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Euphytica 130: 255–260, 2003.
© 2003 Kluwer Academic Publishers. Printed in the Netherlands. 255
A high-throughput DNA extraction method for barley seed
Rebecka von Post1,LarsvonPost
1, Christophe Dayteg1, Marie Nilsson1, Brian P. Forster2&
Stine Tuvesson1,∗
1Svalöf Weibull AB, SE-268 81 Svalöv, Sweden; 2Scottish Crop Research Institute, Invergowrie Dundee DD2 5DA,
U.K.; (∗author for correspondence; e-mail: stine.tuvesson@swseed.se)
Received 16 October 2001; accepted 25 October 2002
Key words: barley, seed, high-throughput, DNA extraction, marker assisted selection
Summary
A non-destructive, quick DNA extraction method for barley seed is described. The method is simple and consists
of drilling out a sample from the seed, adding sodium hydroxide, heating in a microwave oven and neutralizing
with Tris-HCl. The seed DNA extract can be used directly for PCR with extra cycles added to the PCR programme
compared to PCR programmes used for leaf extracts. This protocol was developed in particular for a microsatellite
marker genetically linked to barley yellow mosaic virus resistance, but it can be applied to other markers of interest
for barley breeding. The quick seed extraction protocol makes it possible to handle thousands of samples per day.
Extraction of DNA from seed also facilitates transfer of plant material compared to the long-distance transfer of
leaf samples.
Introduction
In plant breeding selection is performed for desirable
agronomic traits. Traditionally, the selection is based
on phenotype, e.g. selection for disease resistance can
be performed by growing plants in an infected field
and assessing infection.
Genotypic selection, particularly at the DNA level,
is rapidly becoming a preferred alternative, and can
be exploited in Marker Assisted Selection (MAS) to
identify desirable recombinants among segregating
populations. The process is non-destructive and after
selection the plants and the results can be returned to
the breeder who then can progress material speedily in
the breeding programmes.
A high-throughput MAS system requires simple
and rapid DNA extraction methods. Normally, ex-
traction of DNA is performed on leaf tissue, which
requires growing plants. This is a waste of seed, glass-
house and staff resources. In our lab two DNA extrac-
tion protocols are currently used; a standard protocol
(Cheung et al., 1993) that yields high quality DNA
with which one person can extract 48 to 72 samples
per day, and a quick extraction protocol (Dayteg et al.,
1998; Tuvesson et al., 1998) that yields low quality
DNA but nevertheless good enough for routine ana-
lyses. The quick DNA extraction procedure for leaf
samples simply consists of treatment with sodium hy-
droxide (NaOH), heating (on boiling water), crushing
and neutralizing with Tris-HCl, enabling one person
to extract 5000 samples per day.
DNA extraction performed on seed instead of leaf
tissue allows MAS to be carried out independently of
the growth season, and the time and glasshouse space
needed for growing the plants are saved. Most import-
antly, the seed can be analysed during the non-field
season, selected and prepared for the next breeding
cycle. Furthermore it is possible to send seed samples
internationally, this is difficult for leaf samples which
have to be kept on ice or lyophilised. DNA extraction
performed on seed has been described using single dry
seed of wheat and rice (Chungwungse et al., 1993) and
seed of 12 species including wheat, rice and barley
(Kang et al., 1998).
This paper describes the development of a quick
DNA extraction protocol from barley seed.
256
Materials and methods
Plant material
Seed (caryopses) of winter barley cultivars Frost,
Grete, Igri, Jana (rym4), Misato Golden (rym5), Mok-
usekko (rym5)andVixen(Yd2) and spring barley
cultivars Alexis, Alva, Cecilia, Chevron (Rpg1), Cor-
acle (Yd2), Ellice (Rpg1), Filippa, Goldie, Hanka
(Rph7), Lina, Meltan, Mentor, Pongo, and SW 3756-
99 (Rph16). Seed of a F2winter barley population, SW
99692, segregating for rym5.
DNA extraction from seed
A one-mm diameter drill-bit (VellemanRelectric drill
& engraving set. VTHD21B-DC 18V, 12–20000 rpm)
was used to bore out samples from dry barley seed.
