lodhi1994

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.
Extraction of DNA from Grapevine
7
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.
8
Lodhi et al.
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.
Extraction of DNA from Grapevine
9
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-
10
Lodhi et al.
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
Extraction of DNA from Grapevine
11
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~
12
Lodhi et aI.
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.
References
Arumuganathan, K. and E.D. Earle. 1991. Nuclear DNA content of some important plant
species. Plant Mol. Biol. Rep. 9:208-218.
Baribault, TJ., K.G.M. Skene and N.S. Scott. 1989. Genetic transformation of grapevine
cells. Plant Cell Rep. 8:137-140.
Baribault, T.J., K.G.M. Skene, P.A. Cain and N.S. Scott. 1990. Transgenic grapevines:
Regeneration of shoots expressing ~-glucuronidase. J. Exp. Bot. 41:1045-1049.
Bourquin, J.-C., L. Otten and B. Waiter. 1991. Identification of grapevine root-stocks by
RFLP. C.R. Acad. Sci. Paris 312 S6rie i1I:593-598.
Collins, G.G. and R.H. Symons. 1992. Extraction of nuclear DNA from grape vine leaves by
a modified procedure. Plant Mol. Biol. Rept. 10:233-235.
Collins, G.G. and R.H. Symons. 1993. Polymorphisms in grapevine DNA detected by the
RAPD PCR technique. Plant Mol. Biol. Rept. 11:105-112.
Couch, J.A. and P.J. Fritz. 1990. Isolation of DNA from plants high in polyphenolics. Plant
Mol. Biol. Rep. 8:8-12.
Doyle, J.J. and J.L. Doyle. 1987. A rapid DNA isolation procedure from small quantities of
fresh leaf tissues. Phytochem Bull. 19:11-15.
Doyle, J.J. and J.L. Doyle. 1990. Isolation of plant DNA from fresh tissue. Focus 12:13-15.
Fang, G., S. Hammar and R. Rebecca. 1992. A quick and inexpensive method for removing
polysaccharides from plant genomic DNA. BioTechniques 13:52-56.
Hain, R., H.J. Reif, E. Krause, R. Langebartels, H. Kindl, B. Vornam, W. Wiese, E. Schmelzer,
P.H. Schreier, R.H. St6cker and K. Stenzel. 1993. Disease resistance results from foreign
phytoalexin expression in a novel plant. Nature 361:153-156.
H6bert, D., J.R. Kikkert, F.D. Smith and B.1.Reisch. 1993. Optimization of biolistic transformation of embryogenic grape cell suspensions. Plant Cell Rep. 12:585-589.
Howland, D.E., R.P. Oliver and A.J. Davy. 1991. A method of extraction of DNA from birch.
Plant Mol. Biol. Rep. 9:340-344.
Katterman, F.R.H. and V.1.Shattuck. 1983. An effective method of DNA isolation from the
mature leaves of Gossypium species that contain large amounts of phenolic terpenoids
and tannins. Preparative Biochemistry 13:347-359.
Lodhi, M.A., B.1.Reisch and N.F. Weeden. 1992a. Molecular genetic mapping and genome
size of Vitis. Plant Genome I, 9-11 November, San Diego, CA, USA (Abstract).
Lodhi, M.A., B.1. Reisch and N.F. Weeden. 1992b. Molecular genetic mapping of the Vitis
genome. Am. J. Enol. Vitic. 43:393 (Abstract).
Lodhi, M.A., B.I. Reisch and N.F. Weeden. 1993. Molecular genetic mapping and genome
size of Vitis. Hort Science 28:489 (Abstract #289).
Maliyakal, E.J. 1992. An efficient method for isolation of RNA and DNA from plants
containing polyphenolics. Nucleic Acids Res. 20:2381.
Mauro, M.-C., M. Strefeler, N.F. Weeden and B.I. Reisch. 1992. Genetic analysis of restriction fragment length polymorphisms in Vitis. J. Hered. 83:18-21.
Murray, M.G. and W.F. Thompson. 1980. Rapid isolation of high molecular weight DNA.
Nucleic Acids Res. 8:4321-4325.
Richards, E. 1988. Preparation of genomic DNA from plant tissue. In: Current Protocols in
MolecularBiology. (eds. F.M. Ausubel, R.E. Kingston, D.D. Moore, J.A. Smith, J.G.
Seidman and K. Struhl), pp. 2.3.2-2.3.3. Greene Publishing Associates and Wileylnterscience, New York.
Extraction of DNA from Grapevine
13
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.