DNA Extraction from Vitis

A Simple and Efficient Method for DNA Extraction from Grapevine Cultivars and Vitis Species*

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

Additional Index Words: DNA extraction, polyphenols, polysaccharides, RAPD, restriction digestion, temperate fruits, Vitis sp.

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.

* Lodhi, Muhammad A., Guang-Ning Ye, Norman F. Weeden and Bruce I. Reisch. 1994. A simple and efficient method for DNA extraction from grapevine cultivars and Vitis species. Plant Molecular Biology Reporter 12(1): 6-13.

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; Collins and Symons, 1993). The relatively small genome size of V. 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 procedure for the extraction of DNA from grapevine cultivars, Vitis species and from A. brevipedunculata . 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 2.1 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 60°C. Store at 37°C. Add 0.2 % ofbeta-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
RNase 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, 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 microliters TE.

Treat with 1 µL RNase A per 100 µL DNA solution and incubate at 37°C for 15 minutes.

Quantify DNA in a spectrophotometer at A260.

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

Results and Discussion

We have obtained higher yields of clean DNA from grapevine leaves 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 (Table 2.1) with A260/A280 between 1.8 and 2.0. Uncut DNA appears to be high molecular weight with less shearing and easy mobility (Fig. 2.1). 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 (Eco RI, Eco RV and Hin dIII) showed complete digestion (Fig. 2.2). 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. 2.3).

Figure 2.1 Electrophoretogram of uncut genomic DNA extracted from grape cultivars and genera.
Left to right, (lane 1-7) Vitis 'Aurore', Ampelopsis brevipedunculata, V. labrusca , V. cinerea , V. longii , V. berlandieri and V. vinifera 'Cabernet Sauvignon'. Lane 8 shows lambda DNA/Hin dIII size marker. Five µL DNA was run in each lane.

Proper choice of 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.

Figure 2.2 Gel electrophoretogram of restricted DNA of grape species and genera.
The DNA was restriction-digested with Bam HI (left to right; lanes 1-7), Hin dIII (lanes 9-15) and Eco RI (lanes 17-23). Lanes 1, 9, and 17 Vitis sp. 'Aurore'; lanes 2, 10, and 18 Ampelopsis brevipedunculata ; lanes 3, 11, and 19 V. berlandieri ; lanes 4, 12 and 20 V. cinerea ; lanes 5, 13, and 21 V. acerifolia , lanes 6, 14, and 22 V. labrusca ; and lanes 7, 15 and 23 Vitis vinifera . 'Cabernet Sauvignon'. Lane 8 and 16 are one Kb DNA ladder (Cat # 15615-016 Gibco BRL, Life Technologies, Gaithersburg, MD). Each lane contains ~10 µg DNA. DNA was resolved in 0.9% agarose gel, stained with ethidium bromide and visualized under UV light.

Figure 2.3 PCR amplification of DNA from different grape species and genera with ten bases long oligonucleotides.
Lane 1 is 100 bp DNA ladder; lane 2-8 amplification with K5 primer (CGCAGGATGG); Ampelopsis brevipedunculata , V. acerifolia , V. labrusca , V. cinerea , V. vinifera cv. Cabernet Sauvignon, V. berlandieri and Vitis sp. 'Aurore', lanes 9-15 with OD-8 (GTGTGCCCCA) and lanes 16-22 with S69 (CATCGAACCG) shown in the same order as above. Approximately 50 ng DNA was amplified as described in the text; the amplification products separated by agarose-gel electrophoresis, and stained with ethidium bromide.