Field Crops Research 115 (2010) 171–178

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Field Crops Research

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Analysis of starch swelling power in Australian breeding lines of hexaploid

wheat (Triticum aestivum L.)
Omid Ansari a,b,c, Monica Båga a, Ravindra N. Chibbar a,*, Nilufa Sultana b,c, Neil K. Howes b,c
a
Department of Plant Science, University of Saskatchewan, 51 Campus Drive, Saskatoon, Saskatchewan, S7N 5A8 Canada
b
Plant Breeding Institute, The University of Sydney, Cobbitty, NSW, Australia
c
Value Added Wheat CRC, North Ryde, NSW, Australia

A R T I C L E I N F O A B S T R A C T

Article history: Starch is the major component of wheat (Triticum aestivum L.) grain and is composed of two large glucan
Received 30 July 2009 molecules, amylose and amylopectin. The ratio between the two polymers types influences the water
Received in revised form 2 November 2009 absorbing properties of starch upon heating, and thus affects the end-use of grain and purified starch. In
Accepted 5 November 2009
this study, we evaluated the starch swelling power (SSP) values in seven wheat populations developed
from crosses involving low-SSP lines. Analysis of starch produced by the F2 generation plants showed
Keywords: that the largest SSP variation (11.4–16.2) and lowest SSP mean (13.9) was obtained for a population
Starch swelling power
derived from doubled haploid lines SM1028 (SSP = 14.5) and VK306 (SSP = 13.6). The population of 360
Amylose
lines was advanced by single seed descent to the following generations for further studies. Starch
Heritability
Triticum aestivum L. analysis of grain produced by F4 generation lines in two field locations during 2006 and in a greenhouse
environment during 2005 showed that SSP values were relatively stably inherited. The average broad-
sense heritability was 73% and significant (P < 0.001) genotype  genotype and genotype  environ-
environment interactions were seen. Starches with the highest and lowest SSP values were inversely
related to amylose concentration determined by high pressure liquid chromatography (HPLC)–size
exclusion chromatography (SEC) of debranched starch. Developed lines with the lowest SSP values
surpassed 40% in apparent amylose concentration. The study illustrates that screening for SSP in early
generations can be used to develop wheat lines with desired starch swelling characteristics.
ß 2009 Elsevier B.V. All rights reserved.

1. Introduction
Starch is the main component of wheat endosperm and is
composed of two major forms of glucan polymers, amylose and
amylopectin. Amylose is a near linear glucan polymer, in which
glucose units are linked by a-1,4 bonds and a few a-1,6-linkages
that constitute branches on the polymer. Amylopectin is much
larger a-1,4 glucan polymer than amylose and contains a higher
frequency of a-1,6 linkages resulting in a heavily branched
structure. The starch polymers form highly organized granules
(Gallant et al., 1997), which in the Triticeae species are synthesized
in two size classes: the large lenticular A-type and the small
spherical B-type granules. Biosynthesis of starch polymers in
higher plants is catalyzed by at least four enzyme activities: ADP-
Glc pyrophosphorylase (AGPase), starch synthases (SSs), starch
branching enzymes (SBEs), and starch debranching enzymes
* Corresponding author. Tel.: +1 306 966 4969; fax: +1 306 966 5015.
E-mail addresses: omid.ansari@usask.ca (O. Ansari), monica.baga@usask.ca
(M. Båga), ravi.chibbar@usask.ca (R.N. Chibbar), nsul7723@mail.usyd.edu.au
(N. Sultana), n.howes@usyd.edu.au (N.K. Howes).
(DBEs) [see reviews by James et al. (2003), Morell and Myers
(2005), and Tetlow et al. (2004)]. Many isoforms of the starch
biosynthetic enzymes exist and some of these form multisubunit
complexes (Tetlow et al., 2008; Hennen-Bierwagen et al., 2009).
Cereal starches show a relatively constant 1:3 ratio between
amylose and amylopectin, but altered ratios are often seen when
the activity of one or several starch biosynthetic enzymes is
modified (Smith et al., 1997; Morell and Myers, 2005). Some of the
naturally modified starches in cereals have found commercial
utilization. For wheat, partially waxy wheat lines with one or two
null alleles of granule-bound starch synthase I (GBSSI) produce
grain with low-amylose starches (12–22%) that are suitable for
noodle production (Zhao et al., 1998; Demeke et al., 1999;
Yamamori and Quynh, 2000). Grains with very low-amylose or
amylose-free (waxy) starch caused by mutations in all three GBSSI
alleles are used in the baking industries to extend the shelf life of
baked goods (Chibbar and Chakraborty, 2005).
High-amylose starches have lately attracted increased interest,
mainly due to their properties of a dietary fiber when used in cooked
food products (Topping and Clifton, 2001; Topping et al., 2003; Bird
et al., 2008). For wheat, natural mutants lacking SSII expression
produce starches with apparent amylose concentrations ranging

0378-4290/$ – see front matter ß 2009 Elsevier B.V. All rights reserved.
doi:10.1016/j.fcr.2009.11.001
172 O. Ansari et al. / Field Crops Research 115 (2010) 171–178

