Plant Molecular Biology Reporter 17: 1–8, 1999. © 1999 Kluwer Academic Publishers. Printed in the Netherlands.

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An Improved and Rapid Protocol for the Isolation of Polysaccharide- and Polyphenol-Free Sugarcane DNA S. M. ALJANABI∗, L. FORGET and A. DOOKUN Biotechnology Department, Mauritius Sugar Industry Research Institute, Reduit, Mauritius

Abstract. We have optimized a simple and rapid method for isolating milligram quantities of high quality DNA from polysaccharide- and polyphenolic- rich tissue such as sugarcane, lettuce and strawberry. The protocol utilizes fresh tissue without making use of liquid nitrogen or freeze-drying for initial grinding of the tissue and it significantly minimizes the use of lab materials. At least one hundred samples can be processed daily by one person. The isolated DNA is essentially free of polysaccharides, polyphenols, RNA and other major contaminants, as judged by: its clear color, its viscosity, A260/A280 ratio, digestibility with restriction enzymes, and suitability for Restriction Fragment Length Polymorphism (RFLP)and Polymerase Chain Reaction (PCR)- based techniques. Key words: DNA extraction, polyphenols, polysaccharide, RAPD, Saccharum, sugarcane

Introduction Sugarcane is one of the leading crops in the world, producing 70% of the world’s sugar, which amounted to 126 M tons in 1997–1998. This production level closely matches world sugar consumption for the same period. Any step that can add to the understanding of sugarcane genome organization and function will assist in increasing sugar production worldwide. Like many other plant species, sugarcane tissues contain high levels of polysaccharides and polyphenolic compounds, which present a major contamination problem in the purification of plant DNA. When cells are disrupted, these cytoplasmic compounds can come into contact with nuclei and other organelles (Loomis, 1974). In their oxidized forms, polyphenols covalently bind to DNA giving it a brown color and making it useless for most research applications (Katterman and Shattuck, 1983; Guillemaut and ∗ Author for correspondence. e-mail: [email protected]; fax: 230-454-1971;

ph: 230-454-1061.

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Maréchal-Drouard, 1992; our observations). One method commonly used to avoid problems with polyphenols involves freezing the tissue during or prior to homogenization (Katterman and Shattuck, 1983; Leutwiler et al., 1984). The presence of these compounds renders studies difficult due to long and tedious extraction procedures and often does not result in good standards in terms of yield and quality. The need for a rapid and efficient procedure for plants having high polysaccharides and polyphenols is necessary when hundreds of samples need to be analyzed rapidly, such as in genome mapping and marker assisted selection (MAS) programs. High purity DNA is required for PCR and restriction-based techniques such as random amplified polymorphic DNA (RAPD), for microsatellite, RFLP and amplified fragment length polymorphism (AFLP), for genome mapping and DNA fingerprinting. Several protocols have been described for plants containing high amounts of polyphenols and polysaccharides including extraction of DNA from isolated nuclei (Hamilton et al., 1972) and purification by cesium chloride following the classic plant DNA protocol (Murray and Thompson, 1980) using liquid nitrogen. Although these methods yield DNA of high purity, they are tedious and require expensive reagents and equipment such as high-speed or ultracentrifuges. Also, liquid nitrogen may be difficult to obtain in certain regions of the world where most species of Saccharum are found. A rapid DNA extraction method for sugarcane and its relatives (Honeycutt et al., 1992) and recently, a modification of a CTAB DNA extraction protocol for plants containing high polysaccharide and/or polyphenol components (Porebski et al., 1997; Peterson et al., 1997), have been published. These methods give a low yield of DNA and the procedures are relatively long, thereby limiting the number of samples that can be processed per day. In addition, expensive reagents and tedious processes were used in these protocols, such as RNase, spermidine, proteinase K and the filtration of the ground tissue through cheesecloth and/or mira-cloth. In our research, we need large quantities of highly pure sugarcane genomic DNA for genome mapping and marker-assisted selection (MAS). After trying published protocols and failing to obtain DNA that was not contaminated with polysaccharides and polyphenolic compounds, we have optimized an alternative rapid method that yields polysaccharide and polyphenol-free high quality genomic DNA from the meristem cylinder of sugarcane. It also produced high quality DNA from mature fresh leaves of strawberry and lettuce. These tissues contain high levels of polysaccharides and polyphenols and were included for comparison.

