A Simple and Efficient Method for DNA Extraction from ...

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We wish to thank John C. Sanford and Susan K. Brown for their critical ... Plant Cell Rep. 8:137-140. Baribault, T.J., K.G.M. Skene, P.A. Cain and N.S. Scott. 1990 ...
Plant Molecular Biology Reporter 12 (1) 1994

pages 6--13

Commentary

A Simple and Efficient Method for DNA Extraction from Grapevine Cultivars and Vitis Species Muhammad A. Lodhi, Guang-Ning Ye, Norman F. Weeden, and Bruce I. Reisch Department of Horticultural Sciences, New York State Agricultural Experiment Station, Comell University, Geneva, NY 14456, USA Key Words: DNA extraction, Vitis, polyphenols, polysaccharides, RAPD, restric-

tion digestion. Abstract: A quick, simple, and reliable method for the extraction of DNA from

grapevine species, hybrids, and Ampelopsis brevipedunculata(Vitaceae) has been developed. This method, based on that of Doyle and Doyle (1990), is a CTABbased extraction procedure modified by the use of NaC1 to remove polysaccharides and PVP to eliminate polyphenols during DNA purification. The method has also been used successfully for extraction of total DNA from other fruit species such as apple (Malusdomestica),apricot (Prunusarmeniaca),cherry (Prunus avium), peach (Prunus persica), plum (Pmmus domestica), and raspberry (Rubus idaeus).DNA yield from this procedure is high (up to I mg/g of leaf tissue). DNA is completely digestible with restriction endonucleases and amplifiable in the polymerase chain reaction (PCR), indicating freedom from common contaminating compounds.

V

itis vinifera and related species have been the subject of extensive

genetic studies due to their worldwide cultivation and importance. Recently this plant has been used for gene m a p p i n g (Yamamoto et al., 1991; Mauro et al., 1992; Weeden et al., 1992; Lodhi et al., 1992ab; 1993; Hain et al., 1993), genetic transformation (Baribault et al., 1989; Baribault et al., 1990; H6bert et al., 1993), and D N A fingerprinting (Striem et al., 1990; Bourquin et al., 1991; Collins and Symons, 1993). The relatively small genome size of V. vinifera (0.50 p g / C ) compared to

Abbreviations: CTAB, cetyltrimethylammoniumbromide.

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many other perennial plant species (Arumuganathan and Earle, 1991) should facihtate molecular genetic studies of Vitis. DNA extraction from grapevine has, however, been difficult due to the presence of contaminants such as polyphenols and polysaccharides. These compounds have also been reported to cause difficulty in DNA purification in other plant species: polysaccharides (Murray and Thompson, 1980; Fang et al., 1992), polyphenohc compounds (Katterman and Shattuck, 1983; Couch and Fritz, 1990; Howland et al. 1991; Collins and Syrnons, 1992), and sticky and resinous materials (Webb and Knapp, 1990). The presence of these contaminants in DNA preparations often makes the samples viscous and renders DNA unrestrictable in endonuclease digestion and unamplifiable in PCR. The existing DNA extraction protocols often produce unsatisfactory yields a n d / o r quality (Bourquin et aI., 1991; Collins and Symons, 1992). Here we report a simple, inexpensive, and quick procedure for the extraction of DNA from grapevine Vitis species, their cultivars, and A. brevipedunculata. This procedure purifies greater amounts of clean DNA which can be amplified via PCR or digested with endonucleases.

Materials a n d M e t h o d s Plant Material See Table I for the source of plant material used in this study. Solutions

extraction buffer: 20 mM sodium EDTA and 100 mM tris-HC1; adjust pH to 8.0 with HC1, add 1.4 M NaC1 and 2.0% (w / v) CTAB. Dissolve CTAB by heating to 60~ Store at 37~ Add 0.2% of ~-mercaptoethanol just before use. chloroform:octano124:1 (v/v) 5 M NaC1 TE buffer: 10 mM Tris-HC1 and 1 mM EDTA, adjust pH to 8.0 and autoclave RNase A (Sigma R9009:10 m g / m L )

Protocol 9 Collect unexpanded young leaves in liquid nitrogen or on ice and store at or below -70~ until used. Grind 0.5 g of leaves using mortar and pestle in the presence of liquid nitrogen. 1 9 Add 5 mL of extraction buffer to the ground leaves and mix in the mortar.

