Isolation of High-Quality DNA from a Desert Plant Reaumuria soongorica

Xiaohua Wang1, Honglang Xiao1, Xin Zhao2, Caizhi Li1, Juan Ren1, Fang Wang1 and Lei Pang1 1Key Laboratory of Ecohydrology and of Inland River Basin Cold and Arid Regions Environmental and Engineering Research Institute Chinese Academy of Sciences, Lanzhou 2Extreme Stress Resistance and Biotechnology Laboratory Cold and Arid Regions Environmental and Engineering Research Institute Chinese Academy of Sciences, Lanzhou PR China


1. Introduction
The desert plants, widely distributed in arid and semi-arid regions, have the significant ecological functions of combating desertification and maintenance of the ecosystem stability in the desert ecological system, and also have an irreplaceable role in restoration and reconstruction of vegetation in the sand. With the development of modern molecular biology, molecular markers analysis such as amplified fragment length polymorphism (AFLP), random amplified polymorphic DNA (RAPD), simple sequence repeat (ISSR), and especially full genome sequencing technique are widely used in the study of biological diversity, genetic transformation and the conservation of genetic resources of desert plants. The isolation of high molecular weight and high purity genomic DNA is a pre-requisite for these molecular techniques. Now there are many extraction methods of genomic DNA from biomaterials. The commonly employed DNA isolation methods involve the use of enzymes such as lysozyme and proteinase K (Lockhart et al., 1989), cetytrime-thylammonium bromice (CTAB) (Ghosh et al., 2009;Moyo et al., 2008;Khanuja et al., 1999;Novaes et al., 2009;Singh et al., 2000) or sodium dodecyl sulfate (SDS) (Kaufman et al., 1999;Dellaporta et al., 1983) treatment and extraction with organic solvents, detergent-induced lysis in conjunction with proteinase K and lysozyme (Perera et al., 1994) or lysis using guanidinium isothiocyanate (GITC)-containing solutions (Boom et al., 1990;Noordhoek et al., 1995;Chakravorty & Tyagi, 2001), among which, guanidinium thiocyanate has been shown to be a powerful agent in the purification of DNA because of its potential to lyse cells and its potential to inactivate nuclease (Boom et al., 1990;Chomczynski et al., 1987;Zeillinger et al., 1993). However, high amounts of gummy polysaccharides, polyphenols and other various secondary metabolites such as alkaloids, flavonoids, terpenes and tannins in the desert plants usually hamper the DNA isolation procedures and reactions such as DNA restriction, amplification and cloning (Moyo et al., 2008;Khanuja et al., 1999;Pang et al., 2011;Zhang K., 2011;Ji & Li, 2011). The main problems encountered in the isolation and purification of high www.intechopen.com molecular weight DNA from plant species include degradation of DNA due to endonucleases and high levels of contaminants (polyphenols or polysaccharides) that coprecipitate with DNA. Endonucleases released from the vacuoles during the cell lysis process, which are co-isolated with highly viscous polysaccharide, lead to the degradation of DNA and remarkably reduce the yield of extracted DNA (Khanuja et al., 1999. Polyphenols released from the vacuoles during the cell lysis process are oxidized by cellular oxidases and undergo irreversible interactions with nucleic acids causing browning of the DNA (Varma et al., 2007;Moyo et al., 2008;Khanuja et al., 1999;Porebski et al., 1997). The presence of gelling polysaccharides prevents complete dissolution of nucleic acids and imparts a viscous constituency to the DNA making it stick to the wells during gel electrophoresis (Barnell et al., 1998;Diadema et al., 2003;Varma et al., 2007). Furthermore, inhibitor compounds like residual polyphenols, polysaccharides