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Materials and methods Five hundred sugarcane hybrids, their parents, five strawberry and five lettuce plants were used for DNA extraction. Plant samples were transferred from the field to the laboratory in plastic bags without the need to place them on ice and they were processed on the same day. A modification of the Doyle and Doyle (1990) CTAB extraction procedure was adopted, which uses a higher CTAB and NaCl concentration to remove polysaccharides (Lodhe, et al., 1995), thereby preventing their interaction with DNA. PVP and sodium sulfite were also added to prevent the oxidation of phenolic compounds that results in brown colored DNA (Loomis, 1974). All the chemicals used in this experiment were from Sigma Chemical Co., St. Louis MO, USA. Solutions Homogenization buffer: 200 mM Tris-HCl, 50 mM EDTA, 2.2 M NaCl, 2% CTAB, 0.06% sodium sulfite1 , pH 8.0 phenol:chloroform:isoamyl alcohol (25:24:1) 6 M NaCl 10% polyvinylpyrrolidone (PVP) 5% N-lauroyl-sarcosine 20% CTAB Protocol •

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Slice about 10 cm (2–4 g) of sugarcane fresh meristem cylinder (after removing outer leaf sheaths) and place in 50 mL Falcon tubes. Similarly, slice fresh lettuce and strawberry leaves to about 10 mm2 and place in Falcon tubes. Homogenize the fresh tissue with homogenization buffer (4 mL/g fresh tissue) using an Ultra-Turax homogenizer with a large dispersing element, for a few seconds2 . Add 2 mL of 5% N-lauroyl-sarcosine, 2 mL of 10% PVP3 and 2 mL of 20% CTAB. Mix well by inversion. Incubate for 30–60 min at 65 ◦ C in a water bath. Mix samples by inversion 3–4 times during incubation. Take samples from the water bath and cool down to room temperature. Add an equal volume of 25:24:1 phenol:chloroform:isoamyl alcohol, mix by inversion, then centrifuge the tubes at 3000 g for 10 min at 4 ◦ C. Recover aqueous phase4 and transfer to a fresh tube. Add an equal volume of isopropanol followed by 2 mL of 6 M NaCl. Incubate at −20 ◦ C for at least 1 h.

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Figure 1. Agarose gel electrophoresis of total uncut and Hind III digested genomic DNA isolated as described in materials and methods. Five to ten µg of sugarcane hybrids (Saccharum ssp., lanes 2–5), strawberry (Fragaria sp., lanes 6–7) and lettuce (Lactuca sativa. Lanes 8–9) were loaded in each lane. Lane 1, 100 bp molecular weight marker XIV (Boehringer Mannheim).

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Fish out the DNA with a glass pipette and transfer to a fresh tube containing 10 mL of 70% ethanol. Alternatively, spin down the DNA pellet briefly, wash with 10 mL of 70% ethanol, air dry, then resuspend in 2– 3 mL of TE (10 mM Tris-HCl, 1 mM EDTA, pH 8.0).

Notes Prepare just before use. Longer homogenization time is not recommended as it can result in partial degraded DNA. 3. PVP adsorbs polyphenols thereby preventing their interaction with DNA (Loomis, 1974); sodium sulfite inhibits oxidation of polyphenols. 4. Avoid disturbing the interface while pipetting the aqueous phase. 1. 2.

Results and Discussion The protocol we used is a modification of Doyle and Doyle (1987) where fresh leaf tissue is homogenized in an extraction buffer without making use of liquid nitrogen or filtration of the homogenate through cheese cloth or mira-cloth. In addition no RNase treatment is required, as RNA seems to be degraded during extraction. This significantly reduces sample handling time and wasting of laboratory materials, especially when hundreds of samples need to be processed. Two 50 mL Falcon tubes per sample are needed to finish the extraction of the DNA.