from 31 to 44% (Yamamori et al., 2000; Konik-Rose et al., 2007).
Wheat starch with very high-amylose concentration (>70%) has
been produced using transgenic approaches to down-regulate SBEIIa
and SBEIIb expression (Regina et al., 2006), but the commercial
utilization of these lines is hampered by low grain yields and
consumer unacceptability of genetically modified wheat. Commer-
cially grown maize amylose extender mutants with 50–70% amylose
show a manageable reduction in yield as compared to normal corn
(Campbell et al., 2005). Due to the close relationship between maize
and the Triticeae family of cereals, the use of breeding techniques
and high-throughput screening to produce high-amylose lines with
reasonable yields may also be a possibility for wheat.
The concentration of amylose and amylopectin in starch can be
determined by different methods. The more precise measurements
are based on separation of starch polymers by size exclusion
chromatography (SEC) using low-pressure gel-permeation chro-
matography (Wang et al., 1993) or high pressure liquid
chromatography (HPLC), followed by detection by differential
refractometry (Batey and Curtin, 1996). A variant of the HPLC–SEC
method uses debranched starch, where peaks of long and short
glucan chains are quantified as amylose and amylopectin,
respectively (Demeke et al., 1999). An estimation of amylose
concentration in starch can be obtained by the starch iodine
binding method (Morrison and Laignelet, 1983), which measures
the amount of apparent amylose (Takeda and Hizukuri, 1987).
Apparent amylose consists of long-chain glucans from amylose
and may also include long branches present on the amylopectin
(Jane et al., 1999). Analysis of starch swelling power (SSP) is a rapid
method for the estimation of amylose (Tester and Morrison, 1990).
The analysis determines the amount of water absorbed by a starch
sample upon heating followed by cooling. As the SSP analysis
simulates cooking, the test provides some information about the
end-product quality such as starch pasting viscosity (Crosbie,
1991; McCormick et al., 1991; Yamamori et al., 2006). For wheat
samples, the SSP values show a negative correlation with starch
iodine binding values, amylose concentration and amount of
glucan chains longer than 35 residues on amylopectin (Sasaki and
Matsuki, 1998), starch lipid concentration (Tester and Morrison,
1990) and amount of small starch granules (Ansari et al., 2006).
Only a small grain sample is required for SSP determination, and
thus, the test has proven to be cost-effective and reliable for
screening breeding lines for noodle quality (Crosbie, 1991;
McCormick et al., 1991).
In this report, we developed seven wheat populations by crossing
one or two low-SSP parent lines. One cross showed a wide range of
SSP values and transgressive segregation at the low-SSP end in the F2
generation and was selected for further studies. The SSP trait was
relatively stably inherited throughout the generations and selection
of low-SSP lines in early generations allowed for development of
lines with up to 40% amylose as determined by HPLC–SEC.
2. Materials and methods
2.1. Plant material and growth conditions
The wheat lines and cultivars used in the study are listed in
Table 1. Doubled haploid (DH) lines SM1118, SM1126, SM1046
and SM1028 are low-SSP lines derived from a Sunmist  Meering
DH population (Sultana et al., 2006). Both Sunmist and Meering
produce starches with about 40% apparent amylose concentra-
tions (Blazek, 2008). The DH low-SSP line VK306 was derived
from a cross between low-SSP lines Vulcan and Kewell (Ansari et
al., 2006). Cultivars Minto, Banks and Vasco were identified as
high-amylose lines in previous studies (Sultana et al., 2006).
Cultivars EGA Hume and Diamond bird carry GBSSI-4A null
mutations and wheat line WAXVAW1104 (value added wheat
CRC line) is an amylose-free (waxy) line (this study). Cultivar
Lang shows normal SSP value (26% amylose concentrations)
(Sultana et al., 2006).
Seven different wheat populations were developed for evalua-
tion of SSP values and distribution. The parent lines for the crosses
were selected based on their higher than normal amylose
concentration or lower than normal SSP values (Table 1). To
increase the chances of transgressive segregation, the genetic
distance between parent lines was also considered. Crosses OA3,
OA5, OA6 and OA10 were made between a low-SSP line and
cultivar Minto (Fig. 1). The Minto line grown in our study produced
starch with a slightly higher SSP value (16.7) than previously
reported (SSP = 15.2; Sultana et al., 2006), and was considered to
produce normal wheat starch (SSP = 16.5–17.5). The remaining
populations (OA11, OA24 and OA69) were developed using various
low-SSP parent lines (Table 1 and Fig. 1).
A total of 360 seeds derived from six F1 plants of each of the
seven crosses shown in Fig. 1 were advanced by single seed
descent to the F2 generation. Cross OA24 was further developed by
single seed descent to the F6 generations. The lines were grown at
Plant Breeding Institute, Cobbitty, NSW, Australia in a greenhouse
with 25/20 8C day/night temperatures, 570–620 mmol/m2/s light
intensity and 18 h photoperiod. The F4 seeds of the OA24
population were planted along with their parents and grand-
parents at two sites (Landsdown and Karalee) in the 2006 growing
season and in the greenhouse in 2005 at Plant Breeding Institute,

Table 1
List of wheat lines and cultivars used in study.

Cultivar/line Pedigree Starch characteristics Reference

Banks PWTH/Condor’S’//2*Condor SSP = 14.8 Sultana et al. (2006)

Vasco 3Ag14/4*Condor//5*Oxley SSP = 16.2 This study
Lang QT3765/Sunco SSP = 15.9 Sultana et al. (2006)
Vulcan Condor/Pitic 62//Condor’S’ SSP = 14.3 Ansari et al. (2006)
Kewell Olympic mutant/South African 184//Olympic SSP = 13.5 Ansari et al. (2006)
Sunmist Pindar/Thatcher//2*Insignia/3pinnacl SSP = 14.5 Sultana et al. (2006)
Meering Condor Selection SSP = 15.3 Sultana et al. (2006)
Minto Condor/Olympic/Egret SSP = 14.8 Sultana et al. (2006)
SM1028 Sunmist/Meering selection SSP = 12.8 Sultana et al. (2006)
SM1118 Sunmist/Meering selection SSP = 12.9 Sultana et al. (2006)
SM1046 Sunmist/Meering selection SSP = 15.6 This study
SM1126 Sunmist/Meering selection SSP = 13.5 Sultana et al. (2006)
VK306 Vulcan/Kewell selection SSP = 12.6 Ansari et al. (2006)
WAXVAW1104 VAWCRC waxy nursery collection SSP = 20.7 This study
EGA Hume Pelsart/2*Batavia DH SSP = 17.5 Sultana et al. (2006)
Diamond bird Vicam//Ciano/7C/3/KAL/BB SSP = 17.4 Sultana et al. (2006)
Sunvale Cook*2//VPM1/Cook*3 SSP = 15.6 Sultana et al. (2006)
 O. Ansari et al. / Field Crops Research 115 (2010) 171–178 173

Table 2
Analysis of SSP values in OA24 F5 lines grown in three different locations.

