A simple and efficient method for DNA extraction from grapevine cultivars, Vitis species and Ampelopsis.

Lodhi, Muhammad A., Guang-Ning Ye, Norman F. Weeden and Bruce I. Reisch. 1994.

Plant Molecular Biology Reporter 12(1): 6-13.

 

Department of Horticultural Sciences, New York State Agricultural Experiment Station, Cornell University, Geneva, NY 14456.

Key Words: DNA extraction, Vitis sp., polyphenols, polysaccharides, RAPD, restriction digestion.

Abstract

A quick, simple and reliable DNA extraction method for grapevine species, hybrids and Ampelopsis (Vitaceae) has been developed. This method is a modification of Doyle and Doyle (1990). It is a CTAB-based extraction procedure modified by the use of NaCl to remove polysaccharides and PVP to eliminate polyphenols during DNA purification. The method also has been used successfully for extraction of total DNA from other fruit species such as apple (Malus domestica), apricot (Prunus armeniaca), cherry (Prunus avium), peach (Prunus persica), plum (Prunus domestica) and raspberry (Rubus idaeus). DNA yield from this procedure is high (up to 1 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.

Vitis 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 mapping (Yamamoto et al., 1991; Mauro et al., 1992; Weeden et al., 1992; Lodhi et al., 1992a; 1992b;1993; Hain et al., 1993), genetic transformation (Baribault et al., 1989; Baribault et al., 1990; Hébert et al., 1993), and DNA fingerprinting (Striem et al., 1990; Bourquin et al., 1991). The relatively small genome size of Vitis vinifera (0.50 pg/C) compared to many other perennial plant species (Arumuganathan and Earle, 1991) should facilitate molecular genetic studies of Vitis. However, DNA extraction from grapevine has 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); polyphenolic compounds (Katterman and Shattuck, 1983; Couch and Fritz, 1990; Howland et al. 1991; Collins and Symons, 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 and/or quality (Bourquin et al., 1991; Collins and Symons, 1992).

Here we report a simple, inexpensive and quick DNA extraction procedure for grapevine Vitis species, hybrids and Ampelopsis. This procedure purifies greater amounts of clean DNA which can be amplified via PCR or digested with endonucleases.

 

Materials and Methods

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-HCl, adjust pH to 8.0 with HCl, add 1.4 M NaCl and 2.0% (w/v) CTAB (cetyltrimethylammonium bromide). Dissolve CTAB by heating to 60C. Store at 37C. Add 0.2 % of ß-mercaptoethanol just before use.

Chloroform:octanol 24:1 (v/v)

5 M NaCl

TE buffer: 10 mM Tris-HCl and 1 mM EDTA, adjust pH to 8.0 and autoclave

RNAase A (Sigma R9009: 10 mg/mL)

Protocol

• Collect unexpanded young leaves in liquid nitrogen or on ice and store at or below -70°C until used. Avoid thawing before grinding the leaf tissue. Grind 0.5 g of leaves using mortar and pestle in the presence of liquid nitrogen. 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.

• Add 5 mL of extraction buffer to the ground leaves and mix in the mortar.

• Pour the slurry into clean 15 mL polypropylene centrifuge tubes (Laboratory Product Sales, Rochester, New York; LX 4109), rinse the mortar and pestle with 1 mL of extraction buffer and add to the original extract.

• Add 50 mg polyvinylpolypyrrolidone (PVP), (Sigma, P6755) and invert the tubes several times to mix thoroughly with the leaf slurry (100 mg PVP/g leaf tissue).

• Incubate at 60°C for 25 minutes and cool to room temperature.

• Add 6 mL of chloroform:octanol and mix gently by inverting the tubes 20 to 25 times to form an emulsion.

• Spin at 6000 rpm for 15 minutes in a table top centrifuge at room temperature.

• 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.

• Add 0.5 volume of 5M NaCl to the aqueous solution recovered from the previous step and mix well.

