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A rapid method for extraction of cotton (Gossypium Spp.) genomic DNA

suitable for RFLP or PCR analysis

Article  in  Plant Molecular Biology Reporter · June 1993

DOI: 10.1007/BF02670470

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Plant Molecular Biology Reporter pages122-127
Volume 11(2) 1993

Commentary

A Rapid Method for Extraction of Cotton

(Gossypium spp.) Genomic D N A Suitable for
RFLP or PCR Analysis
Andrew H. Paterson, Curt L. Brubaker,
and Jonathan F. Wendel
(AHP) Department of Soil and Crop Sciences, Texas A & M University,
College Station TX 77843-2474, USA, (address for correspondence);
(CLB, JFW) Department of Botany, Iowa State University, Ames IA 50011, USA
fax: 1-409-845-0456

Key Words: cotton, genomic DNA extraction, Gossypium, PCR, RFLP

Abstract: Extraction of high-quality genomic DNA from Gossypium (cotton)

species is difficult due to.high levels of polysaccharide, oxidizable quinones, and
other interfering substances. We describe a procedure that consistently permits
isolation of cotton genomic DNA of satisfactory size and quality for RFLP and
PCR analysis, as well as for most routine cloning applications. Several antioxi-
dants, phenol-binding reagents, and phenol oxidase inhibitors are used through-
out the procedure, and most polysaccharides are eliminated early in the proce-
dure by isolation of nuclei.

he genus Gossypium comprises 50 diploid and tetraploid species

T indigenous to Africa, Central and South America, and Australia

(Fryxell, in press). Four of these, namely G. hirsu turn, G. barbadense,
G. arboreum, and G. herbaceum, are cultivated for their fiber and oil. G.
hirsutum dominates world cotton commerce, with an estimated value of
$5 billion per year in the USA alone.

Abbreviations: CIA, chloroform-isoamyl alcohol; CTAB, hexadecyltrimethyl-

ammonium bromide; DIECA, diethyldithiocarbamic acid; RAPD, random am-
plified polymorphic DNA.
Genomic DNA from Cotton 123

DNA marker-based studies in Gossypium have been hindered by the

lack of an efficient DNA extraction protocol. Presumably, this has been
due to high endogenous levels of polysaccharides, phenolics, and other
organic constituents that interfere with DN A isolation and purification.
Previous protocols for cotton genomic DNA extraction are time-consum-
ing and expensive, usually requiring ultracentrifugation in CsC1 gradi-
ents (e.g., Wendel, 1989). Although these procedures may yield high-
quality DNA, they are not amenable to high-throughput applications,
such as RFLP mapping or DNA marker-assisted selection.
The technique described herein streamlines preparation of cotton
genomic DNA, eliminates the need for CsC1 gradients, and adds the
option of mechanical tissue homogenization rather than hand-grinding
with a mortar and pestle. An individual can routinely extract 5 to 150 ~tg
of DNA from each of 32 samples in about four hours. The DNA is highly
susceptible to restriction enzymes, yields satisfactory results from RFLP
analysis, and is amenable to PCR-based procedures (including RAPDs).

Materials and Methods

For extraction of cotton genomic DNA, we use fresh (or frozen) young
leaves which are less than one week old. Fully expanded leaves yield
much less DNA per unit mass. Fresh tissue quick-frozen in liquid
nitrogen and stored at -20~ gives satisfactory results for at least three
months. Lyophilized tissue has given very poor results with this proce-
dure, thus we recommend the use of fresh or frozen tissue. This protocol
is optimized for a sample of 4 grams, which conveniently fits into a 50-
mL disposable screw-cap centrifuge tube.
Solutions for which recipes are not provided can be found in Sambrook
et al. (1989).
Equipment needed
Tabletop or floor-standing centrifuge, preferably refrigerated. Should
reach 2700xg or higher with swinging bucket rotor, and have
carriers for 50-mL conical screw-cap tubes.
Waterbath, 65~
Microcentrifuge, capable of 10,000xg.
(optional) Tissue homogenizer. Several makes and models are avail-
able. Should reach 10,000 rpm or higher, and endure repeated use.
Solutions
Cotton DNA extraction buffer: 0.35M glucose, 0.1 M Tris-HC1 (pH 8.0),
0.005 M Na2-EDTA (pH 8.0), 2% (w/v) polyvinylpyrolidone (PVP40),
124 Patterson, Brubaker, and Wendel