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9 Pour the slurry into clean 15-mL polypropylene centrifuge tubes. 9 Add 50 mg polyvinylpolypyrrolidone (PVP), (Sigma, P6755) and invert the tubes several times to mix thoroughly with the leaf slurry; the final concentration of PVP is 100 m g / g leaf tissue. 9 Incubate at 60~ for 25 minutes and cool to room temperature. 9 Add 6 mL of chloroform-octanol and mix gently by inverting the tubes 20 to 25 times to form an emulsion. 9 Spin at 6000 rpm for 15 minutes in a tabletop centrifuge at room temperature. 9 Transfer the top aqueous phase to a new 15-mL centrifuge tube with a wide-bore pipette tip. A second chloroform-octanol extraction may be performed if the aqueous phase is cloudy due to the presence of PVP. 9 Add 0.5 volume of 5M NaC1 to the aqueous solution recovered from the previous step and mix well. 9 Add two volumes of cold (-20~ 95% ethanol and refrigerate (4 to 6~ for 15-20 minutes or until DNA strands begin to appear. The solution can be left for one hour or more if necessary. 9 Spin at 3000 rpm for three minutes and then increase speed to 5000 rpm for an additional three minutes at room temperature. 2 9 Pour off supernatant and wash pellet with cold (0 to 4~ 76% ethanol. Completely remove ethanol without drying the DNA pellet by leaving the tubes uncovered at 37~ for 20 to 30 minutes. 9 Dissolve in 200 to 300 ~tL TE. 9 Treat with I ~tL RNase A per 100 ~tL DNA solution and incubate at 37~ for 15 minutes. 9 Quantify DNA in a spectrophotometer at A260. 9 Keep DNA at -70~ for long-term or -20~ for short-term storage. Notes

1. Avoid thawing before grinding the leaf tissue. Although leaves should be thoroughly crushed before adding extraction buffer, it is important not to grind the leaves into a very fine powder, as it results in shearing of DNA. 2. This differential spin helps to keep DNA at the bottom of the centrifuge tube.

Results and Discussion We have obtained higher yields of clean DNA from grapevine leaves by using the modified DNA extraction procedure outlined above. The procedure used for DNA extraction is CTAB-based and is modified from Doyle and Doyle (1990). NaC1 has been used to remove polysaccharides (Fang et al., 1992), and PVP to purge polyphenols (Maliyakal, 1992). This procedure does not involve centrifugation in CsC1 gradients.

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Table I. Sources and DNA yield of the plant material used for DNA extraction.

Genotype

Vitis 'Aurore' Vitisa acerifolia Vitis berlandieri Vitis cinerea Vitis labrusca Vitis rupestris Vitis vinifera cv. Cabernet Sauvignon Ampelopsis brevipedunculata Malus domestica (apple) cv. Red Delicious Prunus armeniaca (apricot) NY 500 Prunus avium (cherry) NY 6476 Prunus persica (peach) cv. Rutgers Red Leaf Prunus domestica (plum) NY 65.363.1 Rubus idaeus (raspberry) NY 83

Source NYSAESa USDA, ARSb USDA, ARS USDA, ARS UDSA, ARS USDA, ARS NYSAES USDA, ARS NYSAES NYSAES NYSAES NYSAES NYSAES NYSAES

DNA yield (~g/g leaf) 1,130 +167c 914 +427c 1,040 +39.6r 796 +153r 542 +60.1c 594 +116c 546 +19.8r 850 +_48.1c 830 935 665 805 1,055 1,135

aNYSAES (New York State Agricultural Experiment Station, Geneva, New York) bUSDA, ARS (National Clonal Germplasm Repository, Geneva, New York) xaverage of two extractions

DNA yields from species of Vitis, Ampelopsis, and other woody perennials by our procedure range from 0.5 to 1.0 m g / g fresh leaf tissues with A260/A2s0between 1.8 and 2.0 (Table I). The procedure is fast and simple, and 30 to 40 DNA samples may be processed in a single day. Results of DNA restriction digestion with three endonucleases (Eco RI, Eco RV and Hind 111) showed complete digestion. It is also evident that the uncut DNA exhibits little shearing and is suitable for Southern (1975) hybridization. The DNA is also amplifiable in PCR using the RAPD technique (Williams et al., 1990) (Fig. 1). Proper choice of the leaf tissue is very important for DNA extraction. The use of very young leaf tissues has resulted in poor yields. We found that partially expanded leaves are the best material. This is consistent with the results reported by Mauro et aL (1992), in which the best results were obtained from rapidly expanding leaves, one to two nodes from the shoot tip. With fully expanded leaves the yield was low and the DNA was not completely digestible. We were, however, able to get equally good results with fully expanded leaves when PVP was added to the extraction buffer. PVP has been used to remove polyphenols from mature, dam-

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Fig 1. PCR amplification of DNA from different species of grape and A. brevipedunculata. M, 100-bp DNA ladder; lanes 2-8: amplification with the 10base oligonucleotide K5 (CGCAGGATGG). Ab, A. brevipedunculata; Va,V. acerifolia; V1, V. labrusca; Vc,V. cinerea; Vv,V. vinifera cv. Cabernet Sauvignon; Vb, V. berlandieri; A, Vitis 'Aurore.' Lanes 5-15: amplification with the 10-base oligonucleotide, OD-8 (GTGTGCCCCA) shown in the same order as above. Approximately 50 ng DNA were amplified in each as described in the text, the amplification products separated by agarose-gel electrophoresis, and stained with ethidium bromide.

aged and improperly stored leaf tissues (Rogers and Bendich, 1985; Doyle and Doyle, 1987; Howland et al., 1991). PVP forms complex hydrogen bonds with polyphenolic compounds that can be separated from DNA by centrifugation (Maliyakal, 1992). Interference by p olyphenolic compounds can be reduced by keeping plant material frozen before extraction and by using PVP in the DNA extraction procedure. The

