c Indian Academy of Sciences
RESEARCH ARTICLE
Marker-assisted selection of high molecular weight glutenin alleles related
to bread-making quality in Iranian common wheat (Triticum aestivum L.)
ALI IZADI-DARBANDI1 ∗ and BAHMAN YAZDI-SAMADI2
1
Department of Agronomy and Plant Breeding sciences, College of Aburaihan, University of Tehran,
P. O. Box 3391653775, Pakdasht, Iran
2
Department of Agronomy and Plant Breeding, College of Agricultural and Natural Resources,
University of Tehran, P. O. Box 31587-77871, Karaj, Iran
Abstract
Bread-making quality in hexaploid wheats is a complex trait. It has been shown that the amount and composition of protein
can influence dough rheological properties. The high-molecular-weight (HMW) glutenins are encoded by a complex locus,
Glu-1, on the long arm of group-1 homoeologus chromosome of the A, B and D genomes. In this work we used PCR-based
DNA markers as a substitution tool to distinguish wheat bread-making quality. We detected PCR-based DNA markers for
coding sequence of Glu-A1x, Glu-B1x and Glu-D1x to be 2300 bp, 2400 bp and 2500 bp respectively. DNA markers related
to coding sequence of Glu-A1y, Glu-B1y and Glu-D1y were; 1800 bp, 2100 bp and 1950 bp, however, the repetitive region of
their coding sequence were shown to be about 1300 bp, 1500 bp and 1600 bp. The results demonstrate that the size variation
was due to different lengths of the central repetitive domain. Good or poor bread-making quality in wheat is associated with
two allelic pairs of Glu-D1, designated 1Dx5-1Dy10 and IDx2-1Dy12. The 1Bx7 allele has moderate-to-good quality score.
The specific DNA markers, of 450 bp, 576 bp, 612 bp and 2400 bp respectively were characterized for 1Dx5, 1Dy10, 1Dy12
and 1Bx7 alleles. These markers are very important in screening of wheat for bread-making quality.
[Izadi-Darbandi A. and Yazdi-Samadi B. 2012 Marker-assisted selection of high molecular weight glutenin alleles related to bread-making
quality in Iranian common wheat (Triticum aestivum L.). J. Genet. 91, 193–198]
Introduction
Wheat grain proteins are typically classified according
to their solubility properties into albumins (water soluble), globulins (salt soluble) and prolamins (gliadins and
glutenins, soluble in alcohol solutions). These latter make
up the so-called gluten, and are chiefly responsible for the
rheological properties of wheat dough (Laino et al. 2007).
The major class of glutenin polypeptides have been identified in wheat endosperm, designated as high-molecularweight glutenins subunits (HMW-GS) and low-molecularweight glutenins subunits (LMW-GS); both classes occur in
flour as cross-linked proteins, resulting from interpolypeptide disulphide linkage. The genes coding for HMW-GS
(Glu-1) and LMW-GS (Glu-3) are located respectively on the
long and short arms of 1A, 1B and 1D chromosomes (Payne
et al. 1980; Gupta et al. 1991). Glu-3 loci are tightly linked
to Gli-1 loci, which encode gamma-gliadins and omega-
∗ For correspondence. E-mail: [email protected].
gliadins (Gupta et al. 1990). It is generally accepted that there
are additive and epistastic interactions between glutenin subunits that affect bread-making quality (Gupta et al. 1989;
Nieto-Taladriz et al. 1994). HMW-GS account for up to
12% of the total protein in the endosperm of common wheat
(Triticum aestivum L.), while their allelic variation explains
about 45% to 70% of the variation in bread-making performance within European wheat cultivars (Branlard and
Dardevet 1985; Payne et al. 1988).
Dough with high elasticity and extensibility is ideal for
bakery products, whereas the high-extension doughs are
used in confectionary and moderate ones are good for flat
bread or pasta. Reconstituted flours with additional glutenin
increased dough strength while additional gliadin and
LMW-GS decreased dough strength (Sissons et al. 2007).