and other secondary metabolites inhibit enzymatic reactions such as restriction endonuclease cleavage (Raina and Chandlee, 1996) or Taq DNA polymerase amplifications (Shioda and Murakami-Murofushi, 1987;Tigst and Adams, 1992;Pandey et al., 1996) or ligase links (Moyo et al., 2008;Khanuja et al., 1999, Weishing et al., 1995. Thus, though several successful genomic DNA isolation protocols for high polyphenol and polysaccharide containing plant species have been developed, none of these are universally applicable to all plants (Varma et al., 2007), because qualitative and quantitative differences in the levels of polysaccharides, phenols and secondary metabolites in various plant tissues significantly alter the efficiency of nucleic acid extraction and purification procedures. Therefore researchers often modify a protocol or blend two or more different procedures to obtain DNA of the desired quality (Varma et al., 2007).
Reaumuria soongorica (Pall.) Maxim, an extreme xeric semi-shrub of Tamaricaceae, is a constructive and dominant species of desert shrub vegetation (Liu et al. 1982;Wang et al., 2011;Bai et al., 2008). It is distributed widely on a large area of sand wasteland (Fig. 1a) and saline land (Fig. 1b) in arid and semiarid regions of central Asia from the western Erdos, Alaskans, Hexi Corridor, Qaidam Basin to Tarim Basin and Jungar Basin (from the east to the west) and forms the vast and distinctive landscape of the salt desert (Liu and Liu, 1996). The distribution of R. soongorica in desert in northwestern China is shown in (Fig. 2) (Ma et al., 2007). It can inhabit on the alluvial plains of piedmont, hilly lands, eroded monadnocks, piedmont gravel mass, gravel alluvial fan and the Gobi. It is distributed on large span, wide range, and complex habitat where there are different climatic conditions among regions, especially with significantly different water conditions, such as the average annual rainfall in Lanzhou with 327.7 mm, Shapotou with 188.2 mm, and Ejina with 35.1 mm as it possesses the characteristics of drought resistance, salt tolerance, barrenness tolerance, and dune fixation. It is such a good candidate of desert plants that it is very significant for us to study its biological diversity and the mechanism of adverse environments resistance. However, the leaves of R. soongorica are evolved into the form of pellets suitable for arid environment, which are very hard in texture and contain high level of polysaccharides, polyphenols and secondary metabolites that co-precipitate with DNA, making DNA isolation difficult.
A good isolation protocol should be simple, rapid and efficient, yielding appreciable levels of high quality DNA suitable for molecular analysis. Krizman et al. (2006) were of the opinion that, among other factors, the amount of plant sample extracted could be critical in keeping an extraction procedure robust. In the present study, our objective was to create an improved DNA extraction procedure amenable for the isolation of high quality DNA in the desert plant R. soongorica. Four methods for extracting DNA were tested in this study and they included the TianGen Plant Genomic DNA Kit, the modified TianGen Plant Genomic DNA Kit, the modified CTAB-A method and the modified CTAB-B method herein promoted by us. The results showed that the modified CTAB-B method was a relatively quick and inexpensive method and it was the best method for extraction DNA from leaf materials containing large quantities of secondary metabolites. Furthermore, it was further tested that the modified CTAB-B method for isolating DNA from leaves of R. soongorica yields DNA in a quantity and quality suitable for PCR amplification, DNA marker analysis and restriction digestion.

