Brachiaria access germplasm distinction using SDS PAGE

Brachiaria is a very important grass used as forage in tropical countries. However, improvement of Brachiaria species is slow and time consuming, being made essentially by introduction of the foreign genetic material. To verify the difference among accesses of different species of Brachiaria, five access of six species, were submitted to SDSPAGE. The accesses of Brachiaria presented variations in almost all the species. Brachiaria jubata showed little variation, meaning that received samples are very homogeneous genetically. The other species present enough variations to separate wild accesses.


Introduction
The savannah ecosystem covers approximately 250 million hectares and it is occupied with Brachiaria grass in over 40 million hectares, especially in Colombia, Paraguay, Venezuela and Brazil.The largest territorial area extension used for grazing is in Brazil, and it is generating a great interest for alternative pastures, different from those native that do not fill all the nutritional requirements, originating low yields in animal breeding.
According to Keller-Grein et al. (1996): "The genus Brachiaria, tribe Paniceae, includes 100 species which occur in the tropical and subtropical regions of Africa, Asia and America, but mostly in the eastern and western hemispheres".Seven perennial species of African origin have been used, B arrecta, B.brizantha, B.decumbens, B.dictyoneura, B.humidicola, B.mutica and of B.ruziziensis as forage plants, particularly in tropical America.
The improvement of the Brachiaria species is slow and time consuming, being made essentially by introduction of the foreign genetic material, frequently collected in tropical Africa countries, and distinction between accesses could allow the improvement of germplasm banks as the monitoring the breeding process.
Electrophoresis could be a useful tool for the determination of the molecular weight of the proteins and characterisation of nucleic acids (DNA, RNA).The recognition of proteins and nucleic acid using electrophoresis is necessary and valuable for taxonomy studies in plants (van den Berg et al., 2000), animals, micro-organisms and the viroids (Alfenas, 1998).
In several cultures the electrophoretic pattern can vary according to the type of protein.Storage proteins are the most stable ones, being constant with variations in environmental conditions (Cooke, 1995).Proteins are the most direct expression of the genes, and the main advantage of its use, in comparison to the classic descriptive techniques, it the direct determination of an organism genetic expression, independent of environmental influences, considering that the patterns of the seed proteins are highly stable (Sanches-Yelamo et al., 1992).
The objective of this work was to verify the possibilities of identification of Brachiaria species accesses using SDS-PAGE.

Material and methods
Lots of six species of Brachiaria seed were donated by Embrapa -CNPGC (Empresa Brasileira de Pesquisa Agrepecuária -Centro Nacional de Gado de Corte) (Table 1).
All seed lots were stored in a cold storage room at 15ºC in the Seed Analysis Laboratory -Unoeste -Presidente Prudente, state of São Paulo.
Electrophoresis was carried out in the Plant Tissue Culture Laboratory -Unoeste and image capture was made using the Chemilmager (4000i-v 4.04) at Cytogenetics and Molecular Biology Laboratory -Unoeste.
A dehulled seed sample (200 mg) of each access or species was ground and introduced into a test tube with 2.0ml of extraction buffer composed of 0.625M Tris-HCl pH 6.8; 2% SDS, 5% 2mercaptoethanol and 20% glycerol.The tubes were shaken and kept for 1 h at room temperature and then boiled for 3min, after which the solution was centrifuged at 9500xg for 10min.The pellet was processed again.The resulting supernatants were mixed and frozen in Eppendorf tubes.Protein was quantified according Bradford (1976).
Electrophoresis was carried out according to Laemli (1970) in a system composed of the running gel containing 10% acrylamide-bisacrylamide (30:0.8),pH 8.8, and a stacking gel with 2.5% acrylamide-bisacrylamide, pH 6.8, at 50V for 30 min and 20mA for 4h.Aliquots of 30 µg of protein were loaded per well.The running buffer was composed of Tris (25mM) :Glycine (38mM): SDS (0.7mM), pH 8.8.The gels were fixed with isopropanol:acetic acid:water (4:1:5) for 30min and stained in the same solution containing 2% Coomassie Blue R250 until the protein bands appeared.If the coloration was too dark, the gel was destained in 10% acetic acid.
The evaluation was made observing the presence/absence of bands of the same apparent molecular weight, and a binary matrix constructed.The grouping of each species was made by PC-ORD (2.0 MJM Software Design) using the nearest neighbour group linkage procedure.