Each Glu-1 locus (Glu-A1, Glu-B1 and Glu-D1) consists of
two genes, tightly linked, designated as x-type and y-type.
The former codes for a subunit of higher molecular weight
with respect to the latter. Both x-type and y-type HMW-GS
consist of three distinct domains: a longer, nonrepetitive
N-terminal and a shorter C-terminal domain flanking a
Keywords. wheat; bread-making; HMW-GS; DNA marker.
Journal of Genetics, Vol. 91, No. 2, August 2012
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Ali Izadi-Darbandi and Bahman Yazdi-Samadi
central repetitive domain based on repeats of hexapeptides
(consensus amino acid sequence PGQGQQ) and nonapeptide
(consensus GYYPTSP/LQQ). In x-type subunits, a tripeptide repeat is also found (consensus GQQ). The C-terminal
domain has one cysteine residue in the x-type subunit and
five cysteine residue in the y-type subunits (Skerritt 1998).
The central domain has an additional cysteine residue that is
present close to the end of the C-terminus in the y-type subunits and, occasionally, close to the N-terminus in the x-type
subunits. The central domain forms a β-turn spiral, while
the two nonrepetitive domains have a globular structure
formed by α-helices (Shewry et al. 1992). The HMW-GS
form intramolecular disulphide bands with other HMW-GS
and LMW-GS (Skerritt and Robson 1990). Glutamine-rich
repetitive sequences that comprise the central part of the
HMW subunits are actually responsible for the elastic properties due to extensive arrays of interchain hydrogen bonds,
and the presence of certain HMW subunits is positively correlated with good bread-making quality (Shewry et al. 1989).
Results obtained so far indicate that gliadins were overexpressed in heat-stressed samples, in particular ω-gliadins, but
the HMW-GS/LMW-GS ratio was not affected (Laino et al.
2007). So far, the structural characteristics of more than 10
HMW-GS alleles have been revealed by DNA sequencing
(Forde et al. 1985; Wan et al. 2002). Point mutation, unequal
crossover or slippage of a parental wheat gene, recombination between two parental genes, and gene shuffling are
new mechanisms for formation of novel HMW-GS gene (Liu
et al. 2007). In hexaploid wheats, six HMW-glutenin genes
are present, but only those coding for subunits 1Bx, 1Dx and
1Dy are always expressed, whereas those coding for 1Ax and
1By are not always expressed and the gene for 1Ay is never
expressed. Results reported so far have shown that the nonexpression of the gene for 1Ay can be caused by nucleotide
changes in the promoter region (Forde et al. 1985) or by presence of a transposon-like insertion in the coding sequence.
However, this allele is expressed in some of the tetraploid
and diploid wheats (Harbred et al. 1987). Allelic variation
of HMW-GS and LMW-GS can be shown by SDS-PAGE.
Differences in molecular mass between various subunits is
derived mainly from differences in number of hexapeptide
and tripeptide repeats (D’ovidio et al. 1994). The relationship between protein composition and bread-making quality showed that the quantities of total flour protein, albumin,
globulin, and HMW/LMW glutenin subunits in flour were
significantly and positively correlated with bread loaf volume. However, the ratio of HMW to LMW glutenin subunits had little association with loaf volume (Wang et al.
2007).
There is a kind of intermediate state that appeared in the
divergence between x-type and y-type genes in HMW-GS
evolution (Liu et al. 2008). Genetic engineering can be used
to manipulate the amount and composition of the HMW subunits, leading to either increased dough strength or more
drastic changes in gluten structure and properties (Shewry
et al. 2001). Identification, isolation, cloning and introducing
194
of desired alleles into high performance cultivars with less
quality is possible.
Because of the importance of HMW-GS to the improvement of wheat processing quality, genes encoding these subunits have been cloned from wheat and wheat-related species
(Forde et al. 1985; Sugiyama et al. 1985; Thompson et al.
1985; Halford et al. 1987; Anderson and Greene 1989;
Reddy and Appels 1993; De Bustos et al. 2001; Wan et al.
