International Journal of Agriculture and Crop Sciences.
Available online at www.ijagcs.com
IJACS/2013/6-2/57-62
ISSN 2227-670X ©2013 IJACS Journal
Genetic Variability Analysis of Grains Fe, Zn and
Beta-carotene Concentration of Prevalent Wheat
Varieties in Iran
Hedieh Badakhshan1*, Namdar Moradi1, Hadi Mohammadzadeh1, Mohammad Reza
Zakeri2
1. Department of Agronomy and Plant breeding, Faculty of Agriculture, University of Kurdistan, Sanandaj, Iran
2. Department of Plant Breeding, Faculty of Advanced Studies, Islamic Azad University of Kermanshah, Iran
* Corresponding author email: [email protected]
ABSTRACT: To improvement of grains quality of wheat and reduction of micronutrient malnutrition
in human populations as a basic method, current wheat varieties in Iran were analyzed for diversity
of micronutrients (Fe, Zn and Beta-carotene) concentrations in grain. Eighty two varieties including
modern, landrace and durum wheat were grown in field condition across two years to determine the
importance of genetic, environment and their interaction effects. Remarkable variation among 82
genotypes exhibited for all micronutrients concentration in grains. Based on the mean of two years,
-1
the concentration of grain Fe, Zn, protein and beta-carotene ranging from 41.36 to 67.67 mg Kg ,
-1
-1
from 36.37 to 73.80 mg Kg , from 7.5 to 15.6 percent and from 0.96 to 1.69 µg g respectively.
Significant differences for Fe, Zn and beta-carotene concentration of grains were existed among
genotypes and years. The interaction of year by genotype was significant for Fe and Zn but not for
beta-carotene. Landraces of wheat found to contain higher Zn concentration than modern and durum
varieties. Highly positive correlations were revealed among Fe and Zn but not for beta-carotene.
Both Fe and Zn were correlated positively with grains protein content (p< 0.001). No significant
correlation was found for spike numbers and the numbers of kernels per spike and micronutrients.
The results of this study showed a considerable variation for micronutrient concentration in grains
between genotypes for breeding purposes such as promoting high quality cultivars with high grains
micronutrient concentration or bifortification.
Keywords: Beta-carotene, Biofortification, Fe, micronutrient, protein, wheat, Zn
INTRODUCTION
Deficiencies of micronutrients (hidden hunger) are a major global health problem and more than 2
billion people in the world are estimated to be deficient in key vitamins and minerals, particularly vitamin A,
iodine, iron and zinc (FAO, 2011).
Mineral elements play numerous beneficial roles due to their direct and indirect effects in both plant
and human metabolism and the deficiencies or insufficient intakes of these nutrients leads to several
dysfunctions and diseases in humans (Welch and Graham, 1999) and also lead to entire failure of crops and
lower content of trace elements in plant parts (Cakmak, 2008).
Several strategies have been suggested as intervention programmers for the reduction of micronutrient
malnutrition in human populations (Ng’uni et al., 2012). They include food fortification, dietary supplementation
by use of complementary tablets. Other strategies are dietary diversification and micronutrient biofortification
programs through plant breeding (Ng’uni et al., 2012). Comparatively, plant breeding has been identified as
been potentially more sustainable and less expensive, since seeds could reach a larger number of people
without necessarily changing consumer’s behavior (Ortiz-Monasterio et al., 2007; Cakmak, 2008; Ng’uni et al.,
2012). This solution, however, requires a comprehensive exploration of potential genetic resources and indepth understanding of the physiological and genetic basis of nutrient- accumulation process in crops tissues
(Chatzav et al., 2010). One major strategy to improve the level of mineral nutrients and vitamins in staple food
crops is to exploit the natural genetic variation in food crops (Ortiz-Monasterio et al., 2007; Cakmak, 2008;
Gomez-Becerra et al., 2010a).