Results
Figure 1 shows us that there is difference among accesses of B. ruziziensis.In most of the materials the presence of bands, indicated by the white arrows, allows to differentiate the materials, access R-100 (line 3/3D) presented just a band of ~77.5KDa, and the absence of others, R-106 (line 4/4D) exhibited a pattern of four bands 77.5, 60.2, 47.7 and 40.9KDa, but did not exhibit the 66KDa or the 32.8KDa; access R-108 (line 5-6/5-6D) presented six bands 77.5, 60.2, 58.8, 52.8, 40.9 and 32.6KDa, R-109 (line 7/7D) showed a four banding pattern 77.5, 66, 52.8 and 40.9KDa and R128 access bands of 77.5, 52.8, 40.9 and 34.2KDa.Access R100 exhibited the highest divergence (Figure 7) from the other accesses; and accesses R109 and R128 were closely related.
In the Figure 4 it is shown the differentiation among accesses of B. decumbens.In most of the materials the absence of bands, indicated by the black arrows, allows to differentiate each material from another, the accesses D-7, with band of 100.4,95.4,86.0,70.1,56.1,44.4,34.8,32.5,27.4KDa,95.4,86.0,49.5,46.2,43.8,39.0,35.2,32.5,27.4KDapresented low divergence.The accesses 84.0,58.0,48.2,33.7,95.4,33.7,27.4KDa),as the access 52.6,47.5,33.7,27.4KDa),differ for the presence / absence of bands.Accesses D58 and D59 are not differentiated by group analysis (Figure 7) and access D53 was the most divergent.In Figure 5 it should be noted that there is differentiation among accesses of B. jubata.In most of the materials the absence of bands, indicated by the black arrows, did not allow us to differentiate the materials very well, but the access J-4 is the most different material of all, suggesting high genetic distance between this material and the others, which were very similar, as is shown in Figure 7.
Still, the refinement of the technique, as for example using different fractions of proteins, becomes necessary, permitting more frequent use of those techniques (Cooke, 1995).Accesses separation could allow the best exploitation of genetic variability.As seed banks are relatively expensive structures to be maintained, the knowledge of what is being conserved is necessary, knowing the physiological and agricultural characters of the stored seeds and the molecular patterns will permit a better identification of each access and the storage of a very good amount of genetic diversity as a core collection.
The accesses of Brachiaria presented variations in almost all the species.Brachiaria jubata showed little variation, meaning that received samples are genetically very homogeneous, in this case more expeditions for collecting material would be done to amplify the variation stored.
The other species present enough variations to separate wild accesses.Although of B. brizantha and B. decumbens are presented as apomitic species, there are some sources of variability, as the collection of new materials, or some escape of the apomixy, which would allow small variation rate.Valle and Savidan (1996) reported that although Brachiaria are considered apomitic, some degree of sexuality is present and varies in the species studied as the following proportion B. jubata (0-47%); B. nigropedata (5-11%); B. decumbens (3-56%); B. brizantha (0-74%) and B. humidicola (3-66%), which should explain part of the variability found in these accesses.It could be inferred observing the patterns of Brachiaria ruzizienzis, the most non apomitic species in this work.So, the higher is the apomixy escape probability, the higher is the recombination rate between genes and elevated the changes in alteration of protein pattern profiles.If it is admitted that collected material was submitted to evolutionary forces, several mutations and some recombination could be inferred, so the variation was just selected and maintained in germplasm banks, and those variations could be easily assessed by SDS-PAGE.

Conclusion
The protein variation between accesses of the same species, shown by the SDS-PAGE, could easily

Figure 3 .Figure 4 .
Figure 3. Protein electrophoresis of five accesses of Brachiaria brizantha.Line 1 and 8, Molecular Sigma Markers Wide range (205 up to 6,5 kDa) line 2 access B-23, line 3 access B-67, lines 4 and 5 access B-112, line 6 B-127 and line 7 access B-158.White arrows indicating differentiating bands for the materials, black arrows showing absence of bands

Figure 7 .
Figure 7. Grouping of six accesses of five species of Brachiaria according to their protein patterns