Plant Breeding 123, 225—228 (2004)

2004 Blackwell Verlag, Berlin

ISSN 0179-9541

Single nucleotide polymorphism genotyping of the barley waxy gene by polymerase

chain reaction with confronting two-pair primers
E . D o m o n 1 , T . Y a n a g i s a w a 2 , A . S a i t o 1 and K . T a k e d a 3
1
National Agricultural Research Organization, National Agricultural Research Center for Kyushu Okinawa Region, 2421 Suya,
Nishigoshi, Kikuchi, Kumamoto 861-1192, Japan, E-mail: domon@affrc.go.jp; 2 National Agricultural Research Organization,
National Agricultural Research Center for Western Region, Shikoku Research Center, 1-3-1 Senyu, Zentsuji, Kagawa 765-8508,
Japan; 3 Research Institute for Bioresources, Okayama University, 2-20-1, Chuo, Kurashiki, Okayama 710-0046, Japan
With 3 figures and 1 table
Received July 23, 2003/Accepted October 29, 2003
Communicated by C. Jung

Abstract (MAS) is now a practical choice for the breeding of an elite

A high-throughput single nucleotide polymorphism (SNP) genotyping
procedure was developed to select amylose-free barley mutants whose
waxy genes had a C- to T-base substitution in exon 5, which converted
Gln-89 of the wild-type gene into a termination codon. An F2
population carrying an amylose-free waxy gene was checked for
segregation. Polymerase chain reaction with confronting two-pair
primers (PCR-CTPP) produced allele-specific PCR products that have
different sizes and are inherited in a co-dominant manner. Two alleles
of the barley waxy gene with SNP were correctly identified in parental
strains using the PCR-CTPP procedure. Segregation of the SNP as
detected by PCR-CTPP in an F2 population fitted the expected
1 : 2 : 1 ratio. The PCR-CTPP procedure can provide a time saving
and cost-effective alternative to derived cleaved amplified polymorphic
sequence in marker-assisted selection.
Key words: Hordeum vulgare — beta-glucan — dCAPS —
DNA marker — marker-assisted selection — PCR-CTPP —
SNP — waxy gene
In the East Asian countries, especially in Japan, Korea and
China, barley has been traditionally used for human food,
developing products such as rice extender, seasoning called
ÔmisoÕ, roasted barley tea and liquor. The endosperm of waxy
barley has a high content of beta-glucan, for an unknown
reason (Izydorczyk et al. 2000), the health benefits of which
have been shown (Jenkins et al. 2002).
The barley waxy gene encodes a granule-bound ADP-
glucose-glucosyl transferase, which catalyses the synthesis of
amylose (Rohde et al. 1988). The starch component in barley
endosperm that carries the Wax allele is composed of about
27% amylose and 73% amylopectin, but homozygous wax
endosperm normally contains 2–10% amylose and 98–90%
amylopectin (Ishikawa et al. 1995). Several waxy barley
cultivars have been registered in Canada and Australia. On
the contrary, non-waxy barley cultivars are commercially used
in Japan. Therefore, the simple way to breed an amylose-free
waxy barley cultivar could be to backcross an amylose-free
waxy mutant into a non-waxy genetic background of an elite
strain.
An amylose-free waxy barley mutant was produced by
Ishikawa et al. (1995), and a recent study revealed a point
mutation in the waxy gene of that mutant (Domon et al. 2002).
The introduction of the amylose-free waxy gene into a non-
waxy genetic background by using marker-assisted selection
amylose-free strain.
Large-scale screening procedures have been developed to
detect single nucleotide polymorphisms (SNPs), such as
Masscode (Kokoris et al. 2000) or TaqMan (Heid et al.
1996). Another SNP detection method, called derived cleaved
amplified polymorphic sequence (dCAPS) (Neff et al. 1998), is
suitable for middle-scale SNP screenings, such as are needed
for plant breeding aspects, although dCAPS requires time-
consuming post-amplification enzymatic cleavage. A simple
SNP detection procedure, called polymerase chain reaction
with confronting two-pair primers (PCR-CTPP) (Hamajima
et al. 2000, 2002), also known as tetra-primer ARMS-PCR
(Ye et al. 2001), is a suitable alternative to dCAPS for MAS.
The PCR-CTPP procedure, a sort of multiplex PCR, produces
allele-specific PCR products of different sizes by adding four
primers into one tube as shown in Fig. 1. Thereafter, the PCR-
CTPP products are separated on an agarose gel by electro-
phoresis. As the allele-specific PCR-CTPP products of differ-
ent sizes are inherited in a co-dominant manner, it should be a
powerful tool for MAS in successive backcrossing. This paper
reports on a feasibility study of the application of PCR-CTPP
for mutant selection in the barley waxy gene.
Materials and Methods
Plant materials: Two hull-less barley (Hordeum vulgare ssp. vulgare)
strains were used. ÔShikoku hadaka 84Õ (ÔSH84Õ) is a two-row, non-waxy
breeding line developed at the Agricultural Research Center for the
Western Region, formerly the Shikoku National Agricultural Experi-
ment Station. ÔShikoku hadaka 97Õ (ÔSH97Õ) is a two-row waxy mutant
with an amylose-free phenotype; it was artificially induced from ÔSH84Õ
by sodium azide treatment (Ishikawa et al. 1995, Yasui et al. 2002). The
amylose-free strain has a base substitution of C to T in exon 5 of the
waxy gene, thereby converting Gln-89 of the wild-type gene into a
termination codon (Domon et al. 2002). An F2 population of 100
individuals, which derived from a cross between ÔYon kei 9311Õ and ÔYon
kei 9456Õ, was checked for the segregation ratio of the SNP in waxy gene.
ÔYon kei 9456Õ has the amylose-free waxy gene derived from ÔSH97Õ.
DNA extraction and PCRs: Total DNA was extracted from green
leaves by a modified cetyltrimethylammonium bromide (CTAB)
procedure (Briard et al. 2000). The DNA concentration was assessed
using fluorescent dye Hoechst 33258 in a spectrofluorometer (F-4010,
Hitachi, Tokyo, Japan).