0.1% ( w / v ) diethyldithiocarbamic acid (DIECA). Bring v o l u m e to

1 liter with water. This is a milky solution that should be stored at
4~ It can be stored for I to 2 weeks before use, if necessary.
I m m e d i a t e l y before using, adjust solution to 0.1% ( w / v ) ascor-
bic acid and 0.2% ( w / v ) mercaptoethanol, and adjust the p H to 7.5.

Cotton nuclei lysis buffer: 0.1 M Tris-HC1 (pH 8.0), 1.4 M. NaCI, 0.02 M
Na2-EDTA (pH 8.0), 2% ( w / v ) CTAB (hexadecyl t r i a m m o n i u m
bromide), 2% ( w / v ) PVP40, 0.1% ( w / v ) DIECA, bring v o l u m e to 1
liter with water. This solution is clear but quickly turns yellow. It
can be stored at r o o m t e m p e r a t u r e for 1-2 weeks before use.
I m m e d i a t e l y before using, adjust solution to 0.1% ( w / v ) ascor-
bic acid and 0.2% ( w / v ) mercaptoethanol. 1
TE, p H 8.0

Note
1. CTAB takes several hours to dissolve, so this solution should be made the night
before you do your preps.

Protocol
Homogenize tissue and pellet nuclei
9 To 4 g tissue in a 50-mL centrifuge tube, on ice, add 20 m L of ice-cold
cotton D N A extraction buffer.
9 H o m o g e n i z e approx. 10,000 r p m for approx. 20 sec. ~
9 Return sample to ice.
9 Repeat first three steps for next sample. 2
9 Spin 20 min at 2700xg and 4~
9 Pour off debris; save pellet (includes nuclei).
Notes
1. Alternately, grind to a fine powder with a mortar and pestle, using liquid
nitrogen and a pinch of sand to help. The ground tissue should then be placed
in the indicated volume of buffer, and put on ice until the next step.
2. We process 16 samples at a time.
3. A temperature of 4~ is not essential, but does maintain DNA quality
somewhat better than unrefrigerated centrifugation.

Lyse nuclei
9 A d d 8 m L cotton nuclei lysis buffer to each sample.
9 R e s u s p e n d pellet (nuclei, etc.) with as m u c h v o r t e x i n g as is neces-
sary to obtain a h o m o g e n e o u s solution.
9 Incubate at 65~ for 20 to 30 min. in a w a t e r bath.
Genomic DNA from Cotton 125

C h l o r o f o r m - i s o a m y l a l c o h o l (24:1) e x t r a c t i o n to r e m o v e p r o t e i n s
A d d 10 m L CIA to each sample.
Cap tubes tightly and invert -50 times to mix.
Spin 5 min at 2700xg to accelerate phase separation.
Transfer u p p e r (aqueous) phase to clean tube.
Repeat first four steps if the interphase is dirty, or the a q u e o u s
(upper) phase is cloudy. 1

I s o p r o p a n o l p r e c i p i t a t i o n a n d r e s u s p e n s i o n of D N A
Transfer s u p e r n a t a n t from final CIA extraction to 15-mL Falcon
tube.
A d d 0.6 vols (about 5.4 mL) ice-cold isopropanol to each tube.
Invert tubes 20 to 30 times until D N A aggregates2

R e s u s p e n d D N A in 65~ bath for 10-30 mill.