Wheat is the major staple food crop in many parts of the world, contributing 28% of the world edible dry
matter production and up to 60% of daily energy intake in several developing countries (Cakmak, 2008; Wang
et al., 2011). Iran has one of the world’s highest rates of wheat flour consumption, more than 300kg per capita
Intl J Agri Crop Sci. Vol., 6 (2), 57-62, 2013
in a year (Hemphill, 2012). Therefore, the composition and nutritional quality of the wheat grain have a
significant impact on human health and well-being, especially in developing countries (Chatzav et al., 2010;
Wang et al., 2011). However, most of the cultivated wheat cultivars have low grain Fe and Zn contents
(Cakmak et al., 2004; Cakmak, 2008; Wang et al., 2011). Cereal grains are inherently poor in concentration of
micronutrients, and rich in compounds depressing the bioavailability of micronutrients such as phytic acid
(Morgounov et al., 2007). The nutritional value of cereals needs to be improved through the general use of less
refined flour and the selection of wheat varieties with high mineral diversity (Oury et al., 2006). A survey of rice
indicated a fourfold variation in grain Zn and Fe concentrations similar variation was found in diverse wheat
accessions, although Fe and Zn concentrations were generally lower and showed less genetic variability
among cultivated tetraploid and hexaploid varieties than among wild diploid wheat and relatives (White and
Broadley, 2005).
Variation in mineral levels of wheat flour and bran has been attributed to genetic and environmental
effects, their interaction, and variation in protein levels (Peterson et al., 1986; Gomez-Becerra et al., 2010a, b).
Since wheat, along with rice, supplies the greatest amount of calories and protein per capita in the world,
therefore represents a useful vehicle for increasing the intake of essential nutrients such as carotenoids (Genc
et al., 2005). The concentration of carotenoids in wheat is small compared with that found in the fruit and
vegetables. However, the daily consumption of wheat based products in populations that rely almost entirely on
this grain for caloric intake, indicate that a small increase in concentration would have a large impact (Genc et
al., 2005). On the other hand, certain organic compounds such as beta-carotene (pro-vitamin A) stimulate the
absorption of essential elements by humans (White and Broadley, 2005). Carotenoids from a plant origin have
an advantage over retinol from animal sources, because excessive may cause a toxic excess in vitamin A,
whereas carotenoids can be converted to meet metabolic requirements (Sramkova et al., 2009).
The objective of this study was to evaluate the genetic diversity of grains Fe, Zn and beta- carotene
concentration in prevalent cultivated wheat varieties of Iran and identify varieties with higher micronutrient
concentration in the grain.
MATERIALS AND METHODS
Plant material and experimental condition
Eighty two wheat varieties including 74 of prevalent wheat varieties in Iran, five of landraces from Iran
and north of Iraq and three of durum wheat varieties were used in this study. The varieties were evaluated for
the grain mineral nutrient concentration of iron (Fe), zinc (Zn) and beta-carotene for two years (2009-2011), in
the research farm of University of Kurdistan (35 16’ E, 47 1’ N; 1375 above sea level) in Iran. Grains protein
content data were available only for second year (2010 -2011). Soil samples were randomly collected from the
top 0- 30 cm soil depth of experimental site and analyzed for micronutrients, organic maters and pH before
sowing (Table 1). The experimental design was complete block design with three replications. Each plot
consisted of three rows 1 m long with 0.2 m between rows.
Determination of grains Fe, Zn, beta-carotene and protein concentration
Grain samples (
were digested in a mixture of Chloridric (HCL) and Perchloridric acid (HCLO4)
according to Singh et al. (1999). Digested samples were analyzed for iron and zinc using flame atomic
absorption spectroscopy (SpectrAA220-Varian Ltd, Mulgrave, Australia). Mineral concentrations were
expressed as mg/kg dry weight, three replications were performed. Appropriate quality controls were performed
for each set of measurements.
Beta-carotene concentration of grains was measured according to Santra et al. (2003). Briefly;
Samples (8 gr) was extracted by 40 ml of water-saturated n-butanol (WSB) and the absorbance of supernatant
was measured at 440 nm by a UV- spectrophotometer. A calibration curve was made from known quantities of
pure beta-carotene.