While drilling, the seed were kept in place with
forceps on an aluminium surface (Figure 1). Note:
caution should be taken during the drilling step.
The homemade aluminium tray was prepared using a
punch and hammer to make indentations in the alu-
minium surface. The drill-bit and the aluminium tray
can be cleaned easily between samples by action in
water. After drilling, the seed was positioned in a
microtitre plate and the flour positioned with a small
spoon in the corresponding well of another plate for
extraction: 40µl 0.15 M NaOH were added to each
sample and heated in a microwave oven at 10% power
of 700 W for one minute. Next, 150µl 0.03M Tris-
HCl, pH 8.0, 1 mM EDTA was added and the samples
were left to settle at +4 ◦C for at least one hour before
PCR. Samples were diluted 1:10 or 1:20 with cold wa-
ter before PCR. One to ten extra cycles were added to
the established PCR programmes for leaf DNA.
PCR conditions
PCR reactions were performed on a Peltier Thermal
Cycler from MJ Research. All PCR experiments took
place in a total volume of 25 µ1 with 2 µl seed DNA
extract (diluted in water or undiluted) and 2.5 µ110×
buffer (10x, 200 mM Tris-HCl, pH 8.4, 500 mM
KCl, Gibco BRL) was used for all experiments. The
stock solutions for PCR were: 50 mM MgCl2(Gibco
BRL), 100 mM d’NTPs (Amersham Pharmacia Bi-
otech), primer F (10 pmol/µ1), primer R (10 pmol/µ1)
and Taq DNA polymerase (5 u/µl, Gibco BRL).
Barley Yellow Mosaic Virus resistance, rym4, rym5
The marker for barley yellow mosaic virus (BaYMV)
resistance, rym4,rym5, (Graner et al., 1999), was
Bmac0029 (primer sequence available from The Scot-
tish Crop Research Institute, R Waugh). The seed
DNA extract was diluted 1:10. From stock solutions
0.75 µlMgCl
2,0.2µl d’NTPs, 0.75 µl of each primer
and 0.15 µlTa q DNA polymerase were added to the
PCR mix. The PCR programme was: 1 cycle of (13
min at 94 ◦C, 1 min at 58 ◦C, 1 min at 72 ◦C), 40
cycles of (30 s at 94 ◦C, 30 s at 58 ◦C, 30 s at 72 ◦C), 5
min at 72 ◦C, 20 ◦C ‘forever’. This is the PCR protocol
established for leaf DNA with 10 additional cycles.
Barley Yellow Dwarf Virus resistance, Yd2 (YLM)
The marker for barley yellow dwarf virus (BYDV)
resistance, Yd2, (YLM) was published by Paltridge et
al., 1998. Undiluted seed DNA extract was used for
PCR. From the stock solutions 0.75 µlMgCl
2,0.2µl
d’NTPs, 1.5 µl of each primer and 0.2 µlTa q DNA
polymerase were added to the PCR mix. The PCR pro-
gramme was: 42 cycles of (30 s at 94 ◦C, 30 s at 58 ◦C,
1minat72◦C), 10 ◦C ‘forever’. This is the PCR
protocol established for leaf DNA with 2 additional
cycles.
Barley Yellow Dwarf Virus resistance, Yd2 (YLPRAS)
The PCR marker for BYDV resistance, Yd2,(YL-
PRAS) was published by Ford et al., 1998. The seed
DNA extract was undiluted. From the stock solutions
2.25 µlMgCl
2,0.3µld’NTPs,0.6µl of each primer
and 0.1 µlTaq DNA polymerase were added to the
PCR mix. The PCR programme was: 39 cycles of (1
min at 94 ◦C, 2 min at 50 ◦C, 3 min at 72 ◦C), 10 ◦C
‘forever’. This is the PCR protocol established for leaf
DNA with 4 additional cycles.