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226 D o m o n , Y a n a g i s a w a , S a i t o and T a k e d a

Each PCR was carried out in a 10 ll mixture of 60 ng DNA

380 bp template, 0.2 lM of the FP primer, 0.2 lM of the RP primer, 0.4 lM of
the WD primer; 0.3 or 0.4 lM of the MD primer; 200 lM of dNTPs, 1x
FP AccuPrime Taq reaction buffer (20 mM Tris–HCl pH 8.4, 50 mM KCl,
Wild-type 1.5 mM MgCl2); and 0.2 ll of AccuPrime Taq DNA polymerase
allele 5' C 3' (Invitrogen, Carlsbad, CA, USA). The GeneAmp PCR system 9700
(Applied Biosystems, Foster City, CA, USA) was used for PCR. Two-
G WD RP temperature cycles were used for PCR: denaturing at 96C for 2 min
323 bp and annealing/extension at XC for 25 s for one cycle, followed by 29
cycles at 96C for 5 s and annealing/extension at XC for 25 s, where
the annealing/extension temperature, X, was changed from 65C to
71C to find the optimal annealing condition. The DNA fragments
FP MD A resulting from the PCR were separated by gel electrophoresis on a 3%
Mutant agarose gel (Invitrogen), 1x TBE (89 mM Tris, 89 mM boric acid and
allele 5' T 3' 2 mM EDTA). The gel was stained with GelStar nucleic acid stain
(Cambrex, Rockland, ME, USA) and photographed on a Dark Reader
RP
109 bp transilluminator (Clare Chemical Research, Dolores, CO, USA).

