Molecular Plant Breeding 2016, Vol.7, No.5, 1-8
http://mpb.biopublisher.ca
Research Article
Open Access
Allelic Distribution of Puroindoline Genes Affecting the Grain Hardness in
Some Iranian Bread Wheat Cultivars
Ali Izanloo
, Sanaz Norouzdokht-Nokhandan, Mohammad Zabet, Mohammad-Ghader Ghaderi
Department of Agronomy and Plant Breeding, Faculty of Agriculture, University of Birjand, Shouth Khorassan, Birjand, Iran
Corresponding authors email: a.izanloo@birjand.ac.ir
Molecular Plant Breeding, 2016, Vol.7, No.05 doi: 10.5376/mpb.2016.07.0005
Received: 17 Nov., 2015
Accepted: 01 Jan., 2016
Published: 03 Jan., 2016
Copyright © 2016 Ali et al., This is an open access article published under the terms of the Creative Commons Attribution License, which permits unrestricted
use, distribution, and reproduction in any medium, provided the original work is properly cited.
Preferred citation for this article:
Ali Izanloo, Sanaz Norouzdokht-Nokhandan, Mohammad Zabet, and Mohammad-Ghader Ghaderi, 2016, Allelic Distribution of Puroindoline Genes Affecting
The Grain Hardness in Some Iranian Bread Wheat Cultivars, Molecular Plant Breeding, 7(05): 1-8 (doi: 10.5376/mpb.2016.07.0005)
Abstract Grain hardness is an important characteristics influencing end-use quality of bread wheat. Wheat with the hard grain
texture has higher protein content and more intensive gluten that leads to production of higher bread quality. However, soft texture
wheat has lower protein content and weaker gluten, which are suitable for cake and candy production. Grain hardness is determined
by the Pina and Pinb genes that are located on the short arm of chromosome 5D. In this study, 83 bread wheat cultivars were
characterized for the allelic distribution of Pina and Pinb genes using allele specific markers. At the Pina locus, Pina-D1a and
Pina-D1b alleles were observed in 64% and 36% of varieties, respectively. However, at the Pinb locus, 90% of varieties showed
Pinb-D1a and 10% had Pinb-D1b allele. Based on the grain hardness index which was measured with the NIR, varieties whit the
value less than 50 were considered as soft while above 50 were considered as hard grain texture. The results of marker-trait
association showed that Pina had the significant effect (P = 0.019) on the grain hardness, as cultivars with the Pina-D1a allele were
significantly softer (49.850.37) that those with the Pina-D1b allele (51.380.50). The results of regression analysis showed that for
the traits including bread volume, grain hardness and water absorption as dependent variables, Pina was entered to the model. The
results of this study can help wheat breeders to effectively select parents for the grain hardness in the MAS breeding programs.
Keywords MAS; Grain hardness; Quality; Bread wheat
Introduction
higher water absorption of damaged starch, while soft
wheat, which has low water absorption due to less
protein and starch damage, is ideal for cookies, cakes
and pastries (Lillemo et al., 2006; Morris and Rose,
1996).
Physical characteristics of wheat grain, including
grain weight, dimensions, shape, uniformity, density
and texture, can affect the storage, transportation,
milling and marketing of bread wheat. Grain hardness
is an important quality characteristic of bread wheat
that determines the end use properties and market
classification of the grain. Wheat is classified into soft,
medium soft, hard, medium hard and extra hard on the
basis of grain hardness (reviewed in Pasha et al.,
2012).
Grain hardness is controlled by one major genetic
factor, the hardness locus (Ha) on the short arm of
chromosome 5D. The locus contains two genes
Puroindoline a (Pina) and Puroindoline b (Pinb)
within a region of about 82.0 bp (Chantret et al., 2005;
Morris, 2002). The Pina and Pinb genes code for
puroindoline a and puroindoline b proteins,
respectively. These genes confer soft endosperm when
they are both in their wild-type allelic states
(Pina-D1a/Pinb-D1a). Hard wheat is the result of
mutations in either Pina or Pinb (Morris, 2002).