Plant materials
Tender R. soongorica leaves were collected from Ejina in Mogo, China and snap-frozen in liquid nitrogen. The frozen leaves were transported in liquid nitrogen and stored at -80ºC upon reaching the laboratory.

Grinding
The frozen fresh samples were transferred into a mortar with liquid nitrogen and the ceramic pestle was pre-chilled for grinding. By providing liquid nitrogen as a cooling jacket, the samples were sprinkled with PVPP and ground vigorously to fine powder using the ceramic pestle. This powder was used for the following extraction protocols of the CTAB methods. And the commercial DNA isolation kit was ground in liquid nitrogen free of adding to PVPP.

DNA extraction
For our modified CTAB method, the steps of this protocol was carried out as follows: 1. The ground powder sample (100 mg) was transferred to 2 ml micro-centrifuge tubes filled with 700 µl of pre-warmed CTAB extraction buffer containing 35 µl βmercaptoethanol, following by incubation at 65°C for 30 min in a warm water bath. The mixture was regularly mixed three to four times by gently inversion during the incubation.. 2. 200 µl 3 mol/L sodium acetate (NaAc) (pH 5.2) was added to the incubated mixture and mixed gently by inversion and incubated on ice for 30 min.
room temperature. The upper aqueous phase was carefully collected from each sample without disturbing the interface. This step was repeated twice. 4. 2.5 volumes of absolute ethanol was added to the recovered supernatant and precipitated 30 min at -20ºC. A precipitate formed at this stage and the mixture was centrifuged at 12,000×g for 10 min at 4°C. DNA pellet was recovered by decanting the supernatant. 5. The crude nucleic pellet was dissolved in 1 ml of 1 M NaCl instead of dissolving it in Tris-EDTA (TE) buffer. The entire solution was transferred to a 2 ml microcentrifuge tube and treated with RNase at 37°C for 1 h. RNase contamination was removed by adding an equal volume of phenol (pH 5.0)/chloroform /isoamyl alcohol (25/24/1) and the aqueous phase was collected in a fresh microcentrifuge tube after centrifugation at 12,000×g for 5 min at room temperature. 6. An equal volume of chloroform/isoamyl alcohol (24/1) was added and mixed thoroughly. The samples were centrifuged at 14,000×g for 5 min at room temperature and the top aqueous phase was transferred to a fresh tube. 7. A double volume of absolute ethanol and 0.1 volumes of 3 M (pH 5.2) sodium acetate were added into the collected aqueous phase and were mixed gently by inversion. The samples were incubated at -80ºC for 30 min, followed by centrifugation at 12,000×g. 8. The DNA pellet was washed with 75% ethanol, absolute ethanol, air-dried and finally the purified DNA pellet was dissolved in 100 µl of TE buffer. and stored at -20°C.
Initial tests for DNA isolation from the leaves of R. soongorica were carried out with the modified CTAB-A method and the Plant Genomic DNA Kit (TIANGEN Biotech Co., Ltd., Beijing). The modified CTAB-A method was modified based on the classical Doyle and Doyle (1987) method. The steps of the modified CTAB-A are similar to those of the CTAB-B method before step 4 (the precipitate of crude nucleic pellet). The main difference is that the crude nucleic pellet in CTAB-A method was solved in TE and extracted by chloroform/isoamyl alcohol (24/1) again instead of being treated with DNase-free RNase. Briefly, the crude nuclei pellet was dissolved in 500 µl of TE buffer, followed by the steps 3 and 4 of the CTAB-B method repeatedly. The protocols for the commercial DNA isolation kit was performed according to the manufacturer' procedures on their website:http://www.tiangen.com/newEbiz1/EbizPortalFG/portal/html/ProductInfoExhi bit.html?ProductInfoExhibit_ProductID=c373e923ec4bc4d68f7efc2e13bcb309&ProductInfoE xhibit_isRefreshParent=false. The protocol of the modified TianGen Plant Genomic DNA Kit was based on those of the TianGen Plant Genomic DNA Kit with some slight modifications. The modifications were listed as follows: (1) The plant materials were ground free of liquid nitrogen, but were added to the cooled sterile mortar and ground with Gp1 buffer poured into the mortar.
(2) The ground tissue was transferred to 2 ml micro-centrifuge tubes prepared a warm (65°C) Gp1extraction buffer, and then the 5% beta-mercaptoethanol and 10 µl DNase-free RNase were added to the mixture immediately and mixed gently by inversion. The other steps are carried out by the instructions of the kit. For each method, three independent experiments were done, and three samples were prepared in each independent experiment.

Testing the quality of the genomic DNA
Three microliters of each genomic DNA sample is examined by electrophoresis and remnant DNA sample is stored at −70°C. Mixing 1 µl of 5× DNA loading buffer (TIANGEN Biotech www.intechopen.com (Beijing) Co. Ltd.) with 3 µl of genomic DNA at room temperature for 1 min. Then the sample was loaded on 0.8% agarose formaldehyde denaturing gels stained with ethidium bromide (EtBr) (Biotechnology grade, Amresco), and run on gels in the 1× formaldehyde electrophoresis buffer at 5-7 V/cm.

Assessment of the purity and the yield of the genomic DNA
Two microliters of each genomic DNA sample was diluted into 200 µl of sterilized ultra pure water (pH 7.0). The absorbance of each diluted genomic DNA sample was evaluated at 260 and 280 nm using a ND-2000C (Thermo, America). The yield of genomic DNA was calculated according to the formula: DNA yield = 50 × OD 260 × dilution factor × volume of sample in milliliters/material weight (g). Measured the values at the wavelengths of 260, 280 nm and 230 nm and calculated the ratios of A260/A280 and the A260/A230.