Leaf rust resistance, Rph16
To the PCR mix for the marker for leaf rust resistance,
Rph16, (MWG2133) (Ivandic et al., 1998) was added
undiluted seed DNA extract. From the stock solutions
0.75 µlMgCl
2,0.2µl d’NTPs, 1.25 µl of each primer
and 0.2 µlTaq DNA polymerase were added to the
PCR mix. The PCR programme was: 5 min at 94 ◦C,
37 cycles of (30 s at 94 ◦C, 30 s at 60 ◦C, 1 min
at 72 ◦C), 5 min at 72 ◦C, 20 ◦C ‘forever’. This is
the PCR protocol established for leaf DNA with 2
additional cycles.
257
Figure 1. a. Samples were taken from barley seed using a one-mm drill-bit and DNA extracted from the flour. The seed can be grown on by the
breeder at any suitable time. b. Necklace of barley individuals homozygous for a BaYMV marker.
Stem rust resistance, Rpg1
To the PCR mix used for the marker for stem rust res-
istance, Rpg1, (ABG077) (Horvath et al., 1995) was
added undiluted seed DNA extract. From the stock
solutions 0.75 µlMgCl
2,0.2µl d’NTPs, 1.25 µlof
each primer and 0.2 µlTa q DNA polymerase were
added to the PCR mix. The PCR programme was: 40
cycles of (30 s at 94 ◦C, 45 s at 50 ◦C, 1 min at 72 ◦C),
20 ◦C ‘forever’. This is the PCR protocol established
for leaf DNA with no additional cycles.
Leaf rust resistance, Rph7
A marker associated with leaf rust resistance, Rph7,
(MWG848), (Mano et al., 1999), was made available
from Institut für Pflanzengenetik und Kulturpflanzen-
forschung, (Dr A. Graner). The seed DNA extract was
diluted 1:10. From the stock solutions 1.0 µlMgCl
2,
0.2 µl d’NTPs, 0.75 µl of each primer and 0.125 µl
Taq DNA polymerase were added to the PCR mix. The
PCR programme was: 5 min at 95 ◦C, 34 cycles of (1
min at 95 ◦C, 1 min at 62 ◦C, 2 min at 72 ◦C), 7 min
at 72 ◦C, 20 ◦C ‘forever’. This is the PCR protocol
established for leaf DNA with 4 additional cycles.
Epi-heterodendrin, Eph
Primer sequences for the marker (Bmac0213) for epi-
heterodendrin (EPH) production, Eph, (Swanston et
al., 1999) was provided by the Scottish Crop Research
Institute, R Waugh. The seed DNA extract was diluted
1:10 . From the stock solutions 0.75 µlMgCl
2,0.2µl
d’NTPs, 0.75 µl of each primer and 0.15 µlTa q DNA
polymerase were added to the PCR mix. The PCR
programme was: 1 cycle of (13 min at 94 ◦C, 1 min
at 58 ◦C, 1 min at 72 ◦C), 34 cycles of (30 s at 94 ◦C,
30 s at 58 ◦C, 30 s at 72 ◦C), 5 min at 72 ◦C, 20 ◦C
‘forever’. This is the PCR protocol established for leaf
DNA with 4 additional cycles.
258
Gel electrophoresis
Bmac0029, Bmac0213 and YLM: The fragments
were separated on 3.5% MetaPhor agarose gel
(BioWhittaker Molecular Applications) in 1 ×TBE
(89mMTris-borate,boricacid89mM,2mMEDTA
pH 8.0) Running buffer was 1 ×TBE and the con-
ditions for electrophoresis were 200 V, 250 mA for
1.3 h.
ABG077, MWG848 and YLPRAS markers: The frag-
ments were separated on 1.4% standard agarose gel
(Saveen) in 1 ×TBE. Running buffer was 1 ×TBE
and the conditions for electrophoresis were 200 V,
250 mA for 1.3 h.
MWG2133: The DNA products were separated on
1.0% standard agarose gel (Saveen) in 1 ×TBE.
Running buffer was 1 ×TBE and the conditions for
electrophoresis were 180 V, 200 mA for 6.3 h.
The 300 ml gels contained 25 µl ethidium brom-
ide (10 mg/ml) and the results were visualised by UV
light.
