Plant Molecular Biology Reporter 21: 177a–177g, June 2003

© 2003 International Society for Plant Molecular Biology. Printed in Canada.

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An Efficient Protocol for Genomic DNA Extraction

From Citrus Species

YUN-JIANG CHENG, WEN-WU GUO, HUA-LIN YI, XIAO-MIN PANG and

XIUXIN DENG*
National Key Laboratory of Crop Genetic Improvement, National Center of Crop
Molecular Breeding, Huazhong Agricultural University, Wuhan 430070, PR China

DNA extraction
Abstract. from Citrus
We describe spp.
a simple and Cheng
efficientetmethod
al. for genomic DNA extraction from
woody fruit crops containing high polysaccharide levels. This method involves a modified
CTAB or SDS procedure employing a purification step to remove polysaccharides by
using water-saturated ether and 1.25 M NaCl. Precipitation with an equal volume of
isopropanol caused a DNA pellet to form. After being washed with 70% ethyl alcohol, the
pellet easily dissolved in TE buffer. Using this method, DNA was extracted from samples
of more than 1000 Citrus spp., including young leaves, old leaves, frosted old leaves, with-
ered old leaves, and callus lines. The average yield of DNA ranged from 50-500 μg/g of
sample. DNA was suitable for PCR and RFLP analyses and long-term storage. Recently,
the procedure was used to isolate DNA from withered old leaves of more than 20 tropical
and subtropical fruit crops.

Full text†: This manuscript, in detail, is available only in the electronic version of the
Plant Molecular Biology Reporter.

Key words: DNA extraction, fruit crops, polysaccharides, RFLP, water-saturated ether

Abbreviations: CTAB, hexadecyltrimethylammonium bromide; MAS, molecular

marker–assisted selection; RFLP, restriction fragment length polymorphism; RT, room
temperature.

Introduction
Isolating high quality DNA is essential for molecular research. Polysaccharide
contamination is a common problem in higher plant DNA extraction. DNA sam-
ples are often contaminated with melicera colloidal hyalosome, which is almost
insolvable in water or TE buffer. This can affect manipulation, inhibit enzyme re-
actions (Fang et al., 1992; Porebski et al., 1997; Schlink and Reski, 2002), and
hinder the downstream work in molecular biology research. DNA samples are

*Author for correspondence. e-mail: xxdeng@mail.hzau.edu.cn;

fax: (+86-27) 8728 0016; ph: (+86-27) 8728 1300.
†
Editor’s note: Although the scientific content of this paper has been reviewed, the full
text WEB document has not been edited in detail.
177b Cheng et al.

unstable for long-time storage (Lodhi et al., 1994; Sharma et al., 2002). Several
plant DNA extraction protocols for removing polysaccharides have been reported
(Möller et al., 1992; Cruz et al., 1997; Porebski et al., 1997; Schlink and Reski,
2002; Sharma et al., 2002). However, in some woody fruit crops that contain high
polysaccharide levels, such as crops of Citrus spp., the protocols could only be
used on vigorous tissue (Luro et al., 1995; Porebski et al., 1997), and the DNA
isolated was not of high enough quality to be used in PCR and RFLP analyses.
We describe a modified protocol using water-saturated ether and 1.25-1.3 M
NaCl. Residual phenols and most polysaccharides were removed, and DNA was
precipitated selectively in the presence of high salt (Fang et al., 1992; Möller et
al., 1992). This method is suitable for genomic DNA isolation from fruit crops
containing high polysaccharide levels.

Materials and Methods

Plant materials
Citrus spp. and its wild relatives were tested, including orange, mandarin, tanger-
ine, grapefruit, pummelo, kumquat, trifoliate orange, Chinese box-orange, and
40 kinds of Citrus somatic hybrids (~300 individuals). Recently, more than
20 tropical and subtropical fruit crops were tested. Leaves were harvested at
different developmental stages (young, mature, old, frosted, withered).

Equipment and reagents

• Mortar and pestle
• Waterbath
• Beckman centrifuge (J6-HC)
• UV-1601 spectrophotometer (SHIMADZU)
• Liquid nitrogen
• Extraction buffer: 100 mM Tris-HCl (pH 8), 1.5 mM NaCl, 50 mM EDTA
(pH 8), 0.5% β-mercaptoethanol, 1.5% (w/v) CTAB (added just before use)
• Chloroform-isoamyl alcohol (24:1)
• Phenol-chloroform-isoamyl alcohol (25:24:1)
• TE buffer (pH 8): 10 mM Tris-HCl, 1 mM EDTA
• 10 mg/mL RNase A (free of DNase)
• Water-saturated ether
• Ethanol
• 5 M NaCl
• 70% ethanol
• Agarose

DNA extraction protocol

• Grind 5-8 g of clean tissue with a mortar and pestle in the presence of liquid
nitrogen.
• Transfer the ground powder into a clean autoclaved 50-mL centrifuge tube and
add 15 mL of boiling extraction buffer.
• Mix well and incubate at 65°C for 60 min with occasional inversion.
DNA extraction from Citrus spp. 177c