2002; Liu et al. 2003; Wang et al. 2004, 2006; Guo et al.
2005; Yan et al. 2006). In particular, allelic variations at
Glu-D1 are largely responsible for differences in breadmaking properties of bread wheat flour (Payne et al. 1982).
A set of oligonucleotide primers specific at Glu-1 of wheat
for 1Dx5 (Anderson and Greene 1989), 1Bx7 (Anderson and
Greene 1989), 1Dy10 and 1Dy12 (Smith et al. 1994), x/y
types, repetitive domain of Y type, 1DxN domain (D’ovidio
et al. 1995) were used to the exploitation of PCR-based DNA
markers to distinguish different HMW-GS which correlated
with bread-making quality. Genes encoding HMW glutenin
subunits, present in bread-wheat lines and cultivars, were
studied by RFLP (restriction fragment length polymorphism)
and PCR analyses (D’ovidio et al. 1994). Development of
STS markers and establishment of multiplex PCR for Glu-A3
alleles in common wheat were done (Wang et al. 2010).
Materials and methods
Storage proteins were extracted from single seeds and
after identification of HMW-GS and LMW-GS profiles for
67 common wheat cultivars (Izadi-Darbandi et al. 2010).
We used nine cultivars containing these allelic compositions: 1Dx2, 1Dx5, 1Dy10, 1Dy12, 1Bx7 and null for 1Bx7
allele (table 1). Extraction of protein from selected cultivar was performed using sequential extraction method
based on Singh and Shephard (1991) by using one-step 1-D
SDS-PAGE through acrylamide gradient gels (8.1%–12.5%).
Payne nomenclature system (Payne et al. 1988) was applied
to detection of HMW-GS. Gabo and Chinese Spring were
used as universal standard cultivars in identification of Glu-1
subunits. DNA extraction for validation of STS-PCR markers were performed by Doyle and Doyle (1990) method. A
set of oligonucleotide primers (table 1) specific for genes at
the Glu-1 loci in wheat enable construction of specific DNA
markers.
PCR reactions were performed using the specific primers
for target alleles: one cycle (94◦ C for 5 min), followed by 45
cycles at 94◦ C for 1 min, 63◦ C for 1 min and 72◦ C for 1 min,
except for primers P5 and P6 where the annealing temperature was 60◦ C for 1 min and extension was 72◦ C for 3 min.
The final extension was kept at 72◦ C for 7 min. Amplification for coding and repetitive regions of the HMW-GS and Nterminal of 1Dx gene were performed with 60◦ C as annealing
temperature. PCR products were separated on 1.8% agarose
gel and visualized by UV after 1 h staining in 0.5 μg/mL
EtBr. DNA markers were verified for specific locus or alleles
Journal of Genetics, Vol. 91, No. 2, August 2012
Glutenin alleles related to bread quality in Iranian wheat
Table 1. Specific primers were designed based on published DNA sequences for Glu-1 loci.
Primer
P1
P2
P3
P4
P5
P6
P7
P8
P9
P10
P11
P12
P13
P14
P15
P16
P17
Sequence
Allele
5 -GCCTAGCAACCTTCACAATC-3
5 -GAAACCTGCTGCGGACAAG-3
5 -GTTGGCCGGTCGGCTGCCATC-3
5 -TGGAGAAGTTGGATAGTACC-3
5 -ATGGCTAAGCGCCTGGTCCT-3
5 -TGCCTGGTCGACAATGCGTCGCTG-3
5 -AGATGACTAAGCGGTTGGTTC-3
5 -CTGGCTGGCCAACAATGCGT-3
5 -ATGGCTAAGCGCCTGGTCCT-3
5 -TGCCTGGTCGACAATGCGTGC-3
5 -ATGGCTAAGCGGTTAGTCCT-3
5 -CTGGCTGGCCGACAATGCGT-3
5 -ATGGCTAAGCGGTTGGTCCT-3
5 -GGCTAGCCGACAATGCGTCG-3
5 -CTGTGTTAACATGGTATGGGTTGTC-3
5 -GGGAACATCTTCACAAAACAGTACAA-3
5 -CTGGCCGTTGCGGAGAAGCT-3
Dx2 & Dx5
Dy10 & Dy12
Bx7
1Ax
1Bx
1Dx
1y type
1y type (repetitive domain)
N-terminal 1Dx
Figure 1. SDS-PAGE analysis of HMW glutenin subunits in some common wheat.