Both genes have various different alleles in hexaploid
wheat. Highly conserved mutations in these two genes
have been identified and named Pina-D1b and
Pinb-D1b, the former being a null allele and the latter
representing a glycine to serine mutation at position
46 in the puroindoline protein (Giroux and Morris,
In soft wheat, the starch granules are loosely bound to
the surrounding protein matrix, the soft grains are easy
to mill and produce fine-textured flour with a high
proportion of undamaged starch granules. In contrast,
in the hard wheat there is a tight linkage between the
starch granules and protein matrix, hard grains resist
mechanical crushing and are more difficult to mill and
more energy is needed during milling. Hence, flour
with coarser texture is produced with higher
proportion of damaged starch granules. Hard wheat is
more suitable for yeast-leavened bread because of the
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1997; 1998). Several more alleles of Pina-D1 and
Pinb-D1 have now been identified. Until recently, 17
Pina and 25 Pinb alleles have been reported in
hexaploid wheat and related species (Chen et al., 2012;
Morris and Bhave, 2008). Recent studies showed that
all hardness mutations do not have equal effect of
grain texture (Giroux et al 2000; Martin et al 2001).
Data of allelic contribution of Pina and Pinb indicated
that those hard wheats with null allele at Pina loci
which lack puroindoline a protein (Pina-D1b
/Pinb-D1a) were somewhat harder than those hard
wheats that possess a normal soft puroindoline a but
have an altered, mutant form of puroindoline b
(Pina-D1a /Pinb-D1b) (Morris and Massa, 2003).
program. Therefore, the objective of this study was to
characterize some Iranian wheat cultivars for the
allelic distribution of puroindoline genes and to study
their association with grain quality characteristics.
1 Results and Discussion
1.1 Grain quality related traits
The descriptive statistics for grain quality traits,
measured with the NIR, showed that the average of
grain protein content was 12.81% with the range of
11.5-14.0 in the studied wheat cultivars. The mean of
hardness index of the grains was 50.41 with the
variation range of 44.0-58.0. Zeleny number was
obtained with an average of 35.09 and variation range
of 32.0-40.0. Bread volume had the average of 498.4
with the variation range from 450 to 570 values.
(Table 1). The phenotypic coefficient of variations for
traits showed the existence of variations between the
studied wheat cultivars, except for water absorption
with 1.3%.
Knowledge on the allelic variability of Pina and Pinb
genes in representative sets of bread wheat germplasm
is of importance for their classification and it can help
better explain the nature and different classes of grain
hardness. It can also assist wheat breeders to precisely
apply this information in a marker-assisted selection
Table 1 Mean, standard deviation, range and phenotypic coefficient of variations of grain quality related traits measured in the
studied wheat cultivars.
Grain quality traits
Protein content (%)
Zeleny Number
Bread Volume
Hardness Index
Water Absorption
Average ±SD.
12.82 ±0.68
35.09 ±1.83
498.42 ±25.8
50.41 ±2.68
64.66 ±0.82
Range
11.5-14.0
32.0-40.0
450-570
44.0-58.0
62.9-66.2
Bivariate correlation analysis showed that grain
hardness had a positive correlation with protein
content (r = 0.59, p < 0.0001) and Zeleny number (r =
0.57, p < 0.0001) that is consistent with the reports of
Grosse et al (2004). According to these reports,
significantly positive correlation was observed
between grain protein content and grain hardness
(Table 2). A significantly positive correlation was also
observed between Zeleny number and protein content
(r = 0.87, p < 0.0001), which was accordance with the
results of Shevery and Tatham (2000), where grain
protein content had a positive correlation with Zeleny
number. It can be concluded that the harder grains had
more water absorption. The correlation analysis of the
studied traits showed that the water absorption had
significantly positive correlation with protein content
(r = 0.42, p < 0.0001), Zeleny number (r = 0.38, p <
0.001) and grain hardness (r = 0.23, p < 0.05).
PCV ()
5.3
5.2
5.2
5.3
1.3
Hard texture wheat had higher protein and more
intensive gluten and thus higher bread quality
compared to soft texture wheat (Bushuk, 1998;
Tipples et al., 1994). High protein content and hard
grains would produce courser flour with more
damaged starch, consequently lead to higher water
absorption.