Flour protein content was determined using near-infrared reflectance (NIR) spectroscopy of flour
samples from each plot according to Singh et al. (1999).
Statistical analysis
Grains micronutrient concentration data from each year were analyzed using SAS Version 9.1 (SAS
2002). Genetic diversity among genotypes was tested by analysis of variance using proc GLM. Combined
analysis of variance was done for evaluate the significance of year and genotype interactions. Associations
between traits were established by the Pearson correlation coefficient.
58
Intl J Agri Crop Sci. Vol., 6 (2), 57-62, 2013
RESULTS AND DISCUSSION
Iron and zinc are two micronutrients that along with pro-vitamin A (beta-carotene) are recognized by
the World Health Organization (WHO) as the most limiting due to their low bioavailability in diets based on
cereals and legumes (WHO, 2002). Breeding for enhanced concentrations of Fe and Zn can be divided into a
number of steps: identification of genetic variability within the range that can influence human nutrition,
intogressing this variation into high yielding genotypes possessing acceptable end-use quality attributes, testing
the stability of Fe and Zn accumulation across the target environment and large scale deployment of seed of
improved cultivars to farmers (Ortiz-Monasterio et al., 2007).
Genetic diversity for grain nutrients
The concentrations of three micronutrients (Fe, Zn and beta-carotene) were determined in wheat
genotypes on dry weight bases across two years and the data are presented on the table 2. The protein
content of grains was measured only in second year (2010-2011) because of some technical constraints.
Among the 81 cultivars of bread wheat, the concentration of grain Fe varied by 1.64 fold, ranging from
-1
-1
41.36 to 67.67 mg Kg , grain Zn by 2.03 fold, from 36.37 to 73.80 mg Kg , grain protein by 2.23 fold, from 7.5
-1
to 15.6 percent and grain beta-carotene 1.76 fold, from 0.96 to 1.69 µg g (based on the mean of two years,
table 2). The range of Fe and Zn concentration of bread wheat in the present study was similar to those
reported in earlier researches (Oury et al., 2006; Morgounov et al., 2007; Zhao et al., 2009). ANOVA showed
highly significant differences between wheat cultivars for grains Fe, Zn, protein and beta-carotene
concentrations in both years (table 3). Studies with rice and wheat, and preliminary studies with wild relatives
and landraces of wheat have demonstrated that considerable variation exists in grain Zn and Fe concentration
(Genc et al., 2005; Gomez-Becerra et al., 2010a, b). It has been suggested that in order to have a measureable
biological impact on human health, grain concentrations of Zn and Fe should be increased by at least 10 and
-1
25 mg Kg (Ortiz-Monasterio et al., 2007; Cakmak, 2008; Chatzav et al., 2010). The targets for Fe and Zn
-1
biofortification in wheat grain are around 60 and 40 mg Kg respectively (Ortiz-Monasterio et al., 2007).
Therefore, 11 of 81 examined varieties and most of the tested varieties contain high levels of Fe and Zn
respectively in this study. According to our knowledge, the variation of carotenoides especially beta-carotene
rarely reported in bread wheat.
Analysis of 15 cultivars of durum and landraces in the present study, showed similar means of grain
Fe to those of modern bread wheat but among the landraces and modern cultivars were significant differences
for Zn and protein concentration confirmed by Orthogonal contrasts tests (p< 0.01; table 2). These results were
consistent with the results reported by Zhao et al. (2009) and Murphy et al. (2008) in bread wheat. No
significant differences between three classes of wheat for beta-carotene (table 2). Blanco et al. (2011) reported
that wheat carotenoids concentrations are higher in cultivated varieties than in their wild counterparts. Highly
remarkable variation existing among wheat varieties for grain micronutrient concentrations and protein
indicated the potential for genetic improvement.
