ABSTRACT The present study was undertaken to evaluate the genetic diversity in gluten subunits and protein content in seed endosperm of wheat for nutritional grading of 17 advanced varietal lines of wheat using SDS-PAGE, which is useful for agricultural development regarding quality food stuffs and breeding purposes. After calculating the standard factor in spectrophotometer for unit absorbance at 595 nm and the amount of protein were calculated. The wheat lines PZ-TS-5, PZ-TS-4 and PZ-TS-9 contained maximum amount of HMW-Gs proteins as compared to rest of the lines. By comparing with the six bands of the standard marker, band numbers 4 were common in all 17 wheat lines, but the other bands showed variation. Most frequent HMW protein bands had mole. Wt. 29.03 KDa marker band, while, in LMW protein bands most frequent bands were among the 14 KDa marker band. In cluster analysis, the above mentioned 3 wheat lines grouped in the same cluster showed that they were originated from the same parental line and genetically less diverged

* Patil

A S Gilande

MANE A N

R S K K Wagh

SHIVANGIKAR H G

V A And Choudhary

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AGRES – An International e-Journal , (2013)Vol. 2, Issue 3: 352-357                            ISSN 2277-9663 
_____________________________________________________________________________________ 
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MOLECULAR CHARACTERIZATION OF DIFFERENT ADVANCED WHEAT 
VARIETAL LINES USING SDS-PAGE ELECTROPHORESIS DATA OF SEED STORAGE 
PROTEINS 
 
*PATIL, A. S., GILANDE, H. G., SHIVANGIKAR, A. N., MANE, V. A. AND  
CHOUDHARY, R. S. 
 
K. K. WAGH COLLEGE OF AGRICULTURAL AND BIOTECHNOLOGY 
SARASWATINAGAR, NASHIK-422 003, MAHARASHTRA, INDIA 
 
*Email: abhinandan_patil25@rediffmail.com 
 
 
ABSTRACT 
 
The present study was undertaken to evaluate the genetic diversity in gluten subunits and 
protein content in seed endosperm of wheat for nutritional grading of 17 advanced varietal lines of 
wheat using SDS-PAGE, which is useful for agricultural development regarding quality food 
stuffs and breeding purposes. After calculating the standard factor in spectrophotometer for unit 
absorbance at 595 nm and the amount of protein were calculated. The wheat lines PZ-TS-5, PZ-
TS-4 and PZ-TS-9 contained maximum amount of HMW-Gs proteins as compared to rest of the 
lines. By comparing with the six bands of the standard marker, band numbers 4 were common in 
all 17
 
wheat lines, but the other bands showed variation. Most frequent HMW protein bands had 
mole. Wt. 29.03 KDa marker band, while, in LMW protein bands most frequent bands were among 
the 14 KDa marker band. In cluster analysis, the above mentioned 3 wheat lines grouped in the 
same cluster showed that they were originated from the same parental line and genetically less 
diverged.  
 