Table 1 Bivariate correlation analysis for some quality related
traits in wheat cultivars. Protein content (PROT), Zeleny
Number (ZELL), Bread Volume (BV), Hardness Index (HI),
Water Absorption (WA)
PROT
ZELL
BV
HI
WA
2
Prot
ZELL
BV
HI
WA
1
0.867**
-0.041
0.589**
0.420**
1
-0.008
0.568**
0.383**
1
-0.040
0.079
1
0.234*
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1.2 Pina and Pinb alleles
A set of co-dominant STS primers (Huang and
Brûlé-Babel, 2011) for identification of the
puroindoline a alleles, amplified two specific bands of
447 and 625 bp (Figure 1) for Pina-D1a and
Pina-D1b alleles, respectively. In this study, Pina-D1a
allele was observed in 64% of the cultivars (54), while
Pina-D1b allele was observed in 36% of the studied
cultivars (30).
100 bp
DNA
Ladder
puroindoline b (Pinb-D1c-d) was detected in the
studied wheat cultivars.
M
1
2
3
4
5
6
7
8
9
10
11 12
13
14 15 16
17
18 19
A
250 bp
B
250 bp
1
2
3
4
5
6
7
8
9
10
11
625 bp
Figure 2 Agarose gel electrophoresis of PCR products for the
allele specific primers of A) Pinb-D1aF and Pinb-D1aR for
identification of Pinb-D1a allele, and B) ( Pinb-D1aF and
Pinb-D1bR for identification of Pinb-D1b allele. M is 100 bp
DNA size marker, numbers from left to right (1-19) were
cultivars of Soomali3, Sistan, Shahpasand, Shahi, Shole,
Shariyar, Shiraz, Shiroodi, Tabasi, Toos, Quds, Ghafghaz,
Gaskogen, Cross Alborz, Cross Falat hamoon, Karaj2, Karaj 3,
Karkhe, Kpphdasht, respectively.
447 bp
Figure 1 The STS primers of Pina-F1c ،Pina-R1b ،Pina-R2a
amplified fragments of 447 bp and 625 bp from for Pina-D1a
and Pina-D1b alleles at the puroindoline a locus, respectively.
The lanes from 1 to 11 representing cultivars of Darya, Arg,
Rooshan, Dez, Zarrin, Tajan. Pishtaz, Chamran, Kohdasht,
Kavir and Maroon, respectively.
In general, 56% of the cultivars had the wild type
alleles (Pina-D1a and Pinb-D1a) at both loci (Table 3).
34.5% of the cultivars showed the mutation allele at
puroindoline a locus (Pina-D1b), but the wild type
allele at the puroindoline b locus. The cultivars with
Pina-D1a and Pinb-D1b were about 8.3% (7), while
only one cultivar was found to have mutation alleles at
both loci (Pina-D1b/Pinb-D1b).
However, primers used for identification of alleles in
the Pinb locus amplified a band of 250 bp for
Pinb-D1a and Pinb-D1b alleles (Figure 2). Pinb-D1a
allele was observed in 90% of the cultivars (76), while
only 10% of the cultivars (8) represented the
Pinb-D1b allele. None of the alternative mutations in
Table 2 Mean squares of quality traits resulted from the analysis of variance for different Pin alleles in the studied wheat cultivars.
Protein content (PROT), Zeleny Number (ZELL), Bread Volume (BV), Hardness Index (HI), Water Absorption (WA)
Pin alleles
Pina
Pinb
Pina.Pinb
Residual
Total
df
1
1
1
76
79
WA
HI
*
3.89
0.06NS
1.33 NS
0.635
0.67
BV
*
ZELL
*
39.54
6.46 NS
0.13 NS
6.83
7.15
4075.0
28.0 NS
1098.0 NS
613.4
665.9
NS
3.84
0.22 NS
0.45 NS
3.46
3.36
PROT
1.19 NS
0.01 NS
1.22 NS
0.446
0.46
*, represent significant differences at 5% significant level
NS, represent non-significant differences
1.3 Marker-trait association
The results of unbalanced analysis of variance for the
quality traits as variate and the Pin alleles as factors
(Table 4) showed that only Pina had a significant
effect on grain hardness (p = 0.019), water absorption
(p = 0.015) and bread volume (p = 0.012). Pinb and
the interaction between Pina and Pinb had not
significant effects on grain quality traits. Mean
comparison between different alleles at the Pina and
Pinb genes as well as allelic combinations at both loci
showed that cultivars with the Pina-null allele
(Pina-D1b) had significantly higher grain hardness
(51.34 ± 0.48) relative to those with the wild-type
(Pina-D1a) allele (49.88±0.36). At this locus, cultivars
with Pina-D1b allele had also significantly higher
water absorption (64.95±0.14) and relatively smaller
bread volume relative to Pina-D1a allele (Table 5).