• Add two volumes of cold (-20°C) 95% ethanol and refrigerate (4 to 6°C) for 15-20 minutes or until DNA strands begin to appear. The solution can be left for one hour or more if necessary.

• Spin at 3000 rpm for three minutes and then increase speed to 5000 rpm for an additional three minutes at room temperature. This differential spinning step helps to keep DNA at the bottom of the centrifuge tube.

• Pour off supernatant and wash pellet with cold (0 to 4°C) 76% ethanol. Completely remove ethanol without drying the DNA pellet by leaving the tubes uncovered at 37°C for 20 to 30 minutes.

• Dissolve in 200 to 300 L TE.

• Treat with 1 L RNAase A per 100 L DNA solution and incubate at 37°C for 15 minutes.

• Quantify DNA in a spectrophotometer at A260.

• Keep DNA at -70C for long term and -20°C for short term storage.

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). NaCl has been used to remove polysaccharides (Fang et al., 1992), and PVP to purge polyphenols (Maliyakal, 1992). This procedure does not involve CsCl density gradient purification steps.

DNA yields from Vitis species, Ampelopsis and other woody perennial plant species by the above mentioned procedure range from 0.5 to 1.0 mg/g fresh leaf tissues with A260/A280 between 1.8 and 2.0 (Table 1). 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 (EcoRI, EcoRV and HindIII) showed complete digestion (Fig. 1a). It is also evident that the uncut DNA exhibits little shearing and is suitable for Southern (1975) hybridization (Fig. 1b). The DNA is also amplifiable in PCR using the RAPD technique (Williams et al., 1990) (Fig. 2).

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. However, we were 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, damaged 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 which can be separated from DNA by centrifugation (Maliyakal, 1992). The presence of polyphenolic compounds can be reduced by keeping plant material frozen before extraction and by using PVP in the DNA extraction procedure. The 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 1 M NaCl 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 NaCl (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, hybrids and Ampelopsis 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 (Bourquin et al., 1991, 5-20 g nuclear DNA /g FW; Collins and Symons, 1992, 10-30 g DNA/g FW; and Thomas et al., 1993, 25-150 g DNA/g FW). Doyle and Doyle (1987) reported DNA yields up to 1 mg/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 which remains usable for at least two years when stored at -20°C.

 

Acknowledgments: We are thankful to Dr. Philip L. Forsline, USDA, ARS, National Clonal Germplasm Repository, Geneva, New York for providing us with Vitis species plant material, Dr. Robert L. Andersen for cherry, apricot, plum and peach, and Mr. Kevin E. Maloney for raspberry. We wish to thank Drs. John C. Sanford and Susan K. Brown for their critical review and valuable suggestions.

 

References

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

Genotype

Source

DNA yield

(g/g leaf)

Vitis sp. Aurore

NYSAES a

1,130167 c

Vitis acerifolia

USDA, ARS b

914427 c

Vitis berlandieri

USDA, ARS

1,04039.6c

Vitis cinerea

USDA, ARS

796153 c

Vitis labrusca

UDSA, ARS

54260.1c

Vitis rupestris

USDA, ARS

594116 c

Vitis vinifera cv. Cabernet Sauvignon

NYSAES

54619.8c

Ampelopsis brevipedunculata

USDA, ARS

85048.1c

Apple (Malus domestica cv. Red Delicious)

NYSAES

830

Apricot (Prunus armeniaca cv. NY 500)

NYSAES

935

Cherry (Prunus avium cv. NY 6476)

NYSAES

665

Peach (Prunus persica cv. Rutgers Red Leaf)

NYSAES

805

Plum (Prunus domestica cv. NY 65.363.1)

NYSAES

1,055

Raspberry (Rubus idaeus cv. NY 83)

NYSAES

1,135

a NYSAES (New York State Agricultural Experiment Station, Geneva, New York)

b USDA, ARS (National Clonal Germplasm Repository, Geneva, New York)

c average of two extractions


 

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