although the DNA extraction was a conventional molecular biology experimental technique, but for the plants with higher polysaccharides, phenol and other secondary metabolites content, high quality DNA acquiring was still a technical problem (Zheng et al., 2015). As a common method for extracting DNA from plants, the greatest characteristic of CTAB was to remove polysaccharides impurities (Ma and Zhang, 2009). Because different plant tissues had different cell wall structures, the contents of cell inclusions, such as phenols, polysaccharides and other secondary metabolites, are different, and the extraction methods of genomic DNA from different parts of the plant were also different (Zhou et al., 2010). The purpose of this study was to solve the problems of thick, oxidizing browning and low purity of DNA solution in fruit DNA extraction, and extract high quality genomic DNA, which would provide a theoretical reference for the subsequent molecular biology research of apple fruit.

The fruit DNA extracted by the method 1 was precipitated as a sticky glue, which was brown, and was difficult to dissolve. There were also many residual impurities in the sample. The DNA strip was blurred, and the PCR amplification strip was dark and couldn’t be cut by the EcoR I enzyme, which showed that after the cells were broken, a large number of secondary metabolites such as polysaccharides and polyphenols were released, causing the extract to become thicker and oxidize to brown. The irreversible binding of DNA to these substances led to poor quality of extraction and couldn’t be used in molecular biology research.

Method 2, on the basis of method 1, using a buffer PW without CTAB to wash the material three times before the nuclear split. It was found that the extracted DNA solution was not sticky, the DNA precipitation was milky white, the impurity of the sample hole was less, the DNA strip was clear (Figure 1), the purity increased, and the PCR amplification strip was clear, which could be digested by EcoRI. The result showed that the buffer PW solution without CTAB solved the problem of the viscosity of the solution and the oxidation of polyphenols, and effectively removed the secondary metabolites, such as polysaccharides and polyphenols in the fruit.

The interferon scavenging solution was optimized for the composition of the buffer PW without CTAB in method 3, and 10% PEG 8000 was added. The extraction results were better than that of method two. The purity of DNA was higher, the strip was clear, the PCR amplification band was bright and it could be completely digested by EcoR I. It showed that the increased 10% PEG 8000 in this method played an important role in the removal of secondary metabolites, such as polysaccharides and polyphenols. It was found that with the increase of PEG 8000 concentration, the difference of the extraction effect was not significant (the result was not listed in this study). According to the concentration, purty iand electrophoretic results of DNA, the concentration of DNA extracted by method 3 was low, but its purity and integrity was good. They all could satisfy the study of both of PCR amplification and restriction enzyme digestion. Therefore, it was determined that the method 3 was suitable for the extraction of genomic DNA from apple fruit.

This method synthesized the characteristics that polysaccharide, polyphenols and other secondary metabolites were mainly distributed in cytoplasm, and increased the concentration of β-mercaptoethanol and PVP to inhibit polyphenol oxidation and reduce multi-step impurity removal steps of polysaccharides dissolution in high salt. Before the nucleus split, we washed material with buffer PW or interferon scavenging fluid without CTAB. The most of polysaccharides, phenolic secondary metabolic substances dissolved in the cytoplasm by plasmolysis, and nuclear was be separated by cryogenic centrifugation (Ding and Lv, 2000). Increasing the concentration of reducing agent β-mercaptoethanol and chelating agent PVP to inhibit the oxidation of polyphenols into quinones and avoid browning of solution (Yang, 2015). And the polysaccharide was dissolved by 3 mol/L NaAC in the process of DNA precipitation, so that the polysaccharide was retained in the solution and then separated from the DNA precipitation. This method was simple and effective, which could effectively overcome the interference caused by polysaccharides and polyphenols on DNA extraction. The quality of DNA obtained was high, which would provide an important basis for future research.

The following problems still existed in the DNA extracted by this method: after washing by buffer PWor interferon scavenging fluid without CTAB, the time consuming was longer than that of the conventional method and the concentration of DNA was reduced. Although the OD260/OD230 value of peel and flesh increased, it was still lower than 2. A small amount of impurity remained in the sample hole. It showed that the extraction method of fruit DNA needed further improvement. The direction of future improvement was not only to improve the concentration of DNA, but also to improve the purity of DNA. Optimizing the components of the interfering clearance could develop a polysaccharide and polyphenol scavenger which could be added to the lysis solution, and removed polysaccharides and polyphenols before DNA was dissolved, which could not only avoid DNA loss, but also save time.

3 Materials and Methods

3.1 Test material

The "Granny Smith" used in the experiment was taken from the Baishuiapple test station of Northwest Agriculture and Forestry University. Six plants with the same growth were selected, and ten fruits were taken from the middle of each tree crown in the mature period of each plant, and 3 times were repeated. Cutting the skin of fruit body (approximately 1 cm wide, 1~2 mm thick) and cutting the flesh. Then put it in the refrigerator at -80℃ after immediately liquid nitrogen freezer. Young leaves were used as control.