KEY WORDS: Cluster analysis, genetic diversity, protein content, SDS-PAGE, wheat 
 
INTRODUCTION 
Wheat is counted among the ‘big three’ 
cereal crops, with over 600 million tonnes 
being harvested worldwide. For example, in 
2011, the total wheat harvest was about 85.93 
million tonnes in India, from the production 
areas 29.25 million hectare. In Maharashtra, 
the yield of wheat was 1730 kg/ha 
(http://www.indiastat.com/table/agriculture/2/
wheat/17195/7400/data.aspx). There is no 
doubt that the adaptability and high yields of 
wheat have contributed to its success, but these 
alone are not sufficient to account for its 
current dominance over much of the temperate 
world. The key characteristic which has given 
it an advantage over other temperate crops is 
the unique properties of doughs formed from 
wheat flours, which allow it to be processed 
into a range of breads and other baked 
products (including cakes and biscuits), pasta 
and noodles, and other processed foods. These 
properties depend on the structures and 
interactions of the grain storage proteins, 
which together form the ‘gluten’ protein 
fraction (Shewry, 2009). It is, therefore, very 
essential to study protein profiling of wheat 
varietal lines. 
Variation is the key for any change, so 
the key for genetic change is genetic variation. 
The high chance of success in a breeding 
AGRES – An International e-Journal , (2013)Vol. 2, Issue 3: 352-357                            ISSN 2277-9663 
_____________________________________________________________________________________ 
www.arkgroup.co.in Page 353 
program follows the availability of a wide 
variety of appropriate materials for the breeder 
to choose from. Selecting appropriate parents 
for breeding purpose requires a good 
understanding on genetic variation and on 
classification of germplasms. 
Wheat is important due to gluten 
features. The sticky part is the endosperm 
strict proteins and it causes the stretching or 
expansion of fermented dough, the term is 
peeling. When peeling is heated to bread 
production, this part of protein has been linked 
and it holds together. Only wheat and lesser 
extent of rye and triticale hold the property 
(Arzani, 2001). 
Glutenin subunits with high molecular 
weight can be used to identify cultivars as a 
supplementary indicator (Maragheh, 2013). 
Subunits can also are applied as an index for 
breeding projects to select parents and results 
with stronger gluten properties (Payne et al., 
1982). In recent years, Polyacrylamide gel 
electrophoresis (PAGE) grains storage proteins 
has been valid method for assessing the 
chemical diversity of the wheat. This method 
may be used in order to collect germplasm 
where have considerable genetic diversity or in 
areas where there is expected a large 
variability (Dominici and Grottanelli, 1984). 
In the quality modification programs, choose 
the lines is permissible based on glutenin 
components with high molecular weight that 
are controlled by different locus. In the present 
investigation 17 wheat lines were evaluated to 
study the genetic diversity based on seed 
storage proteins through constructing the 
dendrogram for high molecular weight 
(HMW) and low molecular weight (LMW) of 
gluten subunit bands.  
MATERIALS AND METHODS 
Seeds of 17 wheat lines obtained from 
the Department of Agriculture Botany, K. K. 
Wagh, College of Agriculture, Nashik, were 
used for molecular characterization. The well 
dried healthy seed was grinded to powdered 
form. Bradford (1976) assay was made to 
determine the wheat endosperm proteins. 
Proteins were extracted by adding 400 μl 
protein extraction buffer (0.05 M Tris-HCL, 
pH 8.0, 2% SDS, 5 M Urea, 1% β- 