Genotype Location-year

Parent: KL-2006 GH-2005 LD-2006

SM1028 15.1  0.3 14.8  0.2 15.7  0.1
VK306 14.8  0.1 13.7  0.1 15.6  0.2

Grandparent:
Sunmist 14.8  0.7 14.2  0.1 15.0  0.6
Meering 14.6  0.1 13.6  0.1 13.8  0.6
Vulcan 15.6  0.5 15.2  0.1 15.1  0.3
Kewell 16.1  0.4 16.7  0.2 15.9  0.1

OA24 F5 plants 11.8–14.8 12.3–18.0 13.6–17.1

Mean  SD 13.5  0.6 15.2  1.6 15.0  0.8

Median 13.4 15.0 15.1

Cobbitty, NSW, Australia (Table 2). Plants grown in the field were
irrigated on a regular basis. The field grown lines were harvested as
F3-derived F5 seed bulks by hand, threshed and kept at 15 8C until
being assayed for starch properties. Six selected lines of F3-derived
F5 plants of the SM1028  VK306 population and control wheat
lines (Table 3) were grown in replicated trials at Horsham, SA, and
Narrabri, NSW, Australia in 2006.

2.2. Isolation of starch

Starch was purified from 10 to 15 well developed kernels

essentially as described (Stoddard, 2003). The kernels were
crushed using pliers and soaked overnight in 1.5 ml 0.5 M NaCl
at 4 8C. The grain material was homogenized into a suspension and
passed through a 100 mm pore diameter nylon mesh. Retained
material was washed twice with 0.5 M NaCl (0.5 ml) and filtrates
were combined and centrifuged at 2300  g for 2 min to pellet the
starch granules. A brown layer positioned on top of granules was
removed and washed twice with ddH2O to re-extract attached
granules. The crude starch preparation was washed with a
sequence of 1 ml of 50% (w/v) CsCl, 1 ml 2% (w/v) sodium dodecyl
sulphate solution, and finally with 1 ml 95% (v/v) ethanol. Two
washes with H2O were performed between each washing stage.
Starch suspensions were centrifuged for 2 min at 2300  g and
resuspended for 2 min at 21 8C, 1.2  103 rpm using a Thermo-
mixer Comfort (Eppendorf, Germany). Lipids were removed from
starch by treatment with 1 ml 85% (v/v) methanol at 65 8C for
20 min, followed by centrifugation at 2300  g for 10 min. The
starch was dried over silica gel in desiccators for 48 h.

2.3. Analysis of starch composition by HPLC–SEC

Determination of apparent amylose concentration in

Fig. 1. Distribution of SSP values in grains of F2 population. SSP range (R), mean (m),
standard deviation (SD) values for each population are given. Vertical arrows
indicate parental values for SSP.
extracted starch was performed as previously described (Demeke
et al., 1999). Briefly, 3.0 mg of starch was suspended in 3.0 ml
ddH2O and incubated for 30 min at 130 8C for gelatinization. An
aliquot (1.0 ml) starch solution was transferred to a 1.5 ml
microfuge tube and mixed with 55 ml 1 M sodium acetate buffer
pH 4.0 and 2.5 ml of bacterial isoamylase (Pseudomonas sp.;
250 U/ml; Megazyme Ltd., Bray, County Wicklow, Ireland).
Debranching of starch was done at 40 8C for 4 h and the enzymatic
reaction was stopped by boiling for 20 min. Debrached samples
were freeze dried and dissolved in 200 ml of 99% (v/v) DMSO.
Particulate material was removed by 10 min centrifugation at
1300  g. A 150 ml aliquot of debranched starch was separated
using an HPLC–SEC system (Waters 600 controller, Waters 610
fluid unit, Waters 717 auto-sampler, Waters differential refract-
ometer and Waters column PLGel 5 mm MiniMix). The data was
analyzed using the Empower data analysis package (Waters
Corporation, Milford, MA, USA).
174 O. Ansari et al. / Field Crops Research 115 (2010) 171–178

Table 3
Analysis of SSP in selected lines grown in two field sites in 2006.

Wheat genotype Comment SSP value

Horsham Narrabri Average

OA24-198-1 F3-derived F6 line of SM1028  VK306 population 13.7  0.1 14.5  0.1 14.1
OA24-198-2 F3-derived F6 line of SM1028  VK306 population 14.0  0.1 14.7  0.3 14.3
OA24-198-3 F3-derived F6 line of SM1028  VK306 population 14.3  0.0 14.5  0.1 14.4
OA24-328-1 F3-derived F6 line of SM1028  VK306 population 14.6  0.2 15.0  0.1 14.8
OA24-328-2 F3-derived F6 line of SM1028  VK306 population 15.0  0.1 15.0  0.2 15.0
OA24-328-3 F3-derived F6 line of SM1028  VK306 population 14.3  0.1 14.8  0.4 14.5
SM1028 Parent line 15.9  0.1 15.2  0.1 15.6
VK306 Parent line 14.0  0.1 15.2  0.5 14.6
Meering Grandparent line 15.7  0.2 15.4  0.0 15.6
Sunmist Grandparent line 15.3  0.1 15.6  0.2 15.5
WAXVAW1104 Waxy wheat line 20.9  0.1 20.6  0.7 20.7
Banks Normal wheat 14.8  0.3 15.1  0.1 15.0
EGA Hume Partially waxy wheat line (null GBSSI-4A) 16.6  0.0 16.9  0.3 16.7
Minto Normal wheat 15.2  0.2 15.2  0.5 15.2
Sunvale Normal wheat 16.9  0.1 16.4  0.2 16.7
SM1046 Sunmist/Meering selection 15.5  0.2 15.7  0.1 15.6
Diamondbird Partially waxy line (null GBSSI-4A) 17.5  0.0 18.0  0.1 17.7
Lang Normal wheat 15.6  0.2 15.7  0.3 15.7
Vasco Normal wheat 16.0  0.0 16.5  0.2 16.2

Maize standard (0% amylose) 21.3  0.4 21.0  0.4 21.1

Maize standard (27% amylose) 16.4  0.2 16.0  0.1 16.2
Maize standard (50% amylose) 7.1  0.1 6.9  0.1 7.0