Although, F-statistic for allelic combinations showed no
significant differences, p-value for HI, WA and BV were
0.09, 0.08 and 0.07, respectively. Cultivars having
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wild-type alleles at both loci (Pina-D1a/Pinb-D1a) had
relatively smaller grain hardness (49.76 ± 0.4), while
cultivars bearing either mutant alleles at each locus or
both loci showed relatively higher harness index
(Table 5).
Table 3 The classification of wheat germplasm based on allelic combination and grain texture
Allelic combination
at Pina/Pinb loci
Phenotype
Hardness index
(Mean  Se)
Cultivars
Soft
49.76  0.39
Hard
51.32  0.49
Pina-D1a/Pinb-D1b
Hard
50.83  0.48
Atrak٫ Arg٫ Arvand mutant٫ Star٫ Akbari٫ Alamoot٫ Omid٫ Owhadi٫
Azar1٫ Azar2٫ Azadi٫ Roshan backcross vint.٫ Roshan backcross Sp.٫
Bam٫ Boolani٫ Bayat٫ Peptik٫ Darya٫ Rasad٫ Roshan٫ Zarrin٫ Syson٫
Sabalaan٫ Sardari٫ Sardari101٫ Soomali 3٫ Sistan٫ Shahpasand٫
Shahi٫ Shahriyar٫ Tabasi٫ Cross Alborz٫ Cross Falat, Hamoon٫ Karaj
2٫ Kouhdasht٫ Gaspard٫ Moqan 2٫ Mahdavi٫ Naz٫ Navid٫ Niknejad٫
Homa٫ Hirmand٫ MV-17٫ S -83- 3
Alvand٫ Arta٫ Bahar (MV-79-7) Pishtaz٫ Tajan Dez٫ Rasool٫ Zagros٫
Sepahan٫ Shole٫ Shiraz٫ Shiroodi٫ Toos٫ Quds٫ Karaj 3٫ Karkhe٫
Kavir٫ Maroon٫ Marvdasht٫ Moqan 3٫ Verinak٫ DN11٫ Excalibur٫
GR٫ GS٫ Kukri٫ Weeble٫ WS-82-9
Inia٫ Bezostaja٫ Rizhav٫ Qafqaz٫ Gascogen٫ Moqan1٫ Gladious
Pina-D1b/Pinb-D1b
Hard
52.00  0.38
Darab 2
Pina-D1a/Pinb-D1a
Pina-D1b/Pinb-D1a
2 Conclusion and Discussion
hardness allele in hard wheat cultivars in North
American and northern European germplasm (Lillemo
and Morris, 2000; Morris et al., 2001). Whereas the
Pinb-D1b allele prevails among the spring and winter
wheats of North America, Europe, China and Australia
(Cane et al., 2004; Lillemo and Morris, 2000; Morris
et al., 2001; Xia et al., 2005), Pinb-D1c and Pinb-D1d
were mostly found in Western Europe (Lillemo and
Morris, 2000). Likewise, the Pina-D1b has been
found to be the primary cause of hardness in Iranian
and Indian wheat (Mohammadi et al., 2013; Ram et al.,
2002).
In this study, 84 wheat cultivars were characterized for
the major alleles of Pina and Pinb genes. The most
frequent allele was Pinb-D1a and the second most
frequent allele was Pina-D1a. This result suggests that
most of the wheat cultivars were soft grain, with
allelic combination of Pina-D1a/Pinb-D1a at both
loci. The Pina-D1b (Null mutation) allele was the
most frequent grain hardness allele which observed in
36% of the cultivars. The hardness allele of Pinb-D1b
was observed only in 10% of the studied wheat
cultivars. Lillemo et al. (2006) also found the
Pina-D1b as the most frequent hardness allele which
presented in 283 of the 328 lines with hard endosperm.
A study of historically important CIMMYT bread
wheat lines showed that Pina-D1b has been the
dominating hardness allele since the inception of the
wheat breeding program in Mexico (Lillemo et al.,
2006). Mohammadi et al. (2013) reported
Pina-D1a/Pinb-D1a allelic combinations as the most
frequent genotypes.
Based on unbalanced analysis of variance to identify
marker-trait association, Pina had a significant effect
on grain hardness, water absorption and bread volume.
