Part I

 Introduction

The following Annex contains a list of characteristics derived by using electrophoresis and a description of the method to be used. Authorities decided to place these characteristics in an Annex to the Test Guidelines, thereby creating a special category of characteristic because authorities are of the view that it is not possible to establish distinctness solely on the basis of a difference found in a characteristic derived by using electrophoresis. Such characteristics should therefore only be used as a complement to other differences in morphological or physiological characteristics. Authority reconfirms that these characteristics are considered useful but that they might not be sufficient on their own to establish distinctness. They should not be used as a routine characteristic but at the request or with the agreement of the applicant of the candidate variety.  

For the analysis of high molecular weight [HMW] glutenins, polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate (SDS PAGE) should be used. Glutenins are encoded by three compound loci, known as Glu-A1, Glu-B1 and Glu-D1 on the long arms of the group 1 chromosomes (Payne, 1987). There are a number of alleles at each locus and the analysis of HMW glutenins is based on the recognition of these alleles from proteins, which appear on gels as a series of well defined bands or patterns of bands. The alleles are described by band numbers according to the definition given to them by Payne and Lawrence, 1983 (see Chapter IX, Literature). The corresponding letters and apparent molecular weights are reproduced in the description of the method used.

Part II

 Characteristics Derived by Using Electrophoresis

Part III

 Description of the Method to be used

Glutenin composition: allele expression at loci Glu-A1(37), Glu-B1(38)  and Glu-D1 (39) SDS PAGE Method for Analysis of HMW Glutenins from T. aestivum

1.  Apparatus and equipment

Any suitable vertical electrophoresis system can be used, provided that the gels can be kept at a constant temperature.  A gel thickness of no more than 1.5 mm is recommended.  The power supply used should be capable of delivering both constant current and constant voltage output.

2.   Chemicals

All chemicals should be of ‘Analytical Reagent’ grade or better.

Acrylamide (specially purified for electrophoresis)

Bisacrylamide (specially purified for electrophoresis)

Tris (hydroxymethyl) methylamine (TRIS)

Sodium dodecyl sulphate (SDS)

Ammonium persulphate (APS)

2-mercaptoethanol

TEMED (NNN’N’ –tetramethlethyl enediamine)

Trichloroacetic acid (TCA)

Hydrochloric acid

Glacial acetic acid

Glycine

n-Butanol

Pyronin Y (or G)

Glycerol (d = 1.256)

Methanol

Coomassie Brilliant Blue R-250 (or equivalent)

Coomassie Brilliant Blue G-250 (or equivalent)

3.  Solutions

3.1             Extraction solution

3.1.1       Extraction of glutenins only

Stock solution:  6.25 ml 1M TRIS HCI buffer, PH 6.8 (see 3.3.2)

12.05 ml distilled water

2g SDS

10 mg Pyronin Y (or G)

10 ml glycerol

This solution can be stored for two months at 4 ⁰C.

Immediately before use, extraction solution is prepared as follows:

4.25 ml stock solution (above) plus 0.75 ml 2-mercaptoethanol made up to 10.0 ml with distilled water.  This solution must be prepared immediately prior to use and cannot be stored.

3.1.2  Extraction of glutenins following gliadins

Solution A – 25 ml 2 – chloroethanol + 50 mg Pyronin Y/G, made up to 100 ml with distilled water.  Solution B – 27.0 g urea, 3.0 ml 2 – mercaptoethanol + 10.0 g SDS, made up to 100 ml with distilled water.

 3.2  Electrophoresis (runing) Buffer

Stock solution:

141.1 g glycine

30.0 g  TRIS

10.0 g  SDS

made up to 11 with distilled water.

Immediately before use, the stock solution is diluted 1:10 with distilled water.

The stock buffer solution can be stored for 2 months at room temperature.  Do not store the diluted buffer more than one week.  The pH of the buffer must be close to 8.3

3.3             Gel preparation solutions

3.3.1       Stock  resolving gel buffer (1M TRIS HCl Ph 8.8)

121.14 g TRIS plus approximately 20 ml HCl (d = 1.19) made up to 11 with distilled water.  This buffer can be stored at 4⁰C for 2 months.