2.4. Determination of SSP 3. Results

SSP analysis was based on the swelling test described by
Konik-Rose et al. (2001) with modifications (Ansari et al., 2006).
Duplicate samples of 25  2 mg starch were added to pre-weighed
microfuge tubes and a 0.6 ml of 0.1% (v/v) silver nitrate was added
to inhibit a-amylase activity. The samples were thoroughly mixed
for 2 min using a Thermomixer Comfort and incubated for 30 min
in a water-bath set at 92.5 8C. The samples were mixed thoroughly
by inversion at 1 min intervals for the first 5 min, at 2.5 min
intervals for the next 5 min and 5 min intervals for the last 20 min.
The tubes were cooled at room temperature for 5 min, centrifuged
for 5 min at 2300  g followed by aspiration of supernatant. The
starch swelling power was calculated based on the formula
SSP = (X A)/Ws where X is the weight of heated starch including
microtube, A is the empty microtube weight and Ws is the weight of
starch sample.
2.5. Statistical analysis of SSP
The SSP values determined for the OA24 population grown in
three environments (field locations at Karalee and Landsdown
2006 and in the greenhouse 2005) and combined locations were
analyzed using the Agrobase Generation II software version
13.2.1 (Agronomix software group, 2005). A randomized
complete block design (RCBD) model in which both environment
and genotypes were assumed to be random effects was applied in
the analysis. A combined RCBD analysis of variance was used to
separate the effect of location (environment), genotype and
genotype-by-environment interaction. Broad-sense heritability
was calculated in each environment by conducting the statistical
analysis separately for each location. The broad-sense herit-
ability formula was presented as H = VG/VP where H is broad-
sense heritability, VG is calculated based on the variance among
genotypes and VP is considered the total phenotypic variance for
starch swelling power values. Goodness of fit for normal
distribution for F5 materials grown in multiple environments
was tested with Anderson–Darling and Cramer–von Mises
methods using GenStat v. 11.1 (VSN International, Hemstead,
UK).
3.1. Analysis of SSP in early generations
A total 10–15 seeds produced from each of 24 randomly
selected F2 lines from each cross were analyzed for SSP values to
assess the phenotypic variance within each population (Fig. 1). A
normal distribution of SSP values was indicated for the OA5 and
OA6 populations, for which the average SSP values were close to
parental values. The OA10 and OA11 populations displayed a more
narrow distribution of SSP values where all lines showed a higher
SSP value than the low-SSP male parent, Banks, common to both
crosses. A relatively wide distribution of SSP values that did not
follow a normal distribution was seen for crosses OA3, OA24 and
OA69. Distribution within populations OA3 and OA69 were
skewed at the low-SSP and at the high-SSP ends, respectively. A
bimodal distribution with a 1:1.4 ratio was indicated for the OA24
population which showed the widest SSP distribution (11.4–16.2)
and the lowest mean (SSP = 13.9) among the populations studied.
About 38% of the F2 lines in the OA24 population showed SSP
values that were lower than parental values. As the OA24
population appeared most promising for development of low-
SSP lines, a total of 360 lines were selected for further studies and
advanced to the following generations by single seed descent.
Analysis of 48 lines in the F3 generation plants of the OA24
population revealed SSP values ranging from 12.3 to 17.9 with a
mean SSP of 14.9 (Fig. 2). The bimodal distribution of trait values
seen in the F2 generation was less pronounced in F3 (Fig. 2). A
comparison of SSP values of 24 lines analyzed in both the F2 and the
F3 generation revealed a strong correlation (r2 = 0.82), which
suggested that SSP variation within the population was caused by
few alleles.
3.2. SSP shows high heritability
The F4 generation seeds from the OA24 cross along with parent
and grandparent lines were planted in the greenhouse during the
year 2005 (GH-2005) and in the field at Karalee (KL-2006) and
Landsdown (LD-2006), NSW, Australia during the 2006 growing
season. Among the parent and grandparental lines, Kewell and
 O. Ansari et al. / Field Crops Research 115 (2010) 171–178
Fig. 2. Distribution of SSP values in grains of F3 plants of OA24 population. Vertical
arrows indicate parental values for SSP.
Meering consistently produced starch with the highest and the
lowest SSP values, respectively, at all three sites (Table 2). The
average SSP values for the OA24 population were 13.5 (KL-2006)
and 15.0 (LD-2006) in the two field trials and 15.2 (GH-2005) for
green house-grown plants. For field trials, the average SSP values
for the population were lower that average SSP values of parent
lines. The bimodal SSP distribution profile seen for the F2
generation plants (Fig. 1) was not seen for the F5 generation
seeds, which showed a normal distribution of SSP values for KL-
2006 and LD-2006 growing sites but was near normal for GH-2005
(Table 4, Fig. 3a).
A combined randomized block design ANOVA analysis of SSP
values revealed that genotypes, environments and genotype-by-
environment interaction effects were highly significant (P < 0.01;
Table 5). Broad-sense heritability was 73% in average (60–84% in
the three different environments).
3.3. Weak correlation between amylose concentration and SSP
Analysis of amylose concentrations for starches produced by the
parent lines, SM1028 and VK306, grown in the greenhouse were 34
and 33%, respectively. These values were about 7% higher than for
Fig. 3. Analysis of starch properties in grain produced by F4 generation plants of OA24 population. (a) Distribution of SSP values in grains harvested from plants grown in the
greenhouse in 2005 (GH-2005) and in field locations Karalee (KL-2006) and Landsdown (LD-2006). Parental values are shown by vertical arrows. (b) Amylose concentration in
starch prepared from plants grown in the greenhouse 2005 (GL-2005). Parental values are shown by vertical arrows.
175
Table 4
Goodness of fit tests for normal distribution.