Gabo (Gb) and Chinese spring (Cs) are as standard cultivars.
according to their protein banding patterns and specific PCR
products.
Results and discussion
The validation of each STS markers was confirmed by the
existing of correspondence protein allele on SDS-PAGE system. The specific DNA marker was detected after recognizing the glutenin alleles on SDS-PAGE (figure 1). The allelic
composition for wheat cultivars subjected to this study is
summarized in table 2.
Though there is high degree of similarity among the
sequences of x and y type alleles located in the same locus,
it was possible to find regions differing between 1Dx2 and
1Dx5, 1Dy10 and 1Dy12, and 1Bx7 and other 1Bx alleles to
design site-specific primers for PCR analysis.
PCR using primers P1 and P2 amplified a specific 450-bp
DNA fragment for wheat cultivars possessing 1Dx5 allele,
while cultivars possessing 1Dx2 or 1Dx3 gave no PCR product. PCR fragments of 576 bp and 612 bp were amplified respectively for cultivars possessing 1Dy10 and 1Dy12
alleles using P3 and P4 primers (figure 2).
In figure 3 (left to right) the first three cultivars on both
side of marker with 1Dx5 + 1Dy10 alleles show their corresponding specific DNA markers as 450 bp + 576 bp. The
latter three cultivars with 1Dx2 or 1Dx3 and 1Dy12 gave
no PCR product with P1 and P2 primers and have a specific
612-bp DNA fragment with P3 and P4 primers. Then these
PCR-based DNA markers can be used for distinguishing and
screening of good or poor bread-making quality in wheat
cultivars. Using P5 and P6 primers a specific PCR product
of around 2400 bp was amplified only for cultivars with
1Bx7 and no product for other alleles in these loci (figure 3).
The results and relationships in this study between the
glutenin subunits and their DNA markers for Iranian wheats
Table 2. Wheat cultivars subjected to this study and the composition of their related allelic HMW glutenin subunits.
HMW-glutenin subunits
Number
1
2
3
4
5
6
7
8
9
Cultivar
Navid
Ghods
Flat
Karaj 3
Chinese spring
Gabo
Shahpasand
Kalak Afgani
Karaj 2
Journal of Genetics, Vol. 91, No. 2, August 2012
1Ax
1Bx
1By
1Dx
1Dy
2∗
Null
1
2∗
Null
2∗
Null
2∗∗
Null
17
17
7
13
7
17
7
6
7
18
18
9
19
8
18
8
8
8
5
5
5
2
2
2
3
Null
5
10
10
10
12
12
12
12
10∗
10
195
Ali Izadi-Darbandi and Bahman Yazdi-Samadi
Figure 2. The amplification of specific PCR product of 450 bp for
1Dx5 allele in cultivars 1–3 (Navid, Gods and Flat) from left to
right and the lack of amplification in cultivars without 1Dx5 allele
in lanes 4–7 (Karaj3, Chinese Spring, Gabo and Shahpasand). Specific PCR fragments of 576 bp and 612 bp for cultivars containing 1Dy10 (Navid, Gods and Flat) and 1Dy12 (Karaj3, Gabo and
Shahpasand) alleles respectively.
Figure 3. The amplification of specific PCR product of around
2400 bp for 1Bx7 allele (Flat, Chinese Spring and Shahpasand cultivars: lanes 3–5) and nothing for cultivars lacking this allele (Navid,
Gods: lanes 1–2).
Figure 5. The amplification of specific PCR product of 396 bp for
the N-terminal of 1Dx locus in cultivars 1–7, from left to right.
are similar to the results for New Zealand wheat (Magboul
2000).