Interactions among genotype and environment
Analysis of the effects of year and interaction of year and genotype on Fe, Zn and beta-carotene
according to combined analysis exhibited that all of the effects were significant with the exception of year by
genotype interaction for beta-carotene, demonstrating the importance of environmental effects on Fe, Zn and
Beta-carotene concentration of grains. Three of eight genotypes and three of five genotypes that ranked
highest for Fe and Zn respectively, were the same in both years (table 4). Of the genotypes used in this study,
-1
in each two years, Gaspard (67.67 mgKg ) and two landraces Shahpasand and Azar2 (61.53 and 62.00 mgKg
1
-1
respectively) were superior in Fe, Superior genotypes in Zn were Sabalan (73.80 mgKg ), Gaspard and
-1
Shahpasand (70.98 and 65.69 mgKg respectively), MV17, Sistan, Golestan and R1-ERW87 (1.69, 1.61, 1.51
-1
-1
and 1.48 µg g respectively) had higher beta-carotene concentration and Bezostaya (15.6 mgKg ), Omid
-1
-1
(14.67 mgKg ) and two landraces Shoaleh and Rashagol (14.77 and 14.50 mgKg ) had significantly higher
protein than other genotypes according to second year data (based on Fisher’s LSD means comparison test;
p< 0.01).
Non-significant interactions among genotype and year for beta-carotene indicated that major changes
in these compounds may not occur in grains of wheat varieties in different years and location. Blanco et al.
(2011) underlined polygenic nature as well as genotypic component of yellow pigment concentration in durum
wheat although, environmental factors can influence carotenoids the genetic component is predominant in
durum wheat. In previous studies, significant genotype × environment interactions for grain nutrient
concentrations such as Fe and Zn were reported for bread wheat varieties (Oury et al., 2006; Morgounov et al.,
2007; Murphy et al., 2008; Wang et al., 2011) as well as for their wild and cultivated relatives (Peleg et al.,
2008; Chatzav et al., 2010; Gomez-Becerra et al., 2010a, b). Because the genotype variance was greater than
variety × year interaction for grains Fe and Zn concentration (table 4) thus, it can be concluded that there are
59
Intl J Agri Crop Sci. Vol., 6 (2), 57-62, 2013
non-cross-over interactions and therefore reasonable advances in selection and breeding can be expected as
suggested by previous studies (Peterson et al., 1986; Peleg et al., 2008; Chatzav et al., 2010).
The complexity of the inheritance of grain Fe and Zn concentrations in wheat, plus the associated low
heritability and the large environment and genotype ×environment interaction effects, slow progress in the
genetic analysis of these traits. However, in spite of these challenges there is evidence that breeding for
increased levels of micronutrients is feasible (Ortiz-Monasterio et al., 2007).
Analysis of correlation among micronutrients
Positive highly significant correlations (p≤0.001) among grains Fe, Zn and protein concentrations were
observed but grains beta-carotene did not significantly correlated to grains Fe, Zn and protein (table 5).
Significant correlation between Fe, Zn and protein were observed in most of the studies (Cakmak et al., 2004;
Morgounov et al., 2007; Demirkiran, 2009; Peleg et al., 2009; Zhao et al., 2009; Chatzav et al., 2010; Wang et
al., 2011). This has implicated for the possibility of combine selection for these micronutrients in a single
agronomic background. Genetic mapping in various wheat populations confirmed QTL co-localization
conferring high Protein, high Zn and high Fe (Peleg et al., 2009). Likewise, co-localization of QTLs for Zn and
Fe concentrations has been reported in rice (Garcia- Oliveira et al., 2009).Some genes like grain protein
content-B1 gene (Tt-NAM-B1; Gpc-B1) which is located on chromosome 6BS, can increase the Fe and Zn
translocation from leaves to seeds resulting in increased Fe and Zn accumulation in seeds (Oury et al., 2006;
Cakmak, 2008; Zhao et al., 2009; Wang et al., 2011). The orthologous gene (Tt- NAM-A1) is located on
chromosome 6AS and the paralogous gene (Tt- NAM-B2) is located on chromosome 2BS (Wang et al., 2011).
