Plant Breeding 120, 354Ð356 (2001)
ã 2001 Blackwell Wissenschafts-Verlag, Berlin
ISSN 0179-9541
Short Communication
A new DNA extraction method for high-throughput marker analysis
in a large-genome species such as Triticum aestivum
N . S T E I N 1 , G . H E R R E N 1 and B . K E L L E R 1,2
1
2
University of ZuÈrich, Institute of Plant Biology, Zollikerstrasse 107, CH±8008 ZuÈrich. E-mail: [email protected];
Corresponding author
With 2 ®gures
Received December 21, 2000/Accepted February 1, 2001
Communicated by C. Jung
Abstract
Gene mapping and marker-assisted selection in complex, polyploid
genomes still relies strongly on restriction fragment length polymorphism (RFLP) analysis, as conversion of RFLP to polymerase chain
reaction (PCR) markers can be very dicult. DNA extraction in
amounts suitable for RFLP analysis represents the most timeconsuming and labour-intensive step in molecular marker analysis of
plant populations. In this paper, a new ¯exible method for plant DNA
extraction is presented. It allows a high-throughput of samples in a
short time without the need for freezing or lyophilizing the plant
material. The method allows the isolation of genomic DNA with a
yield of » 100 lg for a minimal amount of 200 mg of leaf material.
This is sucient for work with large-genome plant species such as
hexaploid wheat, where 20 lg of genomic DNA are required for a
single RFLP analysis.
Key words: Triticum aestivum Ð DNA extraction Ð highthroughput Ð marker-assisted selection Ð RFLP
High-resolution genetic mapping of molecular markers for
map-based cloning of genes or marker-assisted selection
(MAS) of agronomically important traits requires DNA
isolation from a large number of plants in a short time.
Several procedures that allow small-scale DNA extraction
from many samples per day, and per person, and give DNA
compatible with polymerase chain reaction (PCR)-based
technology have been reported (Berthomieu and Meyer 1991,
Oard and Dronavalli 1992, Cheung et al. 1993, Chunwongse
et al. 1993, Wang et al. 1993, Guidet 1994, Thomson and
Henry 1995, Haymes 1996, Lange et al. 1998). In polyploid
species such as hexaploid wheat, however, it is not always
possible to convert restriction fragment length polymorphisms
(RFLPs) into sequence-tagged site (STS) or cleaved ampli®ed
polymorphic sequence (CAPS) markers. Therefore, a simple
and rapid DNA extraction protocol providing reproducibly
sucient yield for several Southern blots (60±100 lg) for
RFLP analysis would be extremely valuable. Standard DNA
extraction procedures generally require lyophilization of leaf
material or freezing in liquid nitrogen followed by mechanical
grinding of the plant material (Rogers and Bendich 1988,
Birren et al. 1997), making them unsuitable for high-throughput applications.
This paper reports the development of a simple and ¯exible
high-throughput DNA extraction protocol for high-resolution
U. S. Copyright Clearance Center Code Statement:
mapping and map-based cloning in hexaploid wheat or other
plant species with complex genomes.