Fig. 1: Schematic representation of polymerase chain reaction with
confronting two-pair primers (PCR-CTPP). The single nucleotide
polymorphism used here as an example is C to T substitution. The
allele specificity of each primer is conferred by a mismatch between the
3¢-terminal base of an allele-specific primer and the template. Two
allele-specific PCR products are generated using two primer pairs: one
an FP/WD pair producing a DNA fragment representing the C allele
of 323 bp, and the other an MD/RP pair producing a DNA fragment
representing the A allele of 109 bp. A DNA fragment of 380 bp from
an outer primer of FP/RP pair is generated in common with each
genotype. By positioning the two outer primers at different distances
from the polymorphic nucleotide, the difference in size of the resulting
PCR-CTPP fragments from each allele makes it easy to discriminate
between two fragments by gel electrophoresis
PCR direct sequencing: The PCR products from a portion of the F2
population were subjected to PCR direct sequencing, in order to
confirm the accuracy of the PCR-CTPP genotyping. The PCR products
were generated with FP and RP primers as described above. Total
DNA from each of four individuals from the F2 population with wild-
type homozygous, heterozygous and mutant homozygous genotypes,
previously scored by PCR-CTPP genotyping, were subjected to PCR
amplification. The resulting PCR products were purified using a PCR
purification kit (Qiagen K.K., Tokyo, Japan) before DNA sequencing.
Direct sequencing of the PCR products was performed on both strands
with FP and RP primers using the Big Dye Terminator ver. 3.1 Cycle
sequence kit (Applied Biosystems, ???, Japan) on a ABI PRISM 3100
DNA sequencer (Applied Biosystems, Foster City, CA, USA).

As shown in Fig. 1, four primers were selected from the genomic

DNA sequence of the barley waxy gene of ÔSH84Õ (DNA Data Bank of
Japan databases accession no. AB088761) and of ÔSH97Õ (AB089162).
The four primers were: FP (5¢-CGC AGA TCA AGG TCG CTG
ACG-3¢; the primer melting temperature (Tm) at primer concentration
50 nM and salt concentration 50 mM was 60.2C); RP (5¢-GGG ACC
AGA AAA GTA GGG GTT GTT GTT GA-3¢, 61.5C); MD
(5¢- CGG CAC GGA CTA CGA GGA CAA CCC GT-3¢, 66.7C);
and WD (5¢-TGC CTG GCA GAG AAG GCT GAA GCG ATG-3¢,
65.5C). The specificity of each allele-specific primer was conferred by
a mismatch between the 3¢-terminal base of the primer and that of the
template. The Tm of each oligonucleotide primer was calculated by the
nearest-neighbour method.
Results
The PCR-CTPP procedure successfully detected a base
substitution of C to T in exon 5 of the barley waxy gene.
As shown in Fig. 1, the four primers consist of FP and WD for
the wild-type allele, giving a 323 bp fragment; and MD and
RP, giving a 109 bp fragment for the mutant allele. FP and RP
produce a 380 bp band in common. In the process of finding a
suitable condition for genotyping via PCR-CTPP, several
annealing temperatures were examined in order to avoid
mismatching the allele-specific primers. Figure 2a shows a gel
image of different annealing conditions. From left to right, the