3.3.2       Stock stacking gel buffer (IM TRIS HCI, ph 6.8)

121.14 g TRIS plus approximately 78 ml HCl (d = 1.19) made up to 11 with distilled water.  This buffer can be stored at 4 ⁰C for 2 months.

3.3.3       10% (w/v) SDS solution

10g of SDS dissolved in distilled water and made up to 100 ml.  This solution can be stored at  4 ⁰C  for 2  months.  Prior to use, stir and heat gently to re-dissolve the SDS, if it comes out of solution.

3.3.4       1% (w/v) ammonium persulphate solution)
 
1g of APS dissolved in distilled water and made up to 10 ml.  This solution must be preapared immediately prior to use.

3.3.5       Stock acrylamide solution
  
40.02g acrylamide made up to 100 ml with distilled water.

3.3.6       Stock bisacrylamide solution
  
0.5198g bisacrylamide made up to 130 ml with distilled water.  

3.4             Staining solutions

3.4.1       0.25g Coomassie Brilliant Blue G-250 plus 0.75g Coomassie Brilliant Blue R-250, made up to 100 ml with water.  

3.4.2       55g TCA, 65 ml glacial acetic acid, 180 ml methanol plus 25 ml solution 3.4.1,  made up to 11 with distilled water.

4.   Procedure

4.1      Protein extraction

4.1.1       Glutenins only
  
Individual seeds are ground using a hammer (or other device).  Ground seed meal is mixed with diluted sample extraction buffer (3.1.1) in a 3 ml polypropylene hemolyse or similar tube with a screw-on or fitted cap.  The ratio of meal/extraction buffer is 50 mg/0.75 ml.  The samples are extracted for 2 hours at room temperature, mixed several times using a vortex mixer, heated in a boiling water bath for 10 minutes and than allowed to cool.  The tubes are centrifuged at 18000g for 5 minutes.

4.1.2       Glutenins following gliadins

If desired, glutenins and gliadins can be analysed from the same grain.  Gliadins are extracted first by adding 0.25 ml of Solution A (3.1.2) to a crushed grain (or half-grain) in a microtiter plate or micro-centrifuge tube and incubating overnight at room temperature.  Following this, glutenins are extracted by adding 0.5 ml of Solution B (3.1.2) to the crushed grain and incubating overnight at room temperature.  

According to the gel thinckness and the size of the wells, the volume of extract loaded can vary.  Between 10 and 25 ul is usually sufficient.

4.2             Preparation of the gel

Clean and dry gel cassettes are assembled, according to the design of the equipment used. If tape is used to seal the cassettes, it is advisable to assemble them at least one day in advance of use, to enable the tape to 'age' and adhere better.
  
4.2.1       Resolving (main) gel (10% acrylamide, Ph 8.8)

To make two slab gels of 180 x 160 x 1.5 mm, the following is required:

20ml stock acrylamide soluation (3.3.5)  

26 ml stock bisacrylamide soluation (3.3.6),  

30. ml stock gel buffer (3.3.1).  

These should be at 4⁰ C. The mixture is de-gassed in a 100 ml Buchner flask for 10 minutes. To this is added.  

2 ml APS [3.3.4],  

0.8 ml SDS [3.3.3],  

40 ul TEMED [use straight from bottle]  

The gels are then carefully poured, avoiding the formation of air bubbles, and polymerisation allowed to take place at room temperature.  

The gel cassettes should not be filled entirely, in order to leave room for a 3-4 cm layer of stacking gel. The gel surface is carefully overlayed with n-butanol [or distilled water] using a syringe. When polymerisation is finished [about 30 min.], the gel surface is carefully rinsed with distilled water and dried with filter paper.
  
4.2.2       Resolving [main] gel [7% acrylamide, pH 8.8]

To resolve the sub-units 2 and 2*, it is necessary to use main gels of 7% acrylamide concentration.  