Environment Anderson–Darling Cramer–von Mises
*
GH-2005 1.388 0.238*
KL-2006 0.365ns 0.054ns
LD-2006 1.88ns 0.027ns
ns: not significant.
*
P < 0.05.
normal wheat and correlated with low-SSP values (Table 2). The
amylose concentration in starches produced by the F5 population
grown in the greenhouse ranged from 17.4 to 40.4% (Fig. 3b). The
lowest values compared to levels seen in partially waxy wheat lines
and the highest levels were similar to amylose concentrations seen
in some wheat SSII mutants (Konik-Rose et al., 2007). When the
amylose concentrations were compared to SSP values, only a very
weak correlation (r2 < 0.2; P < 0.01) was found for the OA24
population. Selection of the 12 lowest and 12 highest lines for
comparison yielded a stronger correlation (P < 0.01) with r-values of
0.42, 0.56 and 0.58 for Karalee (KL-2006), Landsdown (LD-
2006) and greenhouse (GH-2005), respectively.
3.4. Evaluation of advanced breeding lines for SSP
The SSP values for lines grown at Horsham in the year of 2006
ranged from 13.7 (OA24-198-1) to 20.9 (WAXVAW1104), whereas
the SSP range was 14.5 (OA24-198-1 and OA24-198-3) to 20.6
(WAXVAW1104) for the material grown at Narrabri site. Although
the SSP values were higher at the Narrabri site, the order of the
lines was found to be significant (correlation = 0.907***, P < 0.001)
for the two sites.
The waxy line WAXVAW1104 showed the highest SSP value
(average 20.7) that compared relatively well to SSP value
determined for maize amylose-free starch (SSP = 21.1). The lowest
SSP values (average 14.1 and 14.5) were found for lines OA24-198-1
and OA24-328-3 selected from the SM1028  VK306 population. In
176 O. Ansari et al. / Field Crops Research 115 (2010) 171–178
Table 5
Statistical analysis of variance for SSP in three different locations.a.
Source of variation Degrees of freedom
Environment 2 351.2
Genotype 113
Genotype  environment 226
Replications within environments 3 16.3
Residual 339
Total 683
a
Greenhouse trial of SM1028  VK306 population grown in greenhouse in 2005 (GH-2005) and field trials at Karalee (KL-2006) and Landsdown (LD-2006) in 2006, NSW,
Australia. Broad-sense heritability was 84, 72 and 60% for GH-2005, KL-2006 and LD-2006, respectively. Mean = 14.6, R2 = 0.92, C.V. = 3.32%.
**
P < 0.01.
the F3 generation, the SSP values for the OA24-198 and OA24-328
lines were 13.2 and 13.3, respectively. The apparent amylose
concentration for the OA24-198-3 line was in an independent study
determined to be 42% as determined by the iodine binding method
(Blazek, 2008). Thus, by selecting lines with lowest SSP values in the
early generations we were able to develop near homozygous
breeding lines with desired water absorbing properties and
moderately high-amylose (40%) concentrations.
4. Discussion
4.1. Breeding strategy for lower SSP
SSP is an important starch character that can be used in wheat
breeding programs to select for lines with desired starch properties
upon cooking. Breeding for endosperm-associated-traits like SSP
and high-amylose concentration is not straight forward as these
traits are genetically controlled by several alleles in a diploid
genome, as well as, the triploid endosperm genome. However, the
chances of success can be increased if the parents chosen show a
mean trait value close to desired value and developed population
demonstrates a high phenotypic variance for the trait. This strategy
was used in this study to develop lines with lower mean SSP value
than both parents.
4.2. Influence of amylose concentration on SSP
The SSP method used to analyze starch properties measures
weight differences in starch before and after cooking in the
presence of excess water. During a heating step, the amount of
gelatinization depends primarily on the ratio of amylose and
amylopectin. The amylopectin polymers promote swelling,
whereas amylose and lipids complexed with amylose have a
negative effect on water absorption (Tester and Morrison, 1990).
The parents of the OA24 population, SM1028 and VK306,
were selected as low-SSP lines and both lines produce starches
with about 7% higher amylose concentration than normal wheat
as determined by HPLC–SEC analysis of debranched starch
(Fig. 3b). For the parent lines, the increased amylose
concentration correlated with low-SSP, as seen for many other
wheat starches and flours (Sasaki and Matsuki, 1998; Blazek and
Copeland, 2008). In contrast to previous studies (Sasaki and
Matsuki, 1998; Yamamori and Quynh, 2000), the SSP values in
the OA24 population did not correlate well with amylose
concentrations. The biosynthesis of amylose in wheat is
catalyzed by GBSSI, however, variation for the SSP trait in the
grandparent populations, Vulcan  Kewell and Sunmist  Meer-
Meering, is not associated with GBSSI alleles (Ansari et al., 2006;
Schofield, 2006; Sultana et al., 2006). Thus, the increased
amylose concentration seen in the parental and grandparental
lines of the OA24 population appears to be controlled by other
locus/loci than GBSSI.
Sum of squares Mean square F value
175.6 32.23**
281.7 2.5 1.89**
298.6 1.3 5.61**
5.4
79.9 0.2
1027.7
4.3. Possible influence of amylopectin fine structure on SSP
A wide range of SSP values (11.4–16.2) was observed for the
SM1028  VK306 population, but the values did not show any
significant correlation to amylose concentration when all the lines
were taken into consideration. Only for the lowest and the highest
SSP values could a weak but significant correlation with amylose
concentration be seen. The amount of amylose determined by the
HPLC–SEC method used in our study measures the amount of long-
and short-chain glucans, where the longer chains are presumed to
originate from amylose molecules. However, analysis of starches
produced by various wheat and barley starch mutants have shown
that amylose concentration is often overestimated in starches
containing amylopectin with an increased amount of long chains
(Jane et al., 1999). The amylose determination is especially
problematic using the iodine binding method, but also analysis of
debranched starch by HPLC–SEC may overestimate amylose
content in starches with altered amylopectin structure (Gérard