Similar relationship between protein, Fe and Zn has been reported in sorghum (Ng’uni et al., 2012), rice
(Garcia- Oliveira et al., 2009) and bean (Gelin et al., 2007).
No significant correlation were among grains protein, micronutrient concentration (Fe, Zn, betacarotene) and measured yield component traits such as spike length and kernels per spike number along with
plant height and peduncle length (results has not shown). Some earlier studies reported that plant productivity
exhibited positive and significant correlations with grain Zn and Fe (Murphy et al., 2008; Chatzav et al., 2010;
Gomez-Becerra et al., 2010b), but in some previous reported researches, increasing grain yield was found not
to result in lower micronutrient concentrations in wheat grain (Cakmak et al., 2004; Murphy et al., 2008; Wang
et al., 2011). The six genomic regions (2B, 2B, 4A, 4B, 5A, 7B) conferring grain yield as studied by Peleg et al.
(2009) were not significantly associated with QTLs for Zn, Fe or other minerals. Thus, the improve grain protein
and mineral concentration is not necessarily expected to reduce productivity. However, negative significant
correlations occurred between Fe, Zn, yield component and morphological traits reported by some authors
(Oury et al., 2006; Morgounov et al., 2007; Zhao et al., 2009; Wang et al., 2011). So, they concluded that
modern varieties with high grain yield and grain yield component traits tend to have lower concentrations of
micronutrients in the grain.
Estimation of broad sense heritability
2
Estimates of broad sense heritability (h B) ranged from 23.08% for beta- carotene to 90.62% for Fe in
first year and from 38.46% for beta-carotene to 90.90% for Zn in second year. Broad sense heritability
estimation of micronutrients exhibited medium to high for each year separately (table 3). Whereas, based on
combined analysis, broad sense heritability estimates ranged from 14.96% for Fe to 55% for Zn (table 4).
Lower level of estimated broad sense heritability in current study indicated the strong environmental effect and
proportion of phenotypic variance attributable to environmental variance to genotype variance especially for Fe
and beta-carotene (table 4). Heritability estimates are limited to experimental material and setup, and may differ
widely in the same crop and same trait (Garcia- Oliveira et al., 2009). Heritability is a measure of genetic
differences among individuals in a population, not simply of whether or not a trait is inherited (Gomez-Becerra
2
et al., 2010b). Controversy to the results of the present study, moderate to high heritability estimates (h B) for
total as well as for individual carotenoides such as beta-carotene indicated by Blanco et al. (2011) in durum
wheat.
High genetic diversity was fond among cultivated wheat genotypes in two successive years for
micronutrient concentration in grains indicating the remarkable potential for improvement of their grains quality
and introduction of varieties with the higher grains micronutrient concentration. Highly positive significant
correlation between Fe, Zn and protein revealed that concurrently improvement of these nutrients are possible.
Non- significant correlation among yield component traits of wheat and grains micronutrient concentration
exhibited that varieties with high micronutrient concentrations not necessarily tend to produce lower yield. So,
this non-relationship is useful for the biofortification of high yielding wheat varieties.
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Intl J Agri Crop Sci. Vol., 6 (2), 57-62, 2013
Table 1. Major soil properties of the experimental location
Soil texture
Clay- loam
Fe
mg kg-1
Mn
Cd
Zn
mg kg-1
Ni
Ca
pH
Ec
µs/cm
K
N(total)
mg kg-1
0.75
2.5
0
0.33
0
0.64
7.85
249
22.03
0.01
Table 2. Micronutrient concentration in 82 wheat genotypes grains evaluated in two years
Total
n=82
Modern
Bread
wheat
varietie
s n=67
Landrac
es
n=12
Durum
wheat
n=3
min
Max
Mean±
sd
min
First
Fe
mgK-1
36.5
88.00
59.33±11
.24
39.62
year
Zn
mgK-1
38.21
79.22
54.63±7.