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developmental stage of the plant is also important. The optimal time for leaf collection was during the period of active shoot elongation following bud break. Later in the season DNA extraction was difficult and the DNA obtained was unstable for long-term storage. Complete digestion with restriction endonucleases and amplification in PCR indicate the absence of polysaccharides. Polysaccharides are difficult to separate from DNA (Murray and Thompson, 1980). These compounds are easily identifiable in the DNA preparations as they impart a sticky, viscous consistency to the DNA preparations dissolved in TE buffer. Polysaccharides interfere with several biological enzymes such as polymerases, ligases and restriction endonucleases (Shioda et al., 1987; Richards, 1988). We found that when polysaccharides were not removed the DNA would not amplify. PCR amplification of the DNA with Several ten-base-long oligonucleotides and complete DNA restriction results are consistent with these results. DNA amplification was possible due to the absence of contaminants (Webb and Knapp, 1990; Fang et al., 1992). Fang et al. (1992) found that I M NaC1 facilitated the removal of polysaccharides by increasing their solubility in ethanol so that they did not co-precipitate with the DNA. However, we found higher concentrations of NaC1 (more than 2.5 M) were more effective with the species under study. The simplicity of the procedure makes it very practical for DNA extraction especially from Vitis species, various hybrids, and A. brevipedunculata and generally from other plant species such as apple, apricot, peach, plum, and raspberry. Moreover, DNA yield is higher compared with other procedures used for DNA extraction from grapevines: the yields of DNA per g fresh weight reported by Bourquin et al. (1991) was 5 to 20 ~tg;Collins and Symons (1992), 10 to 30 ~tg;and Thomas et al. (1993), 25 to 150 ~tg. Doyle and Doyle (1987) reported DNA yields up to 1 m g / g of fresh leaf tissues from different plant species and this procedure was used by Mauro et al. (1992) for extraction of grapevine DNA. We have not been able to obtain such a high yield when this procedure was used on grapevine (data not shown). However, we found that DNA extracted by that procedure was occasionally brownish in color and difficult to digest with restriction endonucleases. Such samples also were found to have a shorter storage life. Likewise, the procedure of Doyle and Doyle (1987) gave similar results for different Vaccinium sp. (Rowland and Nguyen, 1993). Our modification of Doyle and Doyle (1990) consistently produces high-quality DNA that remains usable for at least two years when stored at -20~

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A c k n o w l e d g m e n t s : We are t h a ~ to Philip L. Forsline, USDA, ARS, Plant Genetic Resources Unit, Geneva, N e w York for p r o v i d i n g us with Vitis species p l a n t material, Robert L. A n d e r s e n for cherry, apricot, p l u m , a n d peach, a n d Kevin E. M a l o n e y for raspberry. We wish to thank John C. Sanford a n d Susan K. Brown for their critical reviews a n d valuable suggestions.

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Rogers, S.O. and A.J. Bendich. 1985. Extraction of DNA from milligram amounts of fresh, herbarium and mummified plant tissues. Plant Mol. Biol. 5:69-76. Rowland, L.J. and B. Nguyen. 1993. Use of polyethylene glycol for purification of DNA from leaf tissue of woody plants. BioTechniques 14:734-736. Shioda, M. and K. Marakami-Muofushi. 1987. Selective inhibition of DNA polymerase by a polysaccharide purified from slime of Physarumpolycephalum.Biochem. Biophys. Res. Commun. 146:61-66. Southern, E.M. 1975. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J. Mol. Biol. 98:503-517. Striem, M.J., P. Spiegel-Roy, G. Ben-Hayyim, J. Beckmann and D. Gidoni. 1990. Genomic fingerprinting of Vitis vinifera by the use of mulfi-loci probes. Vitis 29:223-227. Thomas, M.R., S. Matsumoto, P. Cain and N.S. Scott. 1993. Repetitive DNA of grapevine: classes present and sequences suitable for cultivar identification. Theor. Appl. Genet. 86:173-180. Webb, D.M. and S.J. Knapp. 1990. DNA extraction from a previously recalcitrant plant genus. Plant Mol. Biol. Rep. 8:180-185. Weeden, N.F., G.M. Timmerman, M. Hemmat, B.E. Kneen and M.A. Lodhi. 1992. Inheritance and reliability of RAPD markers. In: Proceedingsof the Joint Plant BreedingSymposium Series.Applicationsof RAPD Technologyto Plant Breeding.Crop Science Society of America, American Society for Horticultural Science and American Genetic Association. pp. 12-17. Williams, J.G.K., A.R. Kubelik, K.J. Livak, J.A. Rafalski and S.V. Tingey. 1990. DNA polymorphisms amplified by arbitrary primers are useful as genetic markers, Nucleic Acids Res. 18:6531-6535. Yamamoto, N., G. Ono, K. Takashima and A. Totsuka. 1991. Restriction fragment length polymorphisms of grapevine DNA with phenylalanine ammonia-lyase cDNA. Jap. J. Breed. 41:365-368.