Mercaptoethanol, 100 µl Methylene green 
dye) in about 10 mg powdered endosperm in 
1.5 ml eppendorf, keep overnight at room 
temperature and spun at 13000 rpm for 15 min 
at room temperature and supernatant was taken 
into new Eppendorf to store at -20°C. Profiling 
of proteins was made by one dimensional 
sodium dodecyl sulfate polyacrylamide gel 
electrophoresis (SDS-PAGE) (30% separating 
gel & 8.9% stacking) following Dual in a mini 
gel apparatus (Damania et al., 1983). The gels 
containing protein samples with protein 
molecular weight markers allowed to run on 
100 V until green line of Methylene Green dye 
reach to the bottom of the gel plates. The 
separating gel was kept overnight in staning 
solution and then destain until bands 
sharpened. Finally the gel was analyzed in gel 
documentation system (Bio Rad Instruments, 
Italy). In the data matrix, the presence of a 
band was scored as 1, whereas the absence of 
the band was coded as 0 in the 
electrophorogram. The basic data structure 
finally consisted of a binary (0/1) data was 
analyzed and genetic diversity was measured. 
Hierarchical cluster analysis was made by the 
complete linkage method with Euclidean 
distance proteins in different wheat lines. 
Cluster analysis of wheat grain storage 
proteins was performed on the results of SDS-
PAGE (Sneath and Sokal, 1973) using the 
software MVSP (UPGMA) to find out the 
diversity among these wheat lines. 
RESULTS AND DISCUSSION 
Wheat has gained tremendous attention 
being the major food crop. A wheat 
improvement activity through molecular 
breeding is very essential for developing and 
releasing superior quality wheat varieties. Also 
the study of genetic behaviour on the basis of 
protein profiles of the parents for high yielding 
wheat lines plays very important role in 
AGRES – An International e-Journal , (2013)Vol. 2, Issue 3: 352-357                            ISSN 2277-9663 
_____________________________________________________________________________________ 
www.arkgroup.co.in Page 354 
selection. It is therefore concluded that seed 
storage protein profiles could be useful marker 
in cultivar identification, registration of new 
varieties, cluster analysis and in studies of 
genetic diversity, thereby improving the 
efficiency of wheat breeding programs in 
cultivar development.  
The amount of proteins was estimated 
in 10 mg of wheat seed sample using 
spectrophotometer, after calculating the 
standard factor for unit absorbance at 595 nm. 
Results revealed a wide variation in seed 
storage proteins in 17 wheat lines (Table 1). 
Protein content of these wheat varieties ranged 
from 32ug/ml to 103ug/ml. The minimum seed 
protein content was found in the variety PZ-
TS-3(32 µg/ml), while the maximum protein 
content was observed in wheat varietyPZ-TS-5 
(103µg/ml). The high protein content was also 
observed  in the variety PZ-TS-14 (93µg/ml) 
and PZ-TS-6 (84µg/ml).  
In this study, SDS-PAGE of grain 
storage proteins was performed in order to 
analyze molecular weight of gluten subunits 
and investigate genetic diversity among 
different wheat lines. Molecular weight 
analysis of proteins in the seeds of wheat 
varieties are given in Table 2 and the 
electrophorogram showing protein banding 
pattern of different wheat varieties are given in 
Figure 1. The seed storage protein patterns of 
each sample were determined by examining 
the molecular weight of the bands and 
intensity of staning. The relative proportion of 
each band was calculated on the basis of 6 
bands of standard marker (in the range of 14.3-
97.4 KDa). By comparing with the six bands 
of the standard marker, band numbers 4 were 
common in all 17
 