With primers (P7 , P8 ), (P9 , P10 ) and (P11 , P12 ) respectively for the x type alleles in Glu-A1, Glu-B1 and Glu-D1.
The PCR products were 2300 bp, 2400 bp and 2500 bp
(figure 4). There were no differences between the sizes of
PCR products at each locus encoding different subunits. It
means that sequence differences in each locus make allelic
variation at protein level. STS-PCR has produced the same
fragment for the null allele with expressed ones, at Gu-A1
locus (lanes 2, 5, 7 and 8 in figure 4a) then their changes
because of mutation or insertion/deletion can be detected by
DNA sequencing.
PCR reaction with primers (P11 , P17 ) amplified a 396-bp
fragment for the N-terminal of 1Dx genes (figure 5). This
specific DNA marker can be used as a unique DNA marker
for identification of D genome of wheat. The amplification
bands of about 1800 bp, 2100 bp and 1950 bp (figure 6) with
primer pair (P13 , P14 ) correspond to the coding sequences
of 1Ay, 1By and 1Dy and bands of about 1300 bp, 1500 bp
and 1600 bp (figure 7) with specific primers (P15 , P16 ) correspond to the repetitive regions of 1Ay, 1By and 1Dy genes.
The lack of amplification products in Gabo cultivar (lane 6)
Figure 4. The amplification of specific PCR product of 2300 bp for 1Ax locus (a), 2400 bp for 1Bx locus (b) and 2500 bp for 1Dx locus (c)
in cultivars 1–8 (referred to table 2), from left to right.
196
Journal of Genetics, Vol. 91, No. 2, August 2012
Glutenin alleles related to bread quality in Iranian wheat
potential quality. DNA markers verified in this study can be
used for both quality classification and accelerating breeding
programme for bread-making quality. Wheat quality identification can be done in early plantlet stage without having to
wait for seeds and analysed their glutenin composition. One
can also identify allelic potential in wheat-related species
using these PCR-based DNA markers.
In this study we introduced STS-PCR markers for the
N-terminal, repetitive domain and full coding sequences of
six different alleles are located on the Glu-1 locus. We can
use these markers as a substitution method in wheat quality
breeding programme for detection poor or good qualities in
seedling stage.
Figure 6. The amplification of specific PCR products of 1800 bp,
2100 bp and 1950 bp respectively for 1Ay, 1By and 1Dy loci in
selected cultivars 1–4 from left to right.
for repetitive region of 1Ay may indicate a point mutation or
insertion of a transposon fragment. Our results demonstrate
that the central repetitive domain was in all cases responsible
for the allelic size variation. Hybridization experiments using
specific probe has confirmed the right size of DNA marker
for each locus (D’ovidio et al. 1995) and other bands do not
correspond to HMW-GS sequences.
SDS-PAGE is one method for identification of allelic components in quality scoring of wheat cultivars, but in this system the mobility of subunits does not exactly correspond
with the size and sometimes makes interpretation of banding
pattern difficult. However, marker-assisted selection can help
avoid misinterpretation of results from SDS-PAGE. Markerassisted selection showed that there is a relationship between
the preset of PCR products and their corresponding 5 + 10
and 2 + 12 glutenin subunits, then it is possible to use from
each of specific DNA markers in screening procedure of
bread-making quality. Specific DNA marker for 1Bx7 can be
used for detection of wheat cultivars from moderate to good
Figure 7. The amplification of specific PCR products of around
1300 bp, 1500 bp and 1600 bp for repetitive region of 1Ay, 1By and
1Dy loci in cultivars 1–7 from right to left. Fragment 1300 bp was
not produced in cultivar no. 6.
Acknowledgements
This research was supported by Department of Agronomy and Plant
Breeding of University of Tehran. Authors would like to appreciate the Institute of Seed and Plant Improvement of Iran for kindly
providing wheat genotypes.
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Received 28 September 2011, in revised form 27 February 2012; accepted 21 May 2012
Published on the Web: 8 August 2012
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