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

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)

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.

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 µg nuclear DNA /g FW; Collins and Symons, 1992, 10-30 µg DNA/g FW; and Thomas et al., 1993, 25-150 µg 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.

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