Fresh leaf tissue of wheat (Triticum aestivum L.) and maize plants
grown under di€erent conditions (greenhouse, climate chamber,
®eld) was used for this study. The ®rst two leaves of 2-week-old
wheat or maize seedlings or the equivalent amount (200±300 mg) of
leaf tissue of older plants (¯owering stage) were used for DNA
extraction. Plants were grown in a 96-well format tray and
identi®cation of individuals was guaranteed by their position instead
of by labelling (Fig. 1A). Leaves were harvested and immediately
transferred into plastic bags by cutting them into smaller pieces
(Fig. 1B). Custom-made polyethylene (PE) bags (45 ´ 95 mm,
Vinora AG, Jona, Switzerland) were used, but bags can also be
made from PE plain tubing (0.075 mm LD-PE transparent, Semadeni AG, Ostermundigen, Switzerland). After inserting leaves into
the four bags, 1.2 ml of cetyltrimethylammonium bromide (CTAB)
extraction bu€er (®nal concentration: 2% (w/v) CTAB, 200 mM
Tris/HCl pH 8.0, 20 mM EDTA pH 8.0, 1.4 M NaCl, 1% (w/v)
polyvinylpyrrolidone (K30), 1% (v/v) b-mercaptoethanol) was added
and air was carefully removed from the bags before they were
double sealed with a household bag sealer. The bags were placed on
a piece of cardboard covered with a layer of PE (0.1 mm LD-PE
transparent, Semadeni AG) attached to the bench and a second layer
of PE foil was placed over the bags. Leaf material was completely
homogenized (Fig. 1C) using a hand homogenizer (model 400010,
Bioreba AG, Reinach, Switzerland) (Fig. 1D), followed by incubation for 1 h at 65°C. The bags were separated from each other and
the solution was transferred into a 2.2 ml microcentrifuge tube after
folding each bag to a ®ne tip, which was cut and inserted into the
tube. The solution was extracted with 800 ll cold dichloromethane±
isoamylalcohol (24 : 1) (Chaves et al. 1995) by vertically rotating on
a shaker (Heidolph REAX2, Schwabach, Germany) for 15 min at
4°C followed by centrifugation at 10 000 g for 15 min. Eighthundred microlitres of the supernatants were pipetted into the slots
of a 2.0 ml polypropylene (PP) deep-well plate (96 wells, Matrix
Technologies, Manchester, UK) and treated with 5 ll RNase A
(1000 U/ml) for 15 min at 37°C. The deep-well plate was sealed for
incubation with a cap mat (for 2-ml deep well plate, Matrix
Technologies). For DNA precipitation a 0.7 volume of isopropanol
(560 ll) was added to each sample, the wells were sealed with the
cap mat and the solutions were mixed by inverting the block ®ve
times. The DNA was pelleted by centrifugation (20 min at 1850 g,
room temperature) and the supernatant removed by inverting the
block. To eliminate traces of the supernatant, the block was ®rmly
tapped upside down on a stack of paper towels. Washing the DNA
pellets was performed by adding 0.7 ml of washing solution 1 (76%
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DNA extraction method for high-throughput marker analysis in a large-genome species
355
Fig. 1: Homogenization of plant
material for DNA extraction
in high-throughput analysis. A.
2-week-old wheat seedlings grown
in a 96-well format tray. The ®rst
two leaves were collected for DNA
extraction. B. Leaf samples were
inserted and sealed into plastic
bags together with the extraction
bu€er. C. Plant sample after manual homogenization: only leaf
veins remained intact. D. Hand
tool for homogenization of the
plant tissue. Rubbing with the tool
across the bags led to even homogenization of the leaves
ethanol, 200 mM sodium acetate), sealing the block with the cap
mat, tilting the block to each side to wash the walls of the wells,
followed by incubation for 5 min at room temperature before
centrifugation (10 min at 1850 g). The supernatant was removed as
described earlier and the washing was repeated with washing
solution 2 (76% ethanol, 10 mM ammonium acetate). The DNA
pellets were subsequently air dried for at least 10 min and ®nally
resuspended in TE bu€er (10 mM Tris-HCl, 1 mM EDTA, pH 8.0).
The objective of this study was to develop a fast and ¯exible
DNA extraction protocol for high-throughput analysis in
hexaploid wheat giving enough yield for RFLP mapping
without the need to freeze leaf material. A method based on
modi®ed existing protocols for plant DNA extraction using an
extraction bu€er containing the non-ionic detergent CTAB was
established (Murray and Thompson 1980, Saghai-Maroof
et al. 1984). Fresh leaf material of di€erent developmental
stages was directly squashed in the extraction bu€er in sealed
plastic bags. While the method was especially designed to use
fresh material, frozen material can also be used if placed
quickly and directly into the extraction bu€er (data not shown).
In contrast to other methods using fresh material (Clarke et al.
