**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 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

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

*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

(Varma et al., 2007).

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.

Fig. 1. The natural habitats of *Reaumuria soongorica*. a Populations of *R. soongorica* in sand wasteland or Go; b Populations of *R. soongorica* in saline land with a white visible salt on the ground

Fig. 2. Distribution map of the study plant *Reaumuria soongorica* in northwest China (the triangle symbol indicates the major distribution area)