In this study, cultivars with the Pina-D1a allele were
significantly harder with more water absorption and
less bread volume. Several studies have indicated that
cultivars with Pina-null alleles (Pina-D1b) are usually
harder than those of Pinb-D1b in bread wheat, so that
Pina-D1b allele gives slightly harder endosperm than
Pinb-D1b, and is associated with lower milling yield
and higher water absorption compared to Pinb-D1b
(Cane et al., 2004; Chen et al., 2013; Chen et al.,
2006; Giroux et al., 2000; Lillemo et al., 2006;
Martin et al., 2001). Morris and Massa (2003) have
also reported that Among the hard red spring wheat
cultivars that possess the Pina- D1b
The most second frequent genotype was
Pina-D1b/Pinb-D1a genotypes. However, Cane et al.
(2004) reported that the Pinb-D1b allele was the major
determinant of hardness in southern Australian wheat
germplasm, so that, this allele was found in 84% of
the cultivars and breeding lines (Cane et al., 2004).
The Pinb-D1b allele has been the most frequent
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Table 4 Means, standard error of means and F-statistics for different traits at different alleles and allelic combinations of Pin genes. Protein content (PROT), Zeleny Number (ZELL), Bread
Volume (BV), Hardness Index (HI), Water Absorption (WA)
Trait
PROT
ZEL
Loci
Alleles/ Allelic
combinations
N
Mean ±SE
F-Statistics
P-value
Mean ±SE
F-Statistics
P-value
Mean ±SE
BV
HI
WA
F-Statistics
P-value
N
Mean ±SE
F-Statistics
P-value
Mean ±SE
Pina
Pina-D1a
40
12.71 ±0.1
Pinb
Pina-D1b
25
12.99 ±0.15
2.66
0.108
34.9 ±0.27
35.4 ±0.4
Pinb-D1a
59
12.8 ±0.09
6
12.93 ±0.16
0.19
0.661
35.05 ±0.25
35.5 ±0.43
0.32
0.572
1.15
0.289
504.68 ±
4.04
488.4 ±4.67
498.97 ±
3.43
6.661
0.012
51
49.88 ±0.36
29
51.34 ±0.48
5.87
0.018
64.49 ±0.12
64.95 ±0.14
Pinb-D1b
493 ±8.46
Pina-D1a/Pinb-D
1a
35
12.66 ±0.11
34.77 ±0.3
Pina/Pinb
Pina-D1a/Pinb-D
Pina-D1b/Pinb-D
1b
1a
24
5
13.02 ±0.15
13.08 ±0.09
1.98
0.13
35.46 ±0.42
35.8 ±0.37
34
494.6 ±10.17
485
6
50.83 ±0.48
1
52
64.53 ±0.21
64.2
1.04
0.38
506.11 ±4.38
488.54 ±4.86
.288
0.593
73
50.36 ±0.32
Pina-D1b/Pinb-D
1b
1
12.2
2.50
0.07
7
51 ±0.44
0.37
0.546
64.67 ±0.1
64.49 ±0.18
45
49.76 ±0.4
28
51.32 ±0.5
2.25
0.09
64.48 ±0.13
64.98 ±0.14
F-Statistics
6.12
0.32
2.32
P-value
0.016
0.571
0.08
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3 Materials and Methods
(a-null) hardness allele were harder than the hard red
winter wheat cultivars that possess the Pinb-D1b allele
based on NIR, PSI, and break flour yield. Malekzadeh
et al. (2008) have reported that among Iranian wheats,
those possessing Pina-D1b allele represented a higher
hardness value than those with Pinb-D1b allele.
However, Pina-D1b may be less preferable from a
milling standpoint as well as processing quality.
Genotypes with Pina-D1b alleles may have a
relatively inferior processing quality for steamed
bread, pan bread, and Chinese noodles than the
Pinb-D1b genotype (Chen et al., 2007; Chen et al.,
2012).
3.1 Plant materials and DNA extraction
A total of 84 wheat cultivars were planted at the
experimental field of Faculty of Agriculture,
University of Birjand, Iran, in the 2013-14 cropping
season according to local management practices. At
the stage of tillering, leaf samples of each individual
were collected and snap-frozen in liquid nitrogen then
stored at -20°C. After harvest, about 50 g of bulked
clean seeds from each individual were used to
measure grain quality traits including grain hardness,
protein content, Water absorption, bread volume,
Zeleny Number using the near-infrared reflectance
(NIR) spectroscopy (Inframatic No. 8100) on ground
grain (Method 39-70A in AACCI, 2002).