Results and discussion
Extraction experiments were carried out on dry seed
of winter and spring barley varieties. The project was
divided into three parts: 1) sampling, 2) extraction
and 3) PCR. During development of the final extrac-
tion protocol the extracts were tested for PCR with
a microsatellite marker, Bmac0029 genetically linked
to BaYMV resistance (Graner et al., 1999). The fi-
nal extraction protocol was verified using six PCR
based markers used for MAS and the PCR procedure
optimised for seed DNA extract. Finally an F2popula-
tion segregating for BaYMV resistance were analysed
from seed and leaf.
Sample preparation
To find the best way of taking a sample from the seed
different methods were tried. Our aim was to find a
way of extracting DNA from barley seed that is quick
and simple, gives DNA that is pure enough for PCR,
does not kill the seed and can be done in the microtiter
(96 well) format. Cutting pieces of varying sizes from
the seed creates the need for a crushing/grinding step
and since barley seed tissue is very hard this cannot be
easily done by hand with a pestle. Various approaches
were tried in order to find a way to crush the seed fairly
quickly and easily: a) crushing seed pieces of varying
sizes between papers with a hammer (which is not very
efficient because insufficient cell walls are broken).
Also, barley seed consist mostly of starch which, when
subjected to sodium hydroxide and heat, becomes a
gluey pulp from which it is difficult to extract DNA. b)
To avoid getting too much starch in the extraction very
thin slices from the centre of the seed were cut out and
subjected to the extraction procedure without crushing
but this did not improve the results. c) In order to grind
the material more finely seed were cut in two and the
piece without the embryo abraded using sandpaper,
which rendered a very fine dust that was collected with
a drop of water or NaOH and a pipette. This method
worked quite well but it is not practical for large scale
screening. d) Another attempt at getting very finely
ground samples was made by drilling a hole through
the centre of a seed with a one-mm diameter drill-bit
and then collecting the flour. Drilling was considered
to be the best method for sampling barley seed con-
sidering both the efficiency of the extraction and the
handling of the samples, and was the method chosen
for the final protocol (Figure 1).
Seed DNA extraction
The quick DNA extraction protocol for leaf tissue cur-
rently used at our lab, in a high-throughput system,
consists of the following steps: Cut leaf samples with
a paper punch and placing them in a microtitre plate.
Add 40 µl 0.25 M NaOH. Hold the plate on a boiling
water bath for one minute, crush the samples with a
‘multi pestle’ and neutralize with 120 µl0.1MTris-
HCl. Use 1.3 µlfora25µl PCR reaction (Dayteg et
al., 1998; Tuvesson et al., 1998).
The quick DNA extraction protocol of seed mater-
ial requires some modifications due to the high starch
content of cereal seed. NaOH is needed to break cell
walls but the boiling step was replaced with incuba-
tion at room temperature or at 50 ◦C (waterbath) for
varying periods of time to prevent geling. Various
concentrations of NaOH were tried. We found that
lowering the NaOH concentration to 0.15 M and in-
cubating at room temperature for 50 minutes works
well. The time can be shortened to 20 minutes if in-
cubation is done at 50 ◦C. The Tris-HCl concentration
was lowered to 0.03 M and the volume added was in-
creased to 150 µl compared to the quick leaf extraction
protocol. Adding 1mM EDTA to the Tris-HCl buf-
fer also improved results slightly. Later in the project
period we tried replacing the boiling step with heating
259
Figure 2. Marker assisted selection based on SSR-PCR with seed DNA extract. PCR products from Bmac0029 primer for BaYMV resistance
(Graner et al., 1999). Segregating individuals from an F2population showing homozygous resistant plants (160 bp fragment), homozygous
susceptible plants (176 bp) and heterozygous plants with both fragments.
in a microwave oven (Saini et al., 1999) and this gave
the best results.
After extraction the samples were left to settle
+4 ◦C for at least one hour before PCR to allow the
starch, which otherwise disturbs the PCR reaction to
sink to the bottom. Then the ‘supernatant’ can be used
for PCR. The sedimentation step cannot be replaced
with centrifugation since it appears that the DNA
goes down with the starch and leaves the supernatant
useless.