1989, Tai and Tanksley 1990, Fulton et al. 1995) this new
technique did not require dehydration and milling, expensive
extra laboratory equipment, or extensive washing of any
apparatus between manipulation of di€erent samples.
Up to 100 lg of DNA were routinely obtained from a
minimal amount of 200 mg of leaf sample of an average
fragment size, which is comparable to DNA obtained by other
standard DNA extraction procedures (data not shown). This is
sucient for about four Southern blots and 200 PCR reactions
in wheat genomic analysis. The DNA quality was suitable for
restriction digests, RFLP and PCR analysis (Fig. 2). Plants
were grown in 96-well format trays which made the labelling of
individual plants unnecessary and reduced space requirements
for large numbers of plants. The DNA samples obtained were
then easily handled with eight-channel pipettes and 96-well
microplates.
The DNA yield between samples generally varied little if
the size of the leaf samples was kept constant. Therefore,
to speed up the procedure, the amount of DNA was
estimated only for some random samples and resuspension
followed in a standardized volume of TE bu€er (data not
shown).
The DNA extraction protocol described above is fast and
o€ers good ¯exibility. Two persons can easily handle 500
plants for DNA extraction in 2±3 days. Recently, the procedure was applied to high-resolution mapping for map-based
cloning in a population of > 3000 F2 wheat plants (Stein et al.
2000). In addition, the method can be interrupted at several
steps without any negative impact on DNA quality or yield:
(1) after squashing, the material can be left in the sealed bags
and samples can be stored for several hours at room
temperature or 4°C before or after the incubation at 65°C;
(2) the samples can be kept for at least 12 h at 4°C after
extraction with dichloromethane±isoamylalcohol before centrifugation; (3) the samples can be stored in washing solution 1
for at least 24 h at 4°C. Although not tested extensively,
storage of the samples at room temperature, at the steps
indicated, should also be possible. The option of interrupting
the procedure allows one to start at any time with a large
number of samples.
A single-plant DNA extraction protocol cannot always
be successfully applied to a broad range of plant species.
While this new method was designed especially for highresolution mapping or MAS of hexaploid wheat, it was
also possible to isolate genomic DNA from maize (data not
shown), barley, oat (C. Feuillet, pers. comm.), and tobacco
(C. Ringli, pers. comm.) for restriction analysis and PCR.
DNA was also obtained for random ampli®ed polymorphic
DNA (RAPD) PCR from Hypericum perforatum L., Cynara
scolymus L. and Valeriana ocinale L. (M. Messmer, pers.
comm.).
The advantage of this procedure over existing methods is the
method for plant tissue homogenization and sample processing. It allows DNA to be isolated in amounts sucient for
RFLP analysis from a large number of samples in a short time.
The ¯exibility of the procedure might also make it attractive
for applications in the ®eld.
356
S T E I N , H E R R E N and K E L L E R
References
Fig. 2: Demonstration of the quality of DNA obtained by the
presented method. A. Restriction digest of wheat genomic DNA.
HindIII-digests (approximately 20 lg of genomic DNA per digest) of
DNA isolated from 18 2-week-old wheat seedlings. B. Restriction
fragment length polymorphism (RFLP) analysis of the above on an
agarose gel after Southern blotting. Hybridization was performed with
a 32P-labelled barley RFLP-probe. The arrowheads indicate a
polymorphic locus detected by the probe. C. Sequence tagged site
(STS)±polymerase chain reaction (PCR) analysis of DNA dilutions of
the same samples. PCR products obtained after ampli®cation with
allele-speci®c STS primers analysed on a 1.5% agarose-gel (arrow
indicates the PCR products). The ®rst two lanes show no PCR
products because of the presence in these two samples of homozygotes
for the null allele
Acknowledgements
The authors thank Drs Catherine Feuillet, Nabila Yahiaoui and
Christoph Ringli for their critical reading of the manuscript. This work
was supported by the Swiss Priority Program Biotechnology of the
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