DNA isolation methodology
Commercial DNA isolation kits are widely used for their single-step methods and the relatively short amount of time required (usually about 1-2 h). These kits have also proven effective for isolating DNA from common plants such as rice, barley and Arabidopsis. We first attempted to isolate DNA from the leaves of R. soongorica using three commercial DNA isolation kit: the Plant Genomic DNA Kit (TIANGEN Biotech Co., Ltd., Beijing) which is designed specifically to extract DNA from plant tissues rich in secondary metabolites. In our hands, this kit was not able to isolate any DNA from the leaves of R. soongorica (data not shown). Then, we carried out the improved kit method according to the suggestion of this company' technical assistance employee, which yielded a small amount of DNA, but it was seriously contaminated (Fig. 3, lanes 1,2). The failure of the kit may be explained by the DNA likely formed a sticky, a glue-like gel in complex with these secondary metabolites and this could not be properly separated into two phases by centrifugation.
During the course of the RNA isolation, none of the kits were able to isolate any RNA from the leaves of R. soongorica (Wang et al. 2011), so we did not try more other commercial DNA kits to isolate DNA, but attempted to use a improved CTAB method to extract DNA from the leaves of R. soongorica. When we carried out the modified CTAB-A method based on the classical Doyle and Doyle (1987) method, which consistently resulted in significant RNA contamination of the DNA samples (Fig. 3, lanes 3,4 ). To remove RNA contaminants, additional purification steps must be performed, which not only reduce DNA yield but also increase the time required for DNA extraction. Fig. 3. Electrophoretic analysis of R. soongorica genomic DNA isolated using various extraction methods. The genomic DNA (3 µl) of each sample was loaded into the different well, and then was run on a 0.8% agarose gel stained with ethidium bromide in 1×TAE buffer. Marker indicates the Molecular weight marker -1 kb DNA ladder. Lanes 1 and 2, the modified TianGen Plant Genomic DNA Kit; lanes 3 and 4, the modified CTAB-A method; lanes 5 and 6, our promoted CTAB-B method. As the TianGen Plant Genomic DNA Kit was able to isolate any DNA from the leaves, the photos were not shown in Fig. 3.
The present study was motivated by the need for better methods of extracting sufficient quantities of high-quality DNA from plant tissue rich in secondary metabolites for use in molecular marker assays. The promoted CTAB-B protocol described here efficiently eliminates most of the interfering molecules, including polyphenols, polysaccharides, and proteins, and it yields translucent and water-soluble DNA pellets without RNA contamination. The main protocols made in this method were grounding PVPP together with the plant material, an increase in the volume of high salt extraction buffer, adding 3 mol/L sodium acetate (NaAc) in extraction buffer, dissolving the crude pellet in 1 M NaCl followed by RNase treatment, the purification of acid phenol extraction (phenol: chloroform: isoamylal alcohol (PCI)=25:24:1) and the use of pre-cooled ethanol and sodium acetate in precipitation and all these modifications helped to remove the interference of secondary metabolites in the DNA isolation. PVPP was sprinkled directly onto the frozen fresh leaf tissue in the mortar and vigorously ground with the leaf tissue in the presence of liquid nitrogen, which can avoid the oxidation of released polyphenols into www.intechopen.com quinines, which in turn bind to nucleic acids and hinder the isolation of high quality DNA. Increase in the volume of extraction buffer can completely break down the cell walls and make more nucleotide acid released resulted in increasing the yield of DNA isolation. Krizman et al. (2006) postulated that the plant tissue amount per volume of extraction buffer has an effect on DNA quality and yield. Since the extraction buffer is responsible for the lysis of membranes and liberation of DNA from cellular organelles (Weising et al., 2005), the smaller the quantity of plant tissue per unit volume, the more optimal the lysis process. Striking the correct balance between plant tissue amount and extraction buffer volume would reduce the probability of co-precipitation of contaminants with the DNA pellet as the saturation concentration during precipitation is less likely reached or exceeded (Krizman et al., 2006). During the extraction, 3 mol/L sodium acetate (NaAc) added combined with chloroform/isoamyl alcohol extraction can reduce markedly the coprecipitation of polysaccharides with the nucleic acids and remove most proteins, polysaccharides, polyphenols and other impurities for the first time. The crude nucleic pellet was dissolved in 1 ml of 1 M NaCl instead of dissolving it in Tris-EDTA (TE), which ensured further reduction of viscosity of the mucilaginous substances (Chen and Chen, 2004;Ghosh et al., 2009). DNase-free RNase was added to crude DNA samples dissolved in1 ml of 1 M NaCl to completely clear residual RNA. After RNase treatment, the DNA solution requires purification with an acid-phenol: chloroform : isoamyl alcohol (25: 24: 1) extraction because small amounts of protein in DNA pellets and salts in the RNase reaction buffer and stop solution both influence downstream molecular procedures such as restriction endonuclease digestion, ISSR-PCR amplification and full the genomic sequencing. Thus, we used acidphenol to remove residual protein and the remaining salts after the RNase treatment. Finally, it is necessary to precipitate DNA simultaneously with sodium acetate (pH 5.2) and absolute ethanol which can completely remove the residual polysaccharides from the DNA sample resulted in increasing the yield of DNA isolated.