(a) (b)
M 1 2 3 4 5 6M 7 8 9 10 11 12 M M 13 14 15 16 17 18 M

380 bp
323 bp

109 bp

CC CT TT CC CT TT CC CT TT CC CT TT CC CT TT CC CT TT
65 67 69 71 WD : MD = 0.4 : 0.4 WD : MD = 0.4 : 0.3

Fig. 2: (a) Gel image showing the different genotypes of the barley waxy gene. Four annealing temperatures were examined: lanes 1–3, 65C;
lanes 4–5, 67C; lanes 7–9, 69C and lanes 10–12, 71C. Polymerase chain reaction was conducted for template DNA from three different
genotypes of CC (ÔSH84Õ), CT (F2 individual) and TT (ÔSH97Õ). (b) Gel image showing simple optimization procedure of the MD primer
concentration. The concentration of the MD primer was reduced from 0.4 lM (lanes 13–15) to 0.3 lM (lanes 16–18). The template DNA from
three different genotype of CC (ÔSH84Õ, lanes 13 and 16), CT (F2 individual, lanes 14 and 17) and TT (ÔSH97Õ, lanes 15 and 18) were used. Lane M
contains a 100-bp DNA ladder
SNP genotyping by PCR-CTPP in barley waxy gene
Table 1: Results of polymerase chain reaction (PCR) direct sequen-
cing and PCR with confronting two-pair primers (CTPP) genotyping
of 12 individuals from an F2 population of a cross between ÔYon kei
9311Õ and ÔYon kei 9456Õ
PCR-CTPP genotyping
PCR direct sequencing W H
Gln codon CAG 4 0
Heterozygous [C/T]AG 0 4
Termination codon TAG 0 0
W, homozygous for wild-type allele; H, heterozygous; M, homozygous
for mutant allele.
annealing temperatures were 65C, 67C, 69C and 71C. In all
annealing/extension temperatures tested, except 65C, three
genotypes CC, CT and TT were correctly and easily distin-
guished. ÔSH84Õ, a wild-type strain possessing the CC genotype,
showed a 380 bp band generated from the outer primers and a
wild-type allele-specific 323 bp band generated from the FP
and WD primers (lanes 1, 4, 7 and 10). ÔSH97Õ, an amylose-free
mutant that has the TT genotype, also showed a 380 bp band
generated from the outer primers, as well as a mutant-specific
109 bp band generated from the RP and MD primers (lanes 3,
6, 9 and 12). The F2-population heterozygote having the CT
genotype showed a 380 bp band, a 323 bp band and a 109 bp
band (lanes 2, 5, 8 and 11, respectively). According to the
manufacturer’s recommendation, the optimal extension tem-
perature for AccuPrime Taq DNA polymerase was set at 68C,
but non-specific amplification from the MD and RP primers of
the 109 bp product was detected in the CC parent, and an
allele-specific fragment of 323 bp for the wild-type allele was
somewhat faint in the CT heterozygote at an annealing/
extension temperature of 68C (data not shown) and below
(Fig. 2a, lanes 1, 4). Because the non-specific 109 bp band in
the CC parent disappeared at 69C (Fig. 2a, lane 7), the
annealing/extension temperature of 69C was used for further
experimentation.
In SNP detection by PCR-CTPP, balanced amplification
between the two allele-specific DNA fragments is important
(Hamajima et al. 2000). At a settled annealing temperature of
CT CT TT
CT CT CT CT CT CC TT
Fig. 3: A gel image showing geno-
types of 24 individuals from an F2
population of a cross between ÔYon
kei 9311Õ and ÔYon kei 9456Õ. CC,
CT and TT represent three differ-
ent genotypes. The genotype of
each individual was confirmed by
polymerase chain reaction direct
sequencing
227
69C, the intensity of the wild-type allele-specific 323 bp band
was weaker than that of a mutant allele-specific 109 bp band in
the CT heterozygote (Fig. 2a). Hence the concentration of the
wild-type allele-specific primer was examined for balanced
amplification. Figure 2b shows the effect of the allele-specific
primer concentration on amplification balance. In lanes 13–15,
M
0.4 lM of the WD and MD primers was used, and in lanes
0 16–18, 0.4 lM WD primer and 0.3 lM MD primer were used.
0 The reduction of the MD primer concentration achieved
4 balanced amplification between the wild-type allele-specific
323 bp band and the mutant allele-specific 109 bp band in the
CT heterozygote (Fig. 2b, lane 17).
In order to confirm the accuracy of PCR-CTPP genotyping,
12 individuals from the F2 population were subjected to PCR
direct sequencing. The DNA fragments generated from the
PCR with FP and RP primers were sequenced on both strands.
Table 1 shows the summarized results of PCR direct sequen-
cing and PCR-CTPP genotyping of the 12 individuals. The
DNA sequences of interest are the three nucleotides coding for
a Gln (CAG) or a termination codon (TAG). The results of
PCR direct sequencing were in accordance with the results of
PCR-CTPP genotyping.
To test the segregation ratio of the SNP in the F2
population, the F2 population derived from a cross between
ÔYon kei 9311Õ and ÔYon kei 9456Õ was twice scored by PCR-
CTPP. The gel image shows the results for 24 individuals from
the F2 population (Fig. 3). The segregation ratio of the F2
population of 100 individuals was 31CC : 50CT : 18TT, which