et al., 2001). Thus, the main determinant for SSP values in the
OA24 population may be due to differences in amylopectin fine
structure rather than amylose content. This hypothesis is
supported by studies showing significant differences in glucan
chain length distribution for the parental lines, SM1028 and VK306
(Blazek, 2008). The percentage of glucan chains with a degree of
polymerization exceeding 35 is about twofold higher in VK306
(1.57%) as compared to SM1028 (0.69%). Inversely, VK306
amylopectin contains less of the short chains as compared to
SM1028. Long chains on amylopectin are known to have a negative
effect on swelling (Sasaki and Matsuki, 1998), and thus, will
contribute towards a lower than normal SSP value. This is
consistent with VK306 producing starches with a lower SSP value
than SM1028 (Table 2).
The fine structure of amylopectin is determined by different
isoforms of SBE and SS (James et al., 2003; Morell and Myers, 2005)
that can participate in various forms of high-molecular weight
complexes (Tetlow et al., 2008; Hennen-Bierwagen et al., 2009).
The GBSSI, SSII and SSIII enzymes extend glucan chains on
amylopectin (Denyer et al., 1996; Gao et al., 1998), and null
mutations in genes encoding these enzymes can cause a relative
increase in short chains on amylopectin, resulting in a higher SSP.
Thus, parental alleles for SS with different affinity for substates and
increasing the length of glucan chains on amylopectin may cause
lower SSP seen in the OA24 population. However, the amount of
long glucan chains on amylopectin may not entirely determine SSP
(Blazek and Copeland, 2008). Recently, enzymatic digestion
studies of maize starch has suggested that there are at least two
types of amylopectin structures that are slowly digestible, e.g.
forming resistant starch (Zhang et al., 2008). One amylopectin type
has more of the longer chains and is less branched, whereas the
second amylopectin type has shorter chains and is more branched.
The differences between the two types of amylopectin structure
may affect starch crystallinity, digestibility and SSP values.
 O. Ansari et al. / Field Crops Research 115 (2010) 171–178
Whether the lines developed in this study differ in their relative
amount of the two amylopectin types will require further studies.
4.4. Starch granule size and SSP
The parental lines used in the study were previously reported to
differ in A-granule content (Ansari et al., 2006; Blazek, 2008),
where the SM1028 produces a slightly higher amount of large
granules (61.7%) than the VK306 parent (57.7%). Small (B-type)
granules have a tendency to absorb water more rapidly and swell
more than A-type granules (Chiotelli and Le Meste, 2002), but at
higher amounts, the B-type granules have a negative effect on
swelling by restricting complete swelling of A-type granules (Soh
et al., 2006). In addition, the amount of lipids trapped within
granules affects swelling of starch (Morrison et al., 1993) and may
have a larger role in controlling swelling properties than granule
size (Ao and Jane, 2007). Thus, the impact of starch granule size
distribution on SSP is complex, and it is difficult to postulate if the
small difference in starch granule size observed between parent
lines would have affected SSP in our study.
4.5. Heritability and environmental impact on SSP
Studies of reciprocal crosses in rice have shown that amylose
content is mostly controlled by the endosperm genotype, involving
both dominant and additive factors as well as maternal factors
(Pooni et al., 1993). Sometimes, the genetic effect controlling
amylose concentration in rice shows different directions in the
diploid and triploid tissue, as demonstrated in a rice recombinant
inbred population (Zheng et al., 2008). In our study, we observed
several different SSP segregation patterns for the developed
populations (Fig. 1), suggesting that the trait was determined
by different types of gene actions in the different crosses. The near
1:2:1 segregation ratio seen for the OA5 and OA6 populations
supported that SSP variation in these crosses was caused by
additive gene actions. Lack of lines with lower SSP values than the
parents seen in the OA5 and OA6 crosses suggested the
involvement of recessive alleles or maternally inherited genes.
High-amylose concentration controlled by recessive genes in
wheat has been demonstrated in Aegilops squarrosa (Watanabe et
al., 1998) and Triticum turgidum (Mohammadkhani et al., 1999)
and thus, may also exist in hexaploid wheat. Maternally inherited
genes are known to have a small effect on starch composition in
wheat as indicated by studies of reciprocal crosses (Mohammad-
khani et al., 1999). Early generations of the OA24 population
displayed a bimodal segregation (Fig. 1) that in subsequent
generations developed into a near normal distribution (Figs. 2 and
3a). The complex genetic interactions between the diploid plant
and the triploid endosperm genomes may underlie the shift in SSP
distribution within the OA24 population as the diploid genome
became more homozygous in later generations.
The SSP trait in the SM1028  VK306 population was shown to
have a moderate-to-high heritability and a highly significant
(P < 0.01) genotype  environment interaction (Table 5). The
differences in SSP at different sites did not affect the ranking of the
genotypes, which was quite consistent. Due to environmental
factors, the relative SSP values are more important than the
absolute values in breeding programs, but require that appro-
priate check varieties are included in each trial for normalization
of SSP values.
About 34% of the variation in SSP values in the OA24 population
grown at three locations was estimated to be determined by the
environmental factors (Table 5). In the field trials, these factors
may have included high temperatures that are known to
negatively affect starch deposition in wheat (Shi et al., 1994)
and barley (Wallwork et al., 1998) endosperm, especially during
177
the first 14 days after anthesis. Heat stress above 25 8C primarily
inactivates genes encoding soluble SSs (Keeling et al., 1994;
Hurkman et al., 2003). Shading of wheat grains during the late