83
38.21
0.71
Second
Fe
mgK-1
27.48
62.38
44.74±7.0
1
27.48
year
Zn
mgK-1
29.52
75.80
46.29±10
.37
29.52
Max
88.00
79.22
2.30
62.38
Mean±
sd
min
59.26±
11.18
41.03
54.20±7.
86
41.58
1.35±0.
33
0.75
Max
Mean±
sd
min
Max
78.41
60.31±10
.74
36.5
76.17
69.74
55.54±7.
7
52.69
68.74
Mean±
sd
57.15±15
.71
60.49±5.
53
B-car
µgg-1
0.71
2.64
1.36
Means
Fe mgK-
of two
Zn mgK-1
41.36
67.67
50.58±5.
71
41.36
36.37
73.80
49.63±7.
31
36.37
years
B-car
µgg-1
0.96
1.69
1.25±0.
14
0.96
0.71
Protein
%
7.5
16.7
11.91±1.
7
7.5
75.80
1.98
16.10
67.67
73.80
1.69
44.25±
6.94b
28.57
44.58±9.
44c
29.85
1.14±
0.24
0.71
11.70±1.
62b
10.2
50.25±5.
58
41.61
48.43±6.
73b
42.23
1.25±0.
15
0.97
2.64
1.42±0.
40
0.94
2.02
59.84
47.13±7.5
40.83
54.02
75.6
52.39±
11.44b
43.75
68.29
1.74
1.12±0.
25
0.69
1.77
16.7
13.14±1.
5a
8.2
15.4
62.00
52.40±6.
47
44.36
56.74
68.38
53.65±7.
96a
55.32
62.29
1.40
1.27±0.
13
1.09
1.32
1.15±0.
33
46.04±4.4
7ab
59.51±6.
93a
1.26±0.
37
11.67±2.
28b
50.48±6.
19
59.90±3.
97a
1.21±0.
11
a
B-car
µgg-1
0.69
1.98
1.19
1
Fe1, iron; Zn, zinc; B-car, beta-carotene
a and b are significant differences at 5%
Table 3. Analysis of variance for grains micronutrients and protein concentration in each year separately
Source
Varieties
df
81
First
Mean
Fe
230.956***
year
of
Zn
98.02***
Squares
B-car
0.04*
Second
Mean
Fe
94.06***
year
of
Zn
273.94***
Squares
B-car
0.038**
Protein
5.16***
Blocks
2
114.969*
124.35*
0.003ns
269.62***
56.96ns
0.548***
39.56***
Error
CV
h2B
162
21.64
7.84
90.62%
23.76
8.92
75.76%
0.03
22.28
23.08%
23.93
10.93
74.55%
24.92
10.78
90.90%
0.024
17.07
38.46%
1.29
9.54
75%
Level of significance:
***
p< 0.001, ** p<0.01, * p<0.05 and
CV: coefficient of Variation
2
h B: Broad sense heritability
ns
non-significant
Table 4. Combined analysis of variance for micronutrients Fe, Zn and Beta –carotene concentration of grains
Year
Block(Year)
Varieties
Year×Varieties
Error
CV
h2B
Mean
Fe
20692.02***
217.98
190.44***
161.95***
23.17
9.52
14.96%
df
1
4
81
81
324
Level of significance:
of
Zn
6753.88***
79.43
232.27***
104.51***
24.53
9.98
55%
***
p< 0.001, ** p<0.01, * p<0.05 and
CV: coefficient of Variation
2
h B: Broad sense heritability
ns
Squares
Beta-carotene
2.00***
0.28
0.05***
0.032ns
0.03
19.51
35.93%
non-significant
Table 5. Correlation analysis among grains micronutrient concentration
Zn
Protein
Beta-carotene
Fe
0.499***
0.339***
0.026
Zn
Protein
0.397***
0.152
Level of significance:
-0.084
***
p< 0.001
61
Intl J Agri Crop Sci. Vol., 6 (2), 57-62, 2013
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