International Journal of Horticulture, 2017, Vol.7, No. 5, 33-39
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plates were fixed with in a casting tray and marked 2 cm from the top. To make sure that there was no leakage;
glass plate set ups was filled with water and placed for some time.
2.1 Electrophoresis
Electrophoresis procedure was carried out using slab type SDS-PAGE with 12.25% polyacrylamide gel. Electrode
buffer solution was put into the bottom pool of the apparatus. Gel plates were placed in the apparatus, here again
air bubble formation was avoided. Electrode buffer solution was also put into the top pool of the apparatus; was
formed by combs were washed with distilled water using a syringe. 6.5 µl of supernatant of seed samples
(Vortexed and centrifuged at 12,000 rpm for 10 minutes), was put into wells with the help of micropipette. Protein
molecular weight marker was put in first well of each glass plate. The numbering of seed samples and wells was
noted to avoid repetition. The apparatus was connected with + (red) and – (black) electrodes of power supply. The
voltage of apparatus was kept constant at 50 V in stacking gel and 80 V at separation gel; apparatus was left until
a blue line of BPB will reach at the bottom (2 mm above bottom) of the gel plates.
2.2 Detection of seed protein
Staining and De-staining of Separation gel
When blue line will reach at the bottom of the gel plates, electric supply was disconnected. Gel plates was taken
out from the apparatus and separated by spatula. Stacking gel was removed with the help of same spatula.
Separation gel was put in the tray which contained staining solution and was put on the shaker for two hours.
Staining solution was exchanged by distaining solution and the tray was shacked gently overnight until the
background of the gel disappeared to absorb excess CBB.
2.3 Drying of separation Gel
Wet filter paper was placed on the plate of gel dryer. Separation gel was carefully placed on the paper and was
covered with a wrap. It was dried in a drier for about 1.5 hours at 60°C. When gel sheet was completely dried it
was taken out. All gels were dried with the same manner. The gels will then be analyzed and photographed.
3 Results and Discussion
Results and discussion comprises of nutritional composition of wheat genotypes, estimation of genetic diversity of
wheat genotypes by using SDS-PAGE of 30 genotypes of wheat grain minerals and nutrients. Each of them is
separately discussed below:
Sodium Dodecylsulphate Poly acrylamide Gel Electrophoresis
The data presented in the Table 2 revealed from SDS-PAGE of 30 wheat genotypes (Figure 1; Figure 2; Figure 3)
showed that the total number of banding fragments were 33, which were differently distributed in all the
genotypes under studied. Advance wheat line 8973 generated 17 scorable bands, Tatara and Mehran-89
produced
16, 03FJ-26, Pirsabak-91 and Zarlashta have 15 bands, 9870, Bakhtawar-93, Noshera-96 and WC-24 produced 14
bands, Shafaq-2006, Shahkar-95, Sarsabz and Kohistan-97 contains 13 bands, Shafaq-2006, Shahkar-95, Sarsabz,
BARS-2009, AS-2002, 98FJ13, Lasani-2008 and WC 13, generated 12 bands, GA-2002, NARC-2009 and
GA-2002
generated 11 scorable bands, genotypes WC-25, WC 26 Pasban-90 contained 10 scorable bands,
genotypes Sahar-2006, WC-4 and WC-11 hade 9 bands, genotypes WC 20 and Inqilab-91 contained 8 and 7
bands respectively.
4 Cluster Analysis
Data revealed from SDS-PAGE of seed storage protein were subjected to the cluster analysis, which is an
appropriated method for the assessment of similar and divergent genotypes by data matrix. Figure 4 showed that
genotypes were grouped in to three clusters on the basis of protein banding pattern.