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To check the DNA quality, 5–10 µg of uncut and Hind III digested genomic DNA from each sample were run on a 1% agarose gel as shown in Figure 1. There was neither RNA contamination nor any sign of degraded DNA in all samples. There was no RNase treatment of any DNA sample when the first batch of samples showed only traces of RNA or none at all on the agarose gel. It seems that RNA is degraded during the process of the extraction. Compounds that adsorb polyphenols or prevent oxidation reactions inhibit the interaction of genomic DNA with oxidized polyphenols, which affects DNA quality. The purity of the DNA was determined from the A260/A280 ratio, which averaged 1.76–1.96 in all samples. The yield of DNA ranged from 0.5 to 0.8 mg/g of fresh weight. This amount of DNA is sufficient to perform hundreds of RFLP experiments, which is still the method of choice in many laboratories, and thousands of PCR-based techniques such as RAPD, AFLPTM and simple sequence repeats (SSR) analyses. Figure 1 also shows that the DNA was completely digested with Hind III. Equivalent results were obtained with the 3 other enzymes and all other sugarcane DNA samples (data not shown). The digested DNA was subjected to Southern blot analyses using the non-radioactive Dig-labeling system (Boehringer Mannheim, Germany). The quality of the Southern blot analysis using the sugarcane DNA extracted by the above method is evident in Figure 2. RAPD analysis of genomic DNA was performed, according to Williams et al. (1990), using 10–30 ng genomic DNA, 200 µM each dNTP, 2 units of Stoffel fragment Taq polymerase (Perkin-Elmer USA), 1× Taq polymerase reaction buffer (supplied by the manufacturer) and 0.22 µM 10-mer random primer (Operon Technologies, USA). Two hundred and forty primers were screened against the parent’s DNA and the ones that detected polymorphisms were selected for screening of the segregating population. Figure 3 shows an example of RAPD analyses of sugarcane DNA, which indicates that the DNA extracted by our method is suitable for PCR-based techniques. Unlike other protocols mentioned above, our protocol does not make use of expensive chemicals or equipment. Homogenization of fresh tissue was carried out inside a 50 mL Falcon tube while other procedures used a mortar and pestle to grind fresh tissues in the presence of liquid nitrogen. A sterile mortar needs to be used for each sample to prevent cross contamination. This becomes impractical when hundreds of samples need to be processed. Unlike other DNA extraction protocols, our method is consistent and produces large amounts of high quality DNA necessary for studies requiring relatively large quantities of DNA, such as RFLP. The extracted DNA was stable and could be amplified by PCR both before and after 12 months of storage at 4 ◦ C.

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Figure 2. Southern blot analysis of sugarcane genomic DNA using the non-radioactive Dig-labeling system. Ten µg of Hind III digested genomic DNA of the parents (R570 and M555/60) and their progeny (M24-1 to M24-17) were loaded on an 0.8% agarose gel then transferred to a nylon membrane and hybridized with the probe CDSR29.

Acknowledgements The authors would like to thank Dr L. J. C. Autrey and Dr S. Saumtally for critical review of the manuscript. Part of this work was funded by the Tertiary Education commission.

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Figure 3. RAPD fingerprints of genomic DNAs from 48 sugarcane hybrids using primers OPB-18 (50 -CCA CAG CAG T-30 ) and 2 units of Stoffel fragment Taq DNA polymerase. The amplification program included an initial denaturation step of 94 ◦ C for 3 min followed by 39 cycles of 94 ◦ C, 1 min; 35 ◦ C, 1 min and 72 ◦ C, 2 min. The last cycle was followed by 5 min incubation at 72 ◦ C and a hold at 15 ◦ C. The amplification products were resolved on a 2% agarose gel dissolved in 0.5× TBE, stained with ethidium bromide and visualized under UV light. M, molecular weight marker X (Boehringer Mannheim).

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Murray MG and Thompson WF (1980) Rapid isolation of high molecular weight plant DNA. Nucleic Acids Res 8: 4321–4325. Peterson DG, Boehm KS, and Stack SM (1997) Isolation of milligram quantities of nuclear DNA from tomato (Lycopersicon esculentum), a plant containing high levels of polyphenolic compounds. Plant Mol Biol 15: 148–153. Porebski, S, Baily LG, and Baum BR (1997) Modification of a CTAB extraction protocol for plant containing polysaccharide and polyphenol components. Plant Mol Biol Reptr 15: 8–15. Williams JGK, Kubelik AR, Livak KJ, Rafalski JA, and Tingey SV (1990) DNA polymorphisms amplified by arbitrary primers are useful as genetic markers. Nucleic Acids Res 18: 6531–6535.