• Cool the mixture to RT. Add 10 mL of chloroform-isoamyl alcohol (24:1) and

invert gently for 10 min.
• Centrifuge in a Beckman centrifuge at 5500 g for 15 min at RT.
• Transfer the top aqueous layer to a fresh 50-mL tube. Precipitate the DNA by
adding an equal volume (~15 mL) of isopropanol, mix by gentle inversion, and
incubate at -20°C for 30 min.
• Deposit DNA by centrifuging at 5000 g for 10 min at RT. Wash the pellet 2-3
times (2-3 h each time) with 3 mL of 70% ethanol.
• Air-dry the pellet for 20-30 min at RT. Add 3 mL TE buffer and 25 μL of
10 mg/mL DNA-free RNase A. Incubate at 37°C for 3 h.
• Transfer the DNA solution into a fresh 10-mL tube. Add 3 mL of phe-
nol-chloroform-isoamyl alcohol (25:24:1), mix by using gentle inversion for
5-10 min, and centrifuge for 15 min at 5000 g at RT.
• Transfer the top aqueous layer into a new 10-mL tube. Add 1 mL of 5 M NaCl
(final concentration of 1.25-1.3 M) and 4 mL water-saturated ether. Mix well
by using gentle inversion and centrifuge for 10 min at 5000 g at RT. (Note:
When adding NaCl and water-saturated ether, the aqueous phase immediately
turned milky. After inversion, the white disappeared, and the mixed solution
turned into a hyaloplasm).
• Discard the top ether layer. Carefully transfer the bottom aqueous layer with a
20-μL sterile pipette tip against inside the rim of the tube. Bend the tip, so as to
form a slot, and pour the bottom aqueous layer from the slot into a new 10-mL
tube.
• Add 4 mL of chilled isopropanol, mix well by using gentle inversion, and
freeze at -20°C for 30 min. Remove the DNA pellet with a hook.
• Wash the pellet 3 times for 1 h each time with 3 mL of 70% ethanol.
• Air-dry the pellet for 20 min at RT. Add 1 mL TE buffer to dissolve the pellet.
• Dilute 15 μL of DNA solution to qualitative assay with a UV-1601
spectrophotometer (SHIMADZU).
• Adjust the concentration of the samples to 200 ng/μL. Add 5 μL of each sample
to test on a 0.8% agarose gel. Store the DNA solution at -20°C until use.
Notes
1. With this treatment, polysaccharides concentrated in the interphase layer
while the DNA still dissolved in the bottom aqueous phase. Most polysaccha-
rides could be removed by discarding the gel-like interphase.
2. Handle carefully to prevent contamination of the bottom aqueous layer by the
interphase.
3. Ether is highly flammable and can cause drowsiness. All manipulations
involving ether should be performed in a well-ventilated fume hood.
4. High concentrations of NaCl may inhibit enzyme activity; thus, DNA solu-
tion purified by means of this method should be deposited and washed with
70% ethanol to remove residual salt.

RFLP analysis
According to the RFLP analysis procedure of Cheng et al. (2003), approximately
10 μg of genomic DNA were used for each enzyme digestion. Five
177d Cheng et al.

restriction enzymes (EcoR I, Hind III, BamH I, Dra I, Pst I) were used to digest
the DNA samples. Digested products were electrophoresed in 0.8% agarose gel.
DNA was blotted onto a Hybond-N+ membrane (Amersham, NJ USA) for
16-20 h in an alkali-downward capillary blotting procedure according to the
Hybond-N+ Nylon membrane manual (1998). Probes were labeled with
d(CTP)-P32. Hybridization was performed in tubes at 65°C overnight, and mem-
branes were washed in high stringency solution (0.1 X SSC, 0.1% SDS) at 65°C
for 4 h. Membranes were exposed to x-ray film at -80°C overnight to a week be-
fore developing the film.