Since the extraction can be carried out in a mi-
crotitre plate the extracts can be used in an automated
high-throughput MAS system.
PCR
During evaluation of the different sampling and ex-
traction methods for seed, PCR was carried out with
2µl extract for a 25 µl reaction with a microsatellite
marker for resistance to BaYMV. This worked well
for comparing extraction methods, but never gave very
strong and reliable bands. After establishing the final
quick extraction protocol the PCR protocol had to be
optimized for seed extracted DNA, which is not very
pure and has a very low concentration of DNA. 1)
Since the extracts contain very low amounts of DNA
the resulting bands are weak. Adding one to ten extra
cycles to the PCR programme solved this problem. 2)
To lessen the effects of PCR inhibiting substances the
extract was diluted 1:10 and 1:20 in cold water after
sedimentation. The undiluted extract gave low repro-
ducibility with either of the PCR programmes, but
the 1:10 dilution gave increasingly strong bands and
reproducibility of the results with each added cycle.
Verification
The presented method was developed in particular for
the marker analysis of BaYMV resistance (Graner et
al., 1999) (Figure 2). Resistance to this viral dis-
ease is one of the most important characters in winter
barley breeding and MAS makes possible fixation of
resistance early in the breeding process.
To verify the extraction protocol and test its use-
fulness in MAS a final test was set up with seven dif-
ferent markers useful in barley breeding including the
BaYMV resistance marker used for the experiments.
YLM and YLPRAS are markers for Yd2 conferring
BYDV resistance (Paltridge et al., 1998; Ford et al.,
1998). This viral disease is devastating to spring and
winter barley in years with heavy aphid (vector) in-
fection. Markers for resistance to rust diseases, which
have fungal causal agents, also responded well to
the method developed for Bmac0029: MWG848 on
barley chromosome 3H (Mano et al., 1999) where
an important leaf rust gene, Rph7, is located and
ABG077 linked to the stem rust gene Rph1 (Hor-
vath et al., 1995) worked well with the seed DNA
extract. MWG2133, linked to a new source of leaf
rust resistance (Rph16) (Ivandic et al., 1998) respon-
ded well.The marker Bmac0213 for epi-heterodendrin
(EPH) production (Swanston et al., 1999) also re-
sponded well to the developed method. EPH is a
precursor for ethyl carbamate, which is an undesirable
trace component in malt whisky distilling and thus
important for breeding malting barley cultivars.
All markers were tested with undiluted barley seed
DNA extract and with dilutions 1:10 and 1:20. The
PCR reactions were run with the original PCR pro-
gramme and with one, two, three, four and ten extra
cycles and the results compared to results for quick
extracted leaf DNA extracts (Dayteg et al., 1998;
260
Tuvesson et al., 1998) from the same individuals. The
different markers responded differently to the various
treatments and the resulting optimised seed DNA ex-
tract dilution and PCR programmes are described in
the ‘Materials and Methods’ section. The experiments
showed that the extract can be used with all the mark-
ers tested and that the seed extract and the leaf extract
give similar results.
Segregating F2population
An issue that was further investigated is whether the
seed coat, which is of maternal origin and/or the
endosperm, which is triploid (2/3 maternal and 1/3
paternal) give rise to false results for the embryo. If so
the technique would not be applicable to heterozygous
material and therefore of limited use to plant breeders.
Using the seed DNA extraction protocol 175 F2seed
from a barley population segregating for BYMV res-
istance were analysed with Bmac0029and the banding
profiles were identical to the results obtained for leaf
DNA after germination.
The successful development of a quick DNA ex-
traction protocol for seed for use in barley breeding
makes possible a much more flexible planning of
marker analysis. The quick DNA extraction protocol
is especially useful for laboratories which offer inter-
national DNA analysis services and which until now
have been dependent upon the transfer of frozen or
lyophilised leaf material.
Acknowledgement
The Swedish Farmer’s Foundation (SLF) is acknow-
ledged for financial support.
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