wheat lines, but the other 
bands showed variation. Most frequent HMW 
protein bands had molecular weight of 29.03 
KDa marker band, while, in LMW most 
frequent protein bands were among the 14 
KDa marker band. These banding patterns 
were confirmed with results of Zeb et al. 
(2006), Khan et al. (2007), Shuaib et al. 
(2007), and Maragheh (2013). 
Dendrogram obtained from cluster 
analysis following UPGMA method in 17 
different wheat promising lines based on 
electrophoresis' banding patterns is given in 
Figure 2. Genetic diversity of European spelts 
wheat was evaluated by constructing the 
dendrogram for HMW and LMW glutenin 
subunit bands (Xueli et al., 2005). The 
dendrogram as a whole revealed low genetic 
diversity at proteins level . The diagram 
revealed two main groups; the group 1 has 
eight lines and group 2 has nine wheat lines. 
At Eucladian distance of 1.75, all the varieties 
showed similarity, but at Eucladian distance of 
1.5, all the wheat lines split into two major 
groups that were further divided into 
subgroups and mixed trend was seen in 
grouping of lines. All these lines posses same 
protein due to which they group into same 
cluster, but only differ in their protein 
concentration (µg/ml) and showing lower 
dissimilarity among them. Similarly, the three 
lines which possessed high protein content 
(PZ-TS-5, PZ-TS-14 and PZ-TS-6) were 
grouped in the same cluster showed that they 
were originated from the same parental line 
and genetically less diverged. Fufa et al. 
(2005) reported that the genetic diversity 
estimates based on seed storage protein were 
lowest because they were the major 
determinants of end-use quality, which is a 
highly selected trait. 
CONCLUSION 
From the results, it can be concluded 
that seed storage protein profiles could be 
useful markers in the studies of genetic 
diversity, and thereby improving the efficiency 
of wheat breeding programs in cultivar 
development.   
REFERENCES 
Arzani, A. 2005. Breeding field crops. Isfahan 
University of Technology. 630. Pp. 
(Translated in Persian).  
AGRES – An International e-Journal , (2013)Vol. 2, Issue 3: 352-357                            ISSN 2277-9663 
_____________________________________________________________________________________ 
www.arkgroup.co.in Page 355 
Bradford, M. M. (1976) A rapid and sensitive 
method for the quantitation of 
microgram quantities of protein 
utilizing the principle of protein-dye 
binding. Anal. Biochem., 72: 248-
254. 
Damania, A. B., Porceddu, E. and Jackson, M. 
T. (1983). A rapid method for the 
evaluation of variation in germplasm 
collections of cereals using 
polyacrylamide gel electrophoresis. 
Euphytica, 32: 877-883. 
Dominici, L. A., and Grottanelli, C. (1984). 
Electrophoretic Variability in 
landraces of durum wheat from 
Ethiopia. J. Rachis. 7: 34-35. 
Fufa, H., Baenziger, P. S., Beecher, I., 
Dweikat, V, Graybosch, R. A. and 
Eskridge, K. M. (2005). Comparison 
of phenotypic and molecular 
marker-based classifications of hard 
red winter wheat cultivars. 
Euphytica, 145: 133-146. 
http://www.indiastat.com/table/agriculture/2/w
heat/17195/7400/data.aspx. 
Khan, I., Shuaib, M., Ali, Z. and Zeb, A. 
(2007). Molecular and 
physiochemical Evaluation of 
wheat. J. Chem. Soc. Pak., 29: 301-
306. 
Maragheh, F. P. (2013). Compare the grouping 
of winter wheat lines using SDS-
PAGE electrophoresis data of seed 
storage proteins and quantitative 
traits data. J. Appl. Environ. Biol. 
Sci., 3(8): 81-87. 
Payne. P. I., Holt, L. M., Worland, A. J. and 
Law, C. N. (1982). Structural and 
genetic studies on the high-
molecular weight subunits of wheat 
glutenin. Theor. Appl. Genet., 63: 
129-138. 
Shewry P. R. (2009). Darwin review, Wheat, 
J. Exp. Bot. 60, (6), 1537–1553.  
Shuaib, M., Zeb, A., Ali, Z., Ali, W., Ahmed, 
T. and Khan, I. (2007) 
Characterization of wheat varieties 
by seed storage protein 
electrophoresis. African J. Biotech. 
6(5): 497-500. 
Sneath, P. H. A. and Sokal, R. R. (1973). 
Numerical taxonomy: The principle 
and practice of numerical 
classification. W.F. Freeman & Co. 
San Francisco. p. 573. 
Xueli, A., Qiaoyun, L., Yueming, Y., Yinghua, 
X., Hsam, S. L. K. and Zeller, F. J. 
(2005). Genetic diversity of 
European spelt wheat (Triticum 
aestivum ssp. spelta L. em. Thell.) 
Revealed by glutenin subunit 
variations at the Glu-1 and Glu-3 
loci. Euphytica, 146(3): 193-201.  
Zeb, A., Zahir, A., Ahmad, T. and 
Abdumanon, A. (2006). 
Physiochemical characteristics of 
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Pakistan. Pak. J. Bio Sci. 9(9): 1823-
1828. 
 