Results and Discussion

Compared with other protocols, this method removed polysaccharides efficiently
before DNA precipitation (Figure 1). White DNA pellets formed (Figure 2) and
were quickly soluble in TE buffer. The A260/280 ratio ranged from 1.7-1.9, and the
A260/230 ratio was greater than 2. The absence of a peak at 270 nm indicates that
residual phenols were removed. DNA samples can be stored at 4°C for 1.5 y.
Results of the agarose gel test and PCR or RFLP analysis indicated that polysac-
charides had been efficiently removed and DNA quality had been enhanced
(Figures 3-5). Classic CTAB (Saghai-Maroof et al., 1984) and SDS (Dellaporta et
al., 1983) protocols, when combined with the NaCl and water-saturated ether
treatment, produced satisfactory results.
Crops of Citrus spp. and other tropical or subtropical fruits are perennial
woody plants. Polysaccharide content, even in young tissue, is higher than in field
crops. Two classic DNA extraction methods, CTAB (Saghai-Maroof et al., 1984)
and SDS (Dellaporta et al., 1983), were inefficient in removing polysaccharides.
Several modified DNA protocols that remove polysaccharides have recently been
reported (Fang et al., 1992; Möller et al., 1992; Cruz et al., 1997; Porebski et al.,
1997). All were unsuccessful in removing polysaccharides from crops of Citrus
spp. and other fruits. Isolating high quality DNA for RFLP analysis from some
materials, such as withered and old frosted Citrus leaves, was difficult. DNA sam-
ples were hyaloplasm gel-like (almost insoluble in TE buffer); A260/280 ratios were
always less than 1.5; and a peak of 270 nm, corresponding to the peak of a com-
bination of phenol and polysaccharides, was usually scanned (Tesniere and Vayda,
1991). When tested on 0.8% agarose gel, the sample concentrated on the well and
appeared bright under UV light. Conducting PCR analysis or enzyme digestion
was difficult because polysaccharides inhibited enzyme activity (Fang et al., 1992;
Porebski et al., 1997; Schlink and Reski, 2002; Sharma et al., 2002). In addition,
the concentration of DNA samples was too low for RFLP analysis. These prob-
lems have been resolved in the molecular analysis of Citrus spp. and other fruit
crops in our laboratory by using this modified DNA extraction protocol.
MAS is a powerful tool for conventional fruit breeding. Breeders occasion-
ally find interesting mutants under extreme weather or environmental conditions
or on some genetically abnormal phenotypes (Cheng et al., 2003). However, vig-
orous tissue and chilling equipment were unavailable, which limited the extraction
of DNA according to previous protocols. Using our modified protocol, DNA was
isolated from several tissue samples, including mature leaves and withered or
DNA extraction from Citrus spp. 177e

Figure 1. After extraction with water-saturated ether and 1.25 M NaCl (final concentration) and
centrifugation at 5000 g for 10 min at RT, hyaloplasm gel-like polysaccharides concentrated in the
interphase layer, and DNA dissolved in the bottom aqueous phase.

Figure 2. White DNA pellet of withered old Citrus leaves formed in isopropanol after
polysaccharides were removed.

frosted old leaves stored in a handbag for a week at RT (Figure 3, lanes 8-9). The
quality was high enough to perform DNA marker analysis (Cheng et al., 2003)
(Figure 5, lanes 1-3 and 9-12).
We have performed this protocol in our laboratory since 2000. In the
past 3 y, more than 1000 DNA samples have been extracted from different
177f Cheng et al.

1 2 3 4 5 6 7 8 9
Figure 3. Quality test of 8 DNA samples of Citrus spp. on 0.8% agarose gel. Lane 1, λDNA / Hind
III DNA marker; 2-7, fresh mature leaves; 8, withered old leaves (stored in a handbag for 1 wk); 9,
frosted old leaves (harvested in January 2001), samples harvested from the field.

1 10 20 30 36
Figure 4. Analysis of 35 samples of Citrus spp. by using RFLPs with the enzyme/probe combination
of Hind III / atpA. Lane 1: λDNA / Hind III DNA marker.

Figure 5. Instability of mtDNA in one Citrus somatic hybrid plant analyzed by using RFLP with the
enzyme/probe combination of Hind III / atpA. Lanes 1-12: Samples were harvested on the first day
of every month, from 1 December 2000 to 1 November 2001. Shoots of the plant exhibited serious
dieback in October 2001, and a loss of mtDNA fragments was detected (arrows on 1 September
sample).

developmental stages of Citrus leaves and callus lines. Recently, good quality
DNA samples were obtained from withered old leaves of other tropical and sub-
tropical fruit crops. These samples included longan (Dimocarpus longan L.), ba-
nana (Musa spp.), litchi (Litchi chinensis S.), passion fruit (Passiflora edulis S.),
DNA extraction from Citrus spp. 177g

macadamia nut (Macadamia integrifolia M. & B.), persimmon (Diospyros kaki L.

f.), chestnut (Castanea mollissima Bl.), fig (Ficus carica L.), pineapple (Ananas
comosus L.), plum (Prunus salicina L.), jujube (Zizyphus mauritiana M.), cili
(Rosa roxburghii T.), Hupeh crabapple (Malus hupehensis R.), ginkgo (Ginkgo
biloba L.), pomegranate (Punica granatum L.), wampee (Clausena lansium [L.]
S.), guava (Psidium guajava L.), mango (Mangifera indica L), papaya (Carica
papaya L.), custard apple (Annona squamosa L.), and wax apple (Syzygium
samarangense). Results prove the reproducibility, reliability, and practicality of
this protocol.

Acknowledgments
This research was financially supported by the National Natural Science Founda-
tion of China (NSFC), the 863 Project of China, the International Foundation for
Science (IFS), and the Chenguang Youth Project of Wuhan City of China.

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