stage of grain development also decreases SSI activity (Mengel
and Judel, 1981). Mutant rice lines exhibiting different levels of SSI
activity show variation in amylopectin structure (Fujita et al.,
2006), that can be related to changes in starch gelatinization
temperature.
4.6. Practical applications of study
The current research was focused on the different wheat lines
selected for the growing areas across New South Wales, Australia.
To determine if these lines could be potential new varieties, further
field tests are needed to fully evaluate the environmental impact
on the trait. Tests in a wide range of environments will be needed
to evaluate the stability of SSP over environments. An important
factor to study is yield, as increases in amylose concentrations has
been associated in general with reduced yield (Jobling, 2004) and
in wheat with reduced seed size and yield (Regina et al., 2006).
Some of the low-SSP lines from OA24 population indicated
reasonable yield in the field trials conducted in Narrabri 2006
(data not shown), and will be further tested in various environ-
ments and back-crossed to high-yielding wheat varieties for
cultivar development.
Acknowledgements
Financial support by Value Added Wheat CRC Ltd., North Ryde,
NSW, Australia and Canada Research Chairs is gratefully acknowl-
edged. The Plant Breeding Institute (University of Sydney,
Australia) is gratefully acknowledged for field and laboratory
facilities.
References
Ansari, O., Turner, M., Sultana, N., Howes, N., 2006. Genotype-environment inter-
action investigation on wheat doubled-haploid lines for starch swelling power.
In: Mercer, C.F. (Ed.), Breeding For Success: Diversity in Action. Proceedings of
the 13th Australasian Plant Breeding Conference, Christchurch, New Zealand,
pp. 235–240.
Ao, Z., Jane, J., 2007. Characterization and modeling of the A- and B-granule starches
of wheat, triticale, and barley. Carbohydr. Polym. 67, 46–55.
Batey, I.L., Curtin, B.M., 1996. Measurement of amylose/amylopectin ratio by high-
performance liquid chromatography. Starch/Stärke 48, 338–344.
Bird, A.R., Vuaran, M.S., King, R.A., Noakes, M., Keogh, J., Morell, M.K., Topping, D.L.,
2008. Wholegrain foods made from a novel high-amylose barley variety
(Himalaya, 292) improve indices of bowel health in human subjects. Br. J. Nutr.
99, 1032–1040.
Blazek, J., 2008. Role of amylose in structure-function relationship in starches from
Australian wheat varieties. PhD thesis. Faculty of Agriculture, Food and Natural
Resources, The University of Sydney, Sydney, NSW, Australia.
Blazek, J., Copeland, L., 2008. Pasting and swelling properties of wheat flour and
starch in relation to amylose content. Carbohydr. Polym. 71, 380–387.
Campbell, M., Bonney, C., Tabartet, L.K., 2005. Development of amylomaize VII
hybrids, potential for resistant starch production and QTL mapping of high-
amylose modifying genes from GEM germplasm. Germplasm enhancement of
maize – 2005 Public Cooperator’s Report. Truman State University, Division of
Science, Kirksville, MO, pp. 1–8 (http://www.public.iastate.edu/usda-gem/
Public_Reports/Yr_2005/GEMPR_2005_Campbell.htm).
Chibbar, R.N., Chakraborty, M., 2005. Waxy wheat. In: Abdel-Aal, E., Wood, P.
(Eds.), Specialty Grains for Food and Feed. American Association of Cereal
Chemists, Inc, St. Paul, MN, USA.
Chiotelli, E., Le Meste, M., 2002. Effect of small and large wheat starch granules on
thermomechanical behavior of starch. Cereal Chem. 79, 286–293.
Crosbie, G.B., 1991. The relationship between starch swelling properties, paste
viscosity and boiled noodle quality in wheat flours. J. Cereal Sci. 13, 145–150.
Demeke, T., Hucl, P., Abdel-Aal, E.S.M., Båga, M., Chibbar, R.N., 1999. Biochemical
characterization of the wheat waxy a protein and its effect on starch properties.
Cereal Chem. 76, 694–698.
Denyer, K., Clarke, B., Hylton, C., Tatge, H., Smith, A.M., 1996. The elongation of
amylose and amylopectin chains in isolated starch granules. Plant J. 10, 1135–
1143.
178 O. Ansari et al. / Field Crops Research 115 (2010) 171–178
Fujita, N., Yoshida, M., Asakura, N., Ohdan, T., Miyao, A., Hirochika, H., Nakamura, Y.,
2006. Function and characterization of starch synthase I using mutants in rice.
Plant Physiol. 140, 1070–1084.
Gallant, D.J., Bouchet, B., Baldwin, P.M., 1997. Microscopy of starch: evidence of a
new level of granule organization. Carbohydr. Polym. 32, 177–191.
Gao, M., Wanat, J., Stinard, P.S., James, M.G., Myers, A.M., 1998. Characterization of
dull1, a maize gene coding for a novel starch synthase. Plant Cell 10, 399–412.
Gérard, C., Barron, C., Colonna, P., Planchot, V., 2001. Amylose determination in
genetically modified starches. Carbohydr. Polym. 44, 19–27.
Hennen-Bierwagen, T.A., Lin, Q., Grimaud, F., Planchot, V., Keeling, P.L., James, M.G.,
Myers, A.M., 2009. Proteins from multiple metabolic pathways associate with
starch biosynthetic enzymes in high molecular weight complexes: a model for
regulation of carbon allocation in maize amyloplasts. Plant Physiol. 149, 1541–
1559.
Hurkman, W.J., McCue, K.F., Altenbach, S.B., Korn, A., Tanaka, C.K., Kothari, K.M.,
Johnson, E.L., Bechtel, D.B., Wilson, J.D., Anderson, O.D., DuPont, F.M., 2003.
Effect of temperature on expression of genes encoding enzymes for starch
biosynthesis in developing wheat endosperm. Plant Sci. 164, 873–881.
James, M.G., Denyer, K., Myers, A.M., 2003. Starch synthesis in the cereal endo-
sperm. Curr. Opin. Plant Biol. 6, 215–222.
Jane, J.L., Chen, Y.Y., Lee, L.F., McPherson, A.E., Wong, K.S., Radosavljevic, M.,
Kasemsuwan, T., 1999. Effects of amylopectin branch chain length and amylose
content on the gelatinization and pasting properties of starch. Cereal Chem. 76,
629–637.
Jobling, S., 2004. Improving starch for food and industrial applications. Curr. Opin.
Plant Biol. 7, 210–218.
Keeling, P.L., Banisadr, R., Barone, L., Wasserman, B.P., Singletary, G.W., 1994. Effect
of temperature on enzymes in the pathway of starch biosynthesis in developing
wheat and maize grain. Aust. J. Agric. Res. 21, 807–827.
Konik-Rose, C.M., Moss, R., Rahman, S., Appels, R., Stoddard, F., McMaster, G., 2001.