 
 
 
 
AGRES – An International e-Journal , (2013)Vol. 2, Issue 3: 352-357                            ISSN 2277-9663 
_____________________________________________________________________________________ 
www.arkgroup.co.in Page 356 
                        Table 1: Protein Estimation by spectrophotometer 
 
Sr. No. lines Absorb At 595 nm Protein (µg/ml) 
1  PZ-TS-1 0.24 56 
2  PZ-TS-2 0.34 80 
3  PZ-TS-3 0.14 32 
4  PZ-TS-4 0.18 42 
5  PZ-TS-5 0.44 103 
6  PZ-TS-6 0.36 84 
7  PZ-TS-7 0.27 63 
8  PZ-TS-8 0.28 65 
9  PZ-TS-9 0.30 70 
10    PZ-TS-10 0.22 52 
11    PZ-TS-11 0.28 65 
12    PZ-TS-12 0.32 75 
13    PZ-TS-14 0.40 93 
14    PZ-TS-15 0.26 60 
15    PZ-TS-16 0.20 47 
16    PZ-TS-17 0.35 82 
17    PZ-TS-18 0.19 44 
 
Table 2: Molecular weight analysis of proteins in the seeds of wheat varieties 
Sr. No. Molecular 
weight 
(KDa) 
Wheat lines 
  
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 
Band1 97.4 1 1 0 1 1 1 1 1 0 1 1 1 0 0 1 1 0 
Band2 66 0 1 0 1 1 1 1 1 1 1 1 0 1 1 0 0 0 
Band3 43 1 1 0 0 0 1 0 0 1 1 1 0 0 0 0 0 0 
Band4 29 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 
Band5 20.1 1 0 1 0 1 1 1 1 0 1 1 0 0 0 0 0 0 
Band6 14.3 1 1 1 1 0 0 1 0 0 0 0 1 1 0 0 0 1 
 
Where,  
1=PZ-TS-1, 2=PZ-TS-2, 3=PZ-TS-3, 4=PZ-TS-4, 5=PZ-TS-5, 6=PZ-TS-6, 7=PZ-TS-7,   8=PZ-TS-8, 9=PZ-TS-9, 
10=PZ-TS-10, 11=PZ-TS-11, 12=PZ-TS-12, 13=PZ-TS-14,    14=PZ-TS-15, 15=PZ-TS-16, 16=PZ-TS-17,  
17=PZ-TS-18. 
 
AGRES – An International e-Journal , (2013)Vol. 2, Issue 3: 352-357                            ISSN 2277-9663 
_____________________________________________________________________________________ 
www.arkgroup.co.in Page 357 
 
 
                               
Fig. 1: Electrophorogram showing banding pattern of wheat proteins and molecular  
                        Weight markers of 17 wheat lines 
 
UPGMA
Euclidean
PZ-TS-1
PZ-TS-2
PZ-TS-4
PZ-TS-7
PZ-TS-5
PZ-TS-8
PZ-TS-6
PZ-TS-10
PZ-TS-11
PZ-TS-3
PZ-TS-18
PZ-TS-14
PZ-TS-12
PZ-TS-16
PZ-TS-17
PZ-TS-9
PZ-TS-15
1.8 1.5 1.2 0.9 0.6 0.3 0
 
           Fig. 2: Dendrogram obtained from cluster analysis following  UPGMA   method  in  
       17 different wheat promising lines based on electrophoresis' banding pattern 
 
[MS received: July 11, 2013 ]                                                                            [MS accepted: August  28,2013]
 
Mol. Wt. 
(KDa) 
97.4 
66 
43 
29 
      20.1 
 
 14.3  
 M    1    2    3    4   5    6                          M  7     8    9   10 11               M   12 13 14 15 16 17 
18 12 


MOLECULAR CHARACTERIZATION OF DIFFERENT ADVANCED WHEAT VARIETAL LINES USING SDS-PAGE ELECTROPHORESIS DATA OF SEED STORAGE PROTEINS

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MOLECULAR CHARACTERIZATION OF DIFFERENT ADVANCED WHEAT VARIETAL LINES USING SDS-PAGE ELECTROPHORESIS DATA OF SEED STORAGE PROTEINS

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