fitted the expected 1 : 2 : 1 ratio for a single locus with two
alleles (v2 ¼ 3.501, P ¼ 0.1737). One individual was not
properly scored because of a weak signal of the PCR products.
Discussion
A feasibility study of MAS in the barley waxy gene in an F2
population is presented here. In this study, a point mutation in
that gene is directly tagged by a C- to T-base substitution,
PCR-CTPP successfully genotyped the alleles in the parental
plant as well as in the F2 progeny in a co-dominant manner. In
cases where a recessive gene is introduced into the genetic
background of an elite strain, MAS could be effective as a
CT CT CT CT TT TT CT TT CT
380 bp
323 bp
109 bp
CC TT CT CC CC
380 bp
323 bp
zzaabp
228
direct or an indirect selection tool for successive backcrossing.
A recessive gene such as the amylose-free waxy gene will be
masked by the wild-type phenotype in the back-cross progeny
and will not be selected. Successive backcrossing leads to a
1 : 1 segregation ratio of the wild-type homozygote and
heterozygote in the progeny population, PCR-CTPP could
exactly genotype individuals in an optimal PCR condition.
Optimal annealing temperatures and PCR primer concen-
trations must be determined empirically. PCR-CTPP also
requires a two-step optimization strategy to achieve balanced
amplification between alleles: the first step is to change the
annealing temperature and the next is to change the primer
concentration. To make it easier to determine the optimal
annealing conditions, Hamajima et al. (2002) used a three-step
cycle for PCR and preferred similar Tm for the four primers at
an annealing temperature of around 60C. In the current
study, on the contrary, the Tm was varied (from 60.2C to
66.7C) among the four primers. The allele-specific primers
WD (66.7C) and MD (65.5C) were designed to have higher
Tm than the outer primer pairs (60.2C and 61.5C) to ensure
allele distinction at the higher annealing temperature of 69C.
The chief concerns in optimizing primer concentrations are to
balance the band intensities of the PCR products and to
suppress non-specific amplification. Waterfall and Cobb (2002)
extensively tested concentrations of primers, dNTPs and
MgCl2, although application of the Taq polymerase designed
to reduce non-specific amplification by preventing non-specific
priming would be a practical shortcut to attain balanced
amplification in multiplex PCR. In this report, balanced
amplification was achieved simply by using AccuPrime Taq
DNA polymerase (Invitrogen), and by reducing the MD
primer concentration from 0.4 to 0.3 lM. An extensive trial for
optimization was not needed.
The 12 previously genotyped progeny from the F2 popula-
tion by PCR-CTPP genotyping were subjected to PCR direct
sequencing. The DNA sequence revealed the genotype of
the individuals, which properly reflected the prediction of
PCR-CTPP genotyping (Table 1). Therefore, the accuracy of
the PCR-CTPP genotyping was unambiguously confirmed
through PCR direct sequencing.
The advantage of the PCR-CTPP procedure lies mainly in
the extreme simplicity of the procedure in comparison with
dCAPS. The SNP genotyping procedure with PCR-CTPP
requires only PCR amplification of allele-specific DNA frag-
ments and agarose gel electrophoresis, whereas dCAPS
demands time-consuming enzymatic digestion of amplified
DNA fragments. Recently, SNP detection by bidirectional
allele-specific amplification (ASA) combined with a real-time
PCR approach was reported (Waterfall and Cobb 2002). The
bidirectional ASA is substantially the same as PCR-CTPP.
Instead of gel electrophoresis after the amplification cycle,
SNP genotyping by real-time PCR conducts a melting curve
analysis. Therefore, the bidirectional ASA combined with a
real-time PCR approach would be an alternative to gel
electrophoresis-based PCR-CTPP or dCAPS. However, a
real-time PCR apparatus is required.
In conclusion, the feasibility study for MAS by PCR-CTPP
demonstrated the technical advantages of procedure in geno-
typing. The co-dominant inheritance of the allele-specific
PCR-CTPP marker enabled the selection of a recessive
genotype in an F2 population, thus implying the feasibility of
using MAS in successive backcrossing. Rapid genotyping by
D o m o n , Y a n a g i s a w a , S a i t o and T a k e d a
PCR-CTPP would be an alternative to dCAPS because of the
simplicity of the procedure. In addition, the Taq DNA
polymerase, which was designed to reduce non-specific ampli-
fication, makes it easy to optimize the annealing temperature
and the primer concentration. This allows greater freedom in
the PCR-CTPP condition, including primer design.
Acknowledgements
The authors thank Dr Shigeo Matsui for invaluable advice and
Dr Naoyuki Ishikawa for development of a novel amylose-free barley
mutant.
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