Evaluation of the 40 mg swelling test for measuring starch functionality. Starch/
Stärke 53, 14–20.
Konik-Rose, C.M., Thistleton, J., Chanvrier, H., Tan, I., Halley, P., Gidley, M., Kosar-
Hashemi, B., Wang, H., Larroque, O., Ikea, J., McMaugh, S., Regina, A., Rahman,
S., Morell, M., Li, Z., 2007. Effects of starch synthase IIa gene dosage on grain,
protein and starch in endosperm of wheat. Theor. Appl. Genet. 115, 1053–
1065.
McCormick, K.M., Panozzo, J.F., Hong, S.H., 1991. A swelling power test for selecting
potential noodle quality wheats. Aust. J. Agric. Res. 42, 317–323.
Mengel, K., Judel, G.-K., 1981. Effect of light intensity on the activity of starch
synthesizing enzymes and starch synthesis in developing wheat grains. Physiol.
Plant. 51, 13–18.
Mohammadkhani, A., Stoddard, F.L., Marshall, D.R., 1999. Amylose content in
segregating populations of einkorn, emmer, and rye. Starch/Stärke 51, 66–73.
Morell, M.K., Myers, A.M., 2005. Towards the rational design of cereal starches. Curr.
Opin. Plant Biol. 8, 204–210.
Morrison, W.R., Laignelet, B., 1983. An improved colorimetric procedure for deter-
mining apparent and total amylose in cereal and other starches. J. Cereal Sci. 1,
9–20.
Morrison, W.R., Tester, R.F., Snape, C.W., Law, R., Gidley, M.J., 1993. Swelling and
gelatinisation of cereal starches. IV. Effects of lipid complexed amylose and free
amylose in waxy and normal barley starches. Cereal Chem. 70, 385–391.
Pooni, H.S., Kumar, I., Khush, G.S., 1993. Genetical control of amylose content in a
diallel set of rice crosses. Heredity 71, 603–613.
Regina, A., Bird, A., Topping, D., Bowden, S., Freeman, J., Barsby, T., Kosar-Hashemi,
B., Li, Z., Rahman, S., Morell, M., 2006. High-amylose wheat generated by RNA
interference improves indices of large-bowl health in rats. Proc. Natl. Acad. Sci.
(U.S.A.) 103, 3546–3551.
Sasaki, T., Matsuki, J., 1998. Effect of wheat starch structure on swelling power.
Cereal Chem. 75, 525–529.
Schofield, P., 2006. The use of antibody-based diagnostics to identify end product
quality traits in Triticale. MSc. thesis. Elizabeth Macarthur Agricultural Insti-
tute. The University of Sydney, Australia, Sydney.
Shi, Y.C., Seib, P.A., Bernardin, J.E., 1994. Effects of temperature during grain-filling
on starches from six wheat cultivars. Cereal Chem. 71, 369–383.
Smith, A.M., Denyer, K., Martin, C., 1997. The synthesis of the starch granule. Annu.
Rev. Plant Phys. 48, 67–87.
Soh, H.N., Sissons, J., Turner, M.A., 2006. Effect of starch granule size distribution and
elevated amylose content on durum dough rheology and spaghetti cooking
quality. Cereal Chem. 5, 513–519.
Stoddard, F.L., 2003. Genetics of starch granule size distribution in tetraploid and
hexaploid wheats. Aust. J. Agric. Res. 54, 637–648.
Sultana, N., Howes, N., Ansari, O., Turner, M., 2006. Variation in starch swelling power
in wheat varieties and a doubled haploid population having the GBSS WxB1a
Allele and molecular markers for this phenotype. In: Mercer, C.F. (Ed.), Breeding
for Success: Diversity in action. Proceedings of the 13th Australasian Plant
Breeding Conference, Christchurch, New Zealand.
Takeda, Y., Hizukuri, S., 1987. Structure of rice amylopectins with low and high
affinities for iodine. Carbohydr. Res. 168, 79–88.
Tester, R.F., Morrison, W.R., 1990. Swelling and gelatinisation of cereal starches. I.
Effects of amylopectin, amylose and lipids. Cereal Chem. 67, 551–557.
Tetlow, I.J., Beisel, K.G., Cameron, S., Makhmoudova, A., Liu, F., Bresolin, N.S., Wait, R.,
Morell, M.K., Emes, M.J., 2008. Analysis of protein complexes in wheat amy-
loplasts reveals functional interactions among starch biosynthetic enzymes.
Plant Physiol. 146, 878–1891.
Tetlow, I.J., Morell, M.K., Emes, M.J., 2004. Recent developments in understanding
the regulation of starch metabolism in higher plants. J. Exp. Bot. 55, 2131–2145.
Topping, D.L., Clifton, P.M., 2001. Short-chain fatty acids and human colonic
function: roles of resistant starch and non starch polysaccharides. Physiol.
Rev. 81, 1031–1064.
Topping, D.L., Morell, M.K., King, R.A., Li, Z., Bird, A.R., Noakes, M., 2003. Resistant
starch and health–Himalaya 292, a novel barley cultivar to deliver benefits to
consumers. Starch/Starke 55, 539–545.
Wallwork, M.A.B., Jenner, C.F., Logue, S.J., Sedgley, M., 1998. Effect of high tem-
perature during grain-filling on the structure of developing and malted barley
grains. Ann. Bot. 82, 587–599.
Wang, Y.-J., White, P., Pollak, L., Jane, J., 1993. Characterization of starch structures
of 17 maize endosperm mutant genotypes with Oh43 inbred line background.
Cereal Chem. 70, 171–179.
Watanabe, N., Noda, Y., Goto, N., Miura, H., 1998. Variation for apparent amylose
content of endosperm starch in Triticum durum and Aegilops squarrosa. Euphy-
tica 101, 283–286.
Yamamori, M., Fujita, S., Hayakawa, K., Matsuki, J., Yasui, T., 2000. Genetic elimina-
tion of a starch granule protein, SGP-1, of wheat generates an altered starch
with apparent high amylose. Theor. Appl. Genet. 101, 21–29.
Yamamori, M., Kato, M., Yui, M., Kawasaki, M., 2006. Resistant starch and starch
pasting properties of a starch synthase IIa-deficient wheat with apparent high
amylose. Aust. J. Agric. Res. 57, 531–535.
Yamamori, M., Quynh, N.T., 2000. Differential effects of Wx-A1.-B1 and -D1 protein
deficiencies on apparent amylose content and starch pasting properties in
common wheat. Theor. Appl. Genet. 100, 32–38.
Zhang, G., Ao, Z., Hamaker, B.R., 2008. Nutritional property of endosperm starches
from maize mutants: a parabolic relationship between slowly digestible starch
and amylopectin fine structure. J. Agric. Food Chem. 56, 4686–4694.
Zhao, X.C., Batey, I.L., Sharp, P.J., Crosbie, G., Barclay, I., Wilson, R., Morell, M.K.,
Appels, R., 1998. A single genetic locus associated with starch granule proper-
ties and noodle quality in wheat. J. Cereal Sci. 27, 7–13.
Zheng, X., Wu, J.G., Lou, X.Y., Xu, H.M., Shi, C.H., 2008. The QTL analysis on maternal
and endosperm genome and their environmental interactions for characters of
cooking quality in rice (Oryza sativa L.). Theor. Appl. Genet. 116, 335–342.