The results of this study showed that the wild type
alleles (Pinb-D1a and Pina-D1a) had the highest
allelic frequency, while mutant alleles (Pina-D1b and
Pinb-D1b) representing hardness characteristic were
less frequent in the tested wheat cultivars. Among
hardness alleles, Pina-D1b was observed in 36% of
cultivars, while only 10% of the wheat cultivars
possessed Pinb-D1b. According to marker-trait association
analysis, hard wheat cultivars having Pina-D1b (null-mutant
allele) were harder in grain texture, with more water
absorption and lesser bread volume. The use of STS
diagnostic markers is a simple and reliable method to
characterize wheat germplasms for Pina and Pinb
allelic variability and distribution. The result of this
study provides useful information for this set of wheat
cultivars, which can be used in the future MAS wheat
quality improvement programs.
Extraction of DNA was performed according to the
method of Pallotta et al. (2003) with minor
modifications. The quality and quantity of the
extracted DNA was assessed using NanoDrop 2000
spectrophotometer (Thermo Scientific).
3.2 PCR amplification of Pina and Pinb
The Pina alleles were amplified using a set of
co-dominant STS primers (Table 6) described by
(Huang and Brûlé-Babel, 2011) yielding products of
~447 bp and ~625 bp for Pina-D1a and Pina-D1b
alleles, respectively. The Pinb-D1a and Pinb-D1b
alleles were identified using allele specific primers
that amplify a fragment of about 250 bp (Giroux and
Morris, 1997; 1998).
Table 6 The primer characteristics with the expected size product and the PCR thermo cycling conditions
Primer name
Allele
Primer sequences (5’-3’(
Pina-F1c
Pina-D1a
CACAACCGCACACAGAAATC
Pina-R1b
Pina-R2a
Pina-D1b
GATCACGCTGAAATCCGAA
TCACCCAATGCTGAAGACAC
Pinb-D1aF
Pinb-D1aR
Pinb-D1aF
Pinb-D1bR
Pinb-D1a
Pinb-D1b
Thermal cycle
ATGAAGACCTTATTCCTCCTA
CTCATGCTCACAGCCGCC
ATGAAGACCTTATTCCTCCTA
CTCATGCTCACAGCCGCT
94°C٫ 5 minute
35 cycle (94°C٫ 30
second- 60°C٫ 30
second-90°C٫ 72
second), 72°C٫10
minute
94°C٫ 3 minute, 35
cycle (94°C٫ 45
second- 57°C٫ 35
second-72°C٫45
second), 72°C٫ 10
minute
Product
size )bp)
447
625
Reference
250
Giroux and (
Morris, 1997;
)1998
250
(Huang
and
Brûlé-Babel,
2011)
in 20 μL volume containing 2 μl of 10X PCR buffer,
1.5 mM MgCl2, 0.4 mM of each dNTP, 1 U of Taq
PCR amplifications were performed in an Eppendorf
gradient thermocycler. PCR reactions were carried out
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DNA polymerase and ~100 ng of genomic DNA.
Thermal cycling conditions for the primer pairs are
presented in Table 6. PCR products were separated on
1.5% agarose gel.
and b, Proceedings of the National Academy of Sciences, 95(11):
6262-6266
http://dx.doi.org/10.1073/pnas.95.11.6262
Giroux M.J., Talbert L., Habernicht D.K., Lanning S., Hemphill A., and
Martin J.M., 2000, Association of puroindoline sequence type and
grain hardness in hard red spring wheat, Crop Science, 40(2): 370-374
3.3 Statistical analysis
The statistical analysis was performed to estimate the
effects of the puroindoline genes on grain quality traits
and to identify effective alleles on quality traits. In
order to compare the effects of different alleles on the
quality related traits, the unbalanced analysis of
variance was carried out by GenStat release 12.1
(Payne et al., 2009). Grain quality related traits were
considered as varieties and Pin alleles and allelic
combinations were considered as factors.
http://dx.doi.org/10.2135/cropsci2000.402370x
Groos C., Bervas E., and Charmet G., 2004, Genetic analysis of grain
protein content٫ grain hardness and dough rheology in hard-hard bread
wheat progeny, Journal of Cereal Science, 40(2): 93-100
http://dx.doi.org/10.1016/j.jcs.2004.08.006
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