After r e s u s p e n d i n g , the sample should be spun for 5 min at 10,000xg
to pellet residual impurities ( p r e s u m a b l y largely polysaccharides).
Transfer the s u p e r n a t a n t (including DNA) to a clean tube, and
discard the pellet.

D N A can be stored in TE at 4~ for several weeks, frozen in TE for a year

or more, or precipitated and stored in 70% ethanol at -20~ indefinitely2 ,4

Notes
1. We generally extract each sample twice with CIA. Spinning the aqueous phase
alone between CIA extractions may remove some contaminants without the
yield loss inherent to chloroform extractions.
2. Typically the DNA can be spooled out onto a glass rod or flame-sealed Pasteur
pipet. If so, spool it out, dip briefly into 70% ethanol, and then place in 500 E.L
sterile TE in a 1.5-mL tube. If the DNA cannot be spooled, spin tube for 10 min
at 10,000xg to pellet DNA. Pour off supernatant, then add 1 mL 70% ethanol,
and gently vortex (setting 5 or less) to wash pellet. Spin (10 rain at 2700xg),
pour off ethanol, and allow any residual ethanol to evaporate off for a few
minutes. Add 500 gLTE and resuspend DNA for 10 to 30 rain at 65~
3. Some additional impurities may precipitate out of solution (when stored in
TE), over time. These can be removed by a brief centrifugation.
4. Occasional samples will be resistant to restriction endonucleases, or fail to
produce reliable RAPD amplification. In most cases, two extractions with
phenol:chloroform (1:1) followed by a third extraction with CIA, reprecipitation
of the DNA by adding 0.1 volumes 3 M sodium acetate (pH 5.2) and 2 volumes
ice-cold 70% ethanol, and resuspension of the DNA in TE removes enough
contaminants to achieve satisfactory endonuclease cleavage or DNA amplifi-
cation.
126 Patterson, Brubaker, and Wendel

Results and Discussion

The steps employed in this protocol, namely homogenization, crude

nuclei isolation, nuclei lysis, chloroform extraction, and DNA precipita-
tion, are similar to those described for other dicotyledonous plants
(Murray and Thompson, 1980; Bernatzky and Tanksley, 1986; Couch and
Fritz, 1990). However, the buffers employed in this protocol have been
designed to minimize problems associated with high endogenous levels
of phenolic compounds. Specifically, glucose has been chosen as an
osmoticum (Katterman and Shattuck, 1983), PVP binds phenolic com-
pounds, DIECA inactivates phenol oxidases (Loomis, 1974), and both
ascorbic acid and 2-mercaptoethanol are general antioxidants.
We have used DNA prepared by this method in a number of molecular
marker-based studies of Gossypium, including characterization of levels
and patterns of DNA polymorphism, mapping of more than 600 RFLP
loci in an F2 population of G. hirsutum x G. barbadense, comparative
analysis of genome organization in diploid and tetraploid cottons, and
mapping of agriculturally important genes in cotton. Alleles up to about
20 kb can be reliably scored. In addition, this procedure has been
successfully used to extract genomic DNA from other Malvaceae, includ-
ing Cienfuegosia, and Hibiscus.
In addition, we routinely use DNA prepared by this method for
RAPD-PCR (Williams et al., 1990). We have screened 400 decamers in a
sample of 8 genotypes including G. hirsutum cultivars and isolines, as
well as G. barbadense, finding levels of RAPD polymorphism generally
concordant with levels of RFLP polymorphism observed in these geno-
types (D. Lan and AHP, unpubl, data).

Acknowledgments:We thank J. Garza for technical assistance and R. Wing for

reviewing a preliminary version of the manuscript. AHP was supported by the
Texas Agricultural Experiment Station, USDA-NRICGP (91-37300-6570) and
Texas Higher Education Coord inating Board (TATP 999902-148), and JFW by the
NSF (BSR-8918041).
References

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Genomic D N A from Cotton 127

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