Assessment of the quality and quantity of the total DNA
The success of an DNA isolation protocol may be judged by the quality and quantity of DNA recovered. The quality of DNA was assessed by gel electrphoresis, spectrophotometry, restriction endonuclease digestion and PCR amplification. The mean yield of DNA extracted by our promoted method was approximately 60.29 ± 20.16 µg/100mg of fresh leaves, which was higher than that of the modified CTAB-A method (35.72 ± 15.41 µg/100mg) and the modified TianGen Plant Genomic DNA Kit (20.54 ± 8.43 µg/100mg) ( Table 1). The DNA isolated by our promoted method also exhibited good purity. DNA absorbs UV light maximally at 260 nm, whereas protein absorbs at 280 nm and other contaminants including carbohydrates, phenol, and aromatic compounds generally absorb around 230 nm. Therefore, the A260/A280 and the A260/A230 ratios are often used as indicators of DNA sample purity. Generally, ratio values of A260/A280 in the range of 1.8-2.0 indicate highpurity DNA; the ratio values of A260/A280 less than 1.8 indicate protein contamination in DNA samples; the ratio values of A260/A280 more than 2.0 indicate much RNA or many DNA fragments in DNA samples. With our method, the A260/A280 and A260/A230 ratios were 1.86 ± 0.16 and 1.92 ± 0.13, respectively, indicating that the DNA was free of protein and polysaccharides/polyphenol contamination (Table 1). In addition, there were no other bands visible in the bands (Fig. 1, lanes 5,6), indicating that the DNA was free of genomic RNA contamination. In contrast, the DNA from the CTAB-A method had poor purity as assessed by A260/A280 (2.12 ± 0.18) and the A260/A230 ratios (1.55 ± 0.36 µg/100mg), and the yield was lower than that obtained with our promoted CTB-B method and the DNA was badly contaminated with RNA because there were two other visible bands on the lane (Fig.  1, lanes 3,4; Table 1). The TianGen Plant Genomic DNA Kit was not able to isolate any DNA from the leaves of R. soongorica. Furthermore, even the improved kit method only yielded a small amount of DNA (20.54 ± 8.43 µg/100mg), and it was seriously contaminated (Fig. 1,  lanes 1,2 ). The above results show that the improved CTAB-B protocol described herein efficiently eliminates most of the interfering molecules (including polyphenols, polysaccharides, proteins and salts), and it also provides a higher yield of DNA pellets that are translucent, water-soluble and lack RNA contamination, indicating that it is superior to the CTAB-A method and the commercial kits.  Fig. 4. ISSR-PCR profiles of the genomic DNAs isolation from different R. soongorica populations by our promoted CTAB-B protocol using the UBC-807 primer were analyzed on a 1.5% agarose gel stained with ethidium bromide in 1×TAE buffer. "M" represents the Molecular weight marker -DNA marker DL2000.
The suitability of extracted DNA for downstream molecular processes was further verified by molecular markers ISSR-PCR amplification. AS shown in Fig. 4, the genomic DNA of five different R. soongorica populations were highly amplifiable by ISSR-PCR as indicated by the amplification products resolved on 1.5% agarose gel. This further confirmed the purity of DNA, free of polysaccharide and polyphenol contamination, which would otherwise inhibit Taq DNA polymerase and restriction endonucleases (Ahmad et al., 2004). Plant molecular applications such as RAPD and AFLP necessitate the successful isolation of high quality DNA (Michiels et al., 2003;Ahmad et al., 2004), devoid of contaminants. Without high quality DNA such downstream molecular manipulations are not feasible (Varma et al., 2007). To confirm the applicability of our method, this DNA extraction method has also been found to be efficient in other desert plants, including Tamarix ramosissima, Nitraria tangutorum and Caragana korshinskii Kom. (data not shown).

Conclusion
Our results showed that the modified CTAB-B method promoted here was of high quality, purity and yield and was suitable for downstream molecular assays. Based on a CTAB method, the protocol has been improved as follows: the more volume of extraction buffer was used to completely break down the cell walls; the samples were ground with PVPP to effectively inhibit the oxidation of phenolics; during the extraction, 3 mol/L sodium acetate (NaAc) was added to reduce markedly the co-precipitation of polysaccharides with the nucleic acids; contaminating RNA was removed with RNase I; acid phenol extraction (phenol: chloroform: isoamylal alcohol (PCI)=25:24:1) was used to effectively removes the residual proteins and inhibitors in the RNase reagent. Thus, despite the high levels of secondary metabolites in the leaves of R soongorica, the high quality DNA is isolated from the nuclei without interference. Moreover, the new protocol is also suitable for isolating genomic DNAs from other desert plant species and tissues that are rich in secondary metabolites.

Acknowledgments
This study was supported by the National Key Technologies R&D Program: The Integration and Experiment Demonstration of Ecological Restoration Technology in Loess Hills-Sand Areas (2011BAC07B05) and by the Ministry of Forestry Commonweal Special Project: Monitoring and Assessment Technologies for Hydrological Regulation Function of Desert Ecosystem (201004010-05).