3.2 Reagents

Anthrone, concentrated sulfuric acid, ethanol, Folin-phenol, gallic acid, sodium carbonate, methanol, cetyl trimethyl ammonium bromide (CTAB), β-mercaptoethanol, polyvinylpyrrolidone (PVP), Tris base, concentrated HCL, ethylene diamine tetraacetic acid (EDTA-Na2), PEG 8000, Tris saturated phenol, chloroform: isoamyl alcohol (24:1), isopropanol, 3 mol/L NaAC (pH 5.2), TE buffer solution (10 mmol/L Tris-HCl, 1 mmol/L EDTA, pH 8), etc. were all domestic analytical pure. Agarose and RNaseA (10 mg/mL) were purchased from Tian Gen biochemical technology (Beijing) Co., Ltd. DL2000 DNA Marker, EcoRI (10 U/μL), 10 x Buffer EcoRI, 2 x Es Taq Master Mix reagents were purchased from Bao Bioengineering (Dalian) Co., Ltd. The ISSR primers were synthesized from Huada in Beijing.

3.3 Determination of the content of polysaccharides and polyphenols

The content of polysaccharide was determined by anthrone sulfuric acid method (Zhao et al., 2007). The content of polyphenol was determined by Folin- phenol method (Wang et al., 2013). Each sample was repeated 3 times.

3.4 DNA extraction method

Method 1: refer to the method reported by Zhao et al. (2010) and then improve it; DNA precipitation step, add 1/10 volume 3 mol/L sodium acetate and 0.7 times volume isopropanol.

Method 2: refer to the methods of Wang (2004) and Zhang et al. (2009), and then improve it: weigh and take 100 mg ground sample, add 1 mL rinse without CTAB (100 mmol/L Tris-HCl (pH 8), 20 mmol/L EDTA (pH 8.0), 5% glycerol, 2% PVP, 2% β-mercapto ethanol) and vortex mixed, ice bath 10 min, 4°C, 10 000 r/min centrifuge for 10 min, discard the supernatant. Repeat three times, and then follow the method 1.

Method 3: refer to the methods of Sánchez-Hernández and Gaytán-Oyarzún (2006) and Chen et al. (2014), and then improve it: using interferon scavenging fluid (100 mmol/L Tris-HCl (pH 8), 20 mmol/L EDTA (pH 8), 5% glycerol, 10% PEG 8000, 2% PVP, 2% β-mercapto ethanol) instead of rinse solution without CTAB in method 2, the rest is the same as method 2.

3.5 Concentration and purity test

The genomic DNA extracted from 1μL was obtained, and the concentration of each sample and the value of OD260/OD280 and OD260/OD230 were measured by the Thermo Nan-oDrop 2000 spectrophotometer.

3.6 Product integrity detection

Take a mixture of 2 μL DNA samples and 1 μL 6 x DNA Loading Buffer, with DL2000 DNA Marker as reference, 100 V electrophoretic for 40 min in 0.8% agarose gel and 1 x TAE buffer solution, and then observe and photograph in Gene Snap gel imaging system.

3.7 PCR amplification detection

Refer to the methods of Zhu (2003) and Guo et al. (2010). The reaction system (25 μL): 12.5 μL 2 x Es Taq Master Mix, 2 μL ISSR primers UBC811/(GA)8C, 1 μL DNA templates. PCR amplification program: predenaturation at 94°C for 7 min, denaturation at 94°C for 45 s, annealed at 52°C for 40 s, extend at 72°C for 1 min, 30 cycles, and extend at 72°C for 7 min.

After the reaction, 10 μL PCR products were taken for electrophoresis in 1% agarose gel, then observe and photograph in Gene Snap gel imaging system.

3.8 Restrictive endonuclease detection

Reaction system (20μL ): 2 uL 10 x Buffer EcoR I, 1 uL EcoR I, 0.5 μL DNA, water bath at 37°C for 2 h, take 10 μL enzyme products for electrophoresis in 1% agarose gel. Then observe and take photos in the DNA gel imaging system.

Authors’ contributions

YJY, MCQ and CB were the executors of experimental design and experimental research in this study; FXG and LZ participated in the analysis of experimental results. YJY, MCQ and YYZ participated in the writing and revision of the paper. ZZY conceived the project and was the person in charge, who guided the design of the experiment, the data analysis, and the revision of the paper. All authors read and approved the final manuscript.

Acknowledgments

This research is funded by the National Natural Science Foundation of China (31471845), the National Apple Industrial Technology System (CARS-27) and the Shaanxi Science and Technology Co-ordination Project (2015ZS-13).

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