To make two slab gels of 180 x 160 x 1.5 mm, the following is required:  

14 ml stock acrylamide solution [3.3.5]  

6 ml distilled water  

26 ml stock bisacrylamide solution [3.3.6],  

30 ml stock gel buffer [3.3.1].  

These should be at 4⁰C. The mixture is de-gassed in a 100 ml Buchner flask for 10 minutes. To this is added:  

2 ml APS [3.3.4],  

0.8 ml SDS [3.3.3],  

40 ul TEMED [use straight from bottle].  

The gels are then carefully poured, avoiding the formation of air bubbles, and polymerisation allowed to take place at room temperature.  

The gels cassettes should not be filled entirely, in order to leave room for a 3-4 cm layer of stacking gel. The gel surface is carefully overlayed with n/butanol [or distilled water] using a syringe. When polymerisation is finished [about 30 min.], the gel surface is carefully rinsed with distilled water and dried with filter paper.
  
4.2.3       Stacking gel [3% acrylamide, pH 6.8]  

In a 50 ml Buchner flask, mix:  

1.50 ml stock acrylamide solution [3.3.5],  

2.15 ml stock bisacrylamide solution [3.3.6]  

2.50 ml stoick gel buffer [3.3.2] and  

13.15 ml distilled water.  

Following de-gassing add :  

0.75 ml APS [3.3.4],  

0.2 ml SDS [3.3.3],  

15 ul TEMED [straight from bottle]  

Mix carefully and immediately pour the stacking gels to the top of the gel cassettes. Insert the well-forming "comb" avoiding air bubbles. Allow to polymerise for about 2 hours at room temperature. The "combs" are then removed carefully from the gel cassettes and the wells rinsed using diluted electrophoresis running buffer [3.2].
  
4.3    Electrophoresis
   
The tank is filled with the appropriate volume of running buffer [3.2], cooled to 15⁰C. Following sample loading, electrophoresis is carried out at a constant current of 8 mA/sq cm [cross-sectional area] of gel until the pyronin Y/G has moved through the stacking gel and then at 16 mA/sq cm of gel [maximum voltage 300V] until the marker is at the bottom of the gel. Then temperature should be maintained at 15⁰

4.4   Fixing and staining  

The gel cassettes are removed from the tank, opened and the gels fixed in 250 ml of 15% [w/v] TCA for at least 30 minutes. The gels are rinsed in distilled water and stained overnight in 250 ml of staining solution [3.4.2] at room temperature. Destaining is not usually necessary but gels should be washed in distilled water before being stored in sealed polythene bags. 

Other staining procedures can be successfully used (eg. Coomassie Brilliant Blue G or equivalent in TCA alone).  The final quality control criterion, both for gel preparation and gel staining, is to analyse the suggested example varieties on each batch of gels.  The separation of the suggested bands, and their relative electrophoretic mobilities (molecular weights) must be clear in order for the procedures to be judged satisfactory.

Recognition of Glutenin Alleles

This Table is designed to illustrate the alleles described above and to assist in the recognition of the different bands.  It depicts the position and molecular weight of all of the glutenin bands from each locus, compared to those found in the Example Variety Courtot, along with the band numbers using the nomenclature of payne; the letter given to each allele following Payne and Lawrence (1983) is also given.  

Sub-Units of HMW Glutenins: nomenclature of the individual bands and recognition of the corresponding alleles

Characteristic37: Glu-A1 locus

  
Characteristic 38: Glu-B1 locus

Characteristic39: Glu-D1 locus

Note:  Certain bands (e.g. bands 9 and 10) have similar molecular weights.  This leads to the fact that in the presence of bands 5 + 10 of characteristic 29 two states of expression of characteristic 38, band 7 and bands 7 + 9, cannot be differentiated from one another. Therefore, in the presence of bands 5 + 10 of characteristic 39, note 4 of characteristics 38 could be either one another by their known association with other bands. For characteristic 38, band 13 is always associated with band 16 and band 14 with band 15 while band 40 remains alone.