Varietal/Cultivar/Genotype

Identification through Electrophoresis

 Early, distinctness, uniformity and stability (DUS) of any cultivar only
based on morphological methods, which are influenced by
environmental conditions.
 Further, the morphological makers were not quite enough to expose
the genetic diversity between the morphologically similar cultivars.
 The advent of the electrophoresis as an analytical tool provide an
indirect methods to study structural variations in enzymes or other
proteins.
 Electrophoresis is a process of “separation/movement of different
charged biomolecules under the influence of electric field” and has
been successfully applied for the identification of Cultivars.
 Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS-
PAGE) is most economical simple and extensively used biochemical
technique for genetic structure analysis of germplasm.
 The uniformity of seed protein and not much influenced by the
environmental conditions makes it a unique and powerful tool for
genotype identification.
 Intrinsic changes in the plant such as chromosomal rearrangements or
even doubling of the chromosome numbers have no or very small
effect on the seed proteins profile.
 They are also independent of cultivar morphology and physiology,
hence proteins can be regarded as markers for the structural genes.
 The analysis of protein composition can be considered to be an analysis
of gene expression and can be used as an ideal means of varietal
discrimination.
 It is a rapid and relatively cheap method and eliminates the condition
of plants to grow upto maturity.
What is electrophoresis
 The term electrophoresis describes the “migration of a charged
particles under the influence of an electric field”
 Various essential biological molecules, such as amino acids,
peptides, proteins, nucleic acids, nucleotides, have ionizable group,
which at given pH exist in a solution as electrically charged species
either as cation (+ve) and anion (-ve) are separated by
electrophoresis
 Under the influence of electric field these charged particles will
migrates either to cathode or anode depending on the nature of
their net charge
General principle of electrophoresis:
 When a voltage potential difference is applied, the molecules with
different overall charge will begin to separate owing to their different
electrophoretic mobility.
 Even the molecules with similar charge will begins to separate if they
have different molecular sizes, since they will experience different
frictional forces.
 Therefore, some form of electrophoresis rely almost totally on the
different charges on the molecules for separation while some other
form exploits difference in size (molecular size) of molecules.
 Charge of a molecule is influenced by the following:
• The type, concentration and pH of the buffer
• The temperature
• Strength of the electric field
• The nature of the support material (gel matrix) used for
electrophoresis.
Types of support material (gel matrix) used in electrophoresis
Agarose gel:
 Agarose- a linear polysaccharide (M.W. 12000 Da) made up of the basic
repeat unit of agarobiose .
 It is one of the components of agar, that is a mixture of
polysaccharides obtained from seaweeds.
 It is used at a concentration between 1-3%.
Polyacrylamide gel:
 Cross-linked polysaccharide gel are formed from the polymerization of
acrylamide monomer in the presence of small amount of N,N’-
methylene bis acrylamide (aka- bis-acrylamide).
 The acrylamide gel can be made with a content between 3-30%
acrylamide.
 Gels between 10- 20% acrylamide are used in SDS-gel electrophoresis.
Procedure of protein electrophoresis
Preparation of sample:
 Seeds of each variety are crushed in mortar and pestle and treated with
defattening solution (mixture of methanol, chloroform and acetone in
the ratio of 2:1:1) to remove oil.
 Extraction buffer (0.1M phosphate buffer) is added and the samples are
centrifuged.
 The supernatant is used for electrophoresis.
Protein profiling through SDS PAGE:
 The extracted seed proteins are separated using SDS PAGE.
 Protein samples are treated with SDS (Sodium dodecyl sulphate) for 2-3
minutes to ensure complete interaction.
 The samples are loaded in the gel-well with a micro syringe/pipette.
 Electrophoresis is done under room temperature at constant and
specified voltage supply for a particular time period in a buffer solution.
Procedure of protein electrophoresis, contd……….

Fixing and staining

 At the end of the electrophoresis the gels are removed and immersed
in staining solution (0.1g Comassie Brilliant Blue R 250, 40% methanol,
10% glacial acetic acid, 50% distilled water) for overnight.
 After proper staining the gel is transferred to destainer (40%
methanol, 10% glacial acetic acid, 50% distilled water) with gentle
shaking.
 The destainer is changed frequently till appropriate visibility of the
bands on gel and it is photographed.
Procedure of protein electrophoresis, contd……….
Evaluation and documentation
 The distance moved by the tracking dye from the point of loading is
measured on the gel.
 Then the distance traveled by each band is also measured.
 The complete gel imprints were made on the transparency sheets to
determine their intensity.
 Based on the relative front value and intensity of band, electrophoregram
are prepared for each gel.
 Relative front (Rf) of each band is calculated as follows:
Distance travelled by the band
Rf = ----------------------------------------------
Distance travelled by the tracking dye
 Bands are numbered in the order of increasing Rf values.
 Apart from these, recording presence or absence of a band and the
intensity of the band in each cultivar is also critically observed for
discriminating the cultivars.
Total seed protein profile of 11 oats cultivars
through SDS PAGE
Electrophoregram of total seed protein profile
Merits of eletrophoresis method:
1) The universal distribution of the proteins so that there is no
theoretical limits to using the electrophoretic markers.
2) The electrophoretic markers are less effective to the
environmental fluctuations.
3) They are too proximity to the primary genetic information (third
hand copy of DNA).
4) The discriminatory power of electrophoresis is usually very high
and distinction between genotype can often be achieved with
less effort and with the analysis of fewer individuals than the
morphologically based system.
5) The operating costs are relatively low.
The main demerits of eletrophoresis method are:
1) It requires international system for band nomenclature,
especially for the seed proteins of the plants which are not
fully characterized.
2) Electrophoretic markers are profoundly influenced by tissue
specificity.
3) Electrophoretic techniques require technically complement
and trained laboratory staff, particularly for gel evaluation.
Usage of the electrophoresis of the seed storage proteins to:
1) identify between cultivars
2) to check species identification
3) to assist biosystematic analysis
4) to study phylogenetic relationships of the species
5) to generate pertinent information to complement
evaluation and passport data.
So, that to increase the knowledge of the genetic
diversity of the plant materials in the germplasm collections.