Abstract: Genetic variation in germplasm has an important role in identification of varieties. Electrophoretic patterns of the protein fractions are directly related to the genetic background of the proteins and can be used to certify the genetic makeup. SDS-PAGE is a valid technique increasingly being utilized as an approach for species identification. Each variety or a group of varieties exhibit characteristic protein banding patterns. On the basis of these patterns they can be identified accordingly. Twenty-nine accessions of Cicer arietinum(Chickpea) germplasm were analysed for total seed protein profile using sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE) to ascertain the extent of genetic variation and its geographical distribution. A considerable variation in protein banding pattern was observed which was localised to various geographical regions. Inter-specific variation was more as compared to intra-specific variation.
INTRODUCTION
Chick pea or “Channa” (Cicer arietinum L.) is an important legume crop. It is valued for its nutritive seed with high protein content 25.3-28.9% after dehulling (Hulse, 1991). In Pakistan among legumes Chickpea is the most important crop cultivated under rain fed areas of Thal desert. The area under cultivation is 10,779,000 ha which is three times of other pulses (Anonymous, 1999). Increase in yield could be attained by the use of germplasm /wild relatives, for new combination of favourable genes already existing (Muchlbauer et al., 1988). Recent advances in molecular biology have allowed population geneticists to make genetic comparisons across species as well as within species. Such molecular information has proven to be an important tool in systematics and in reconstructing phylogenies. (Avise, 1994).
Identification of chickpea cultivar using PAGE of storage seed proteins was done by Singh et al. (1991). They had extracted the proteins soluble in sodium chloride from mature seeds of nine chickpea cultivars and analysed by non-denaturing PAGE. Most cultivars had 2-3 major bands and a variable number of minor bands. They also analysed the G1 protein fraction by non-denaturing PAGE and SDS-PAGE. They concluded that the combined use of above three methods were effective of cultivar identification. Genetic relationship in the genus Cicer L. was revealed by polyacrylamide gel electrophoresis of seed storage proteins (Ahmad et al., 1992). Total seed storage proteins of the cultivated, C. arietinum and eight other wild annual species were separated and compared by SDS-PAGE. The seed protein profile was a conservative and species–specific trait.
Kharkwal (1999) conducted another study to find intra-specific relationships in Cicer arietinum. Electrophoretic analyses were conducted on seed protein extracts of four chickpea varieties. The results indicated that the desi and kabuli types were varieties of the same species. Additionally no evidence was obtained to indicate that kabuli types should be classified as Cicer kabulicum. All four varieties were highly homologous; the number of bands resolved were 9 in each case and the pattern was also the same with the exception of minor differences in position and the distance of bands.
Phylogenetic differentiation and geographical distribution of genetic variation for total seed protein in two hundred and seventeen accessions of Setaria italica from Europe and Asia were investigated by SDS-PAGE (Afzal et al., 1994). Total seed storage protein electrophoregrams were characterized and classified into six types which are distributed in different geographical regions of the world.
Another important study of seed protein profile of forage sorghum (Sorghum bicolor L. var. Moench) was done by Chauhan et al. (2002). Twelve varieties of forage sorghum were analysed by SDS-PAGE for identification or characterization on the basis of protein bands. All varieties could be distinguished on the basis of presence/absence of specific band, but HC-171 and HC-308 had same number of bands and could only be differentiated on the basis of intensity/thickness of similar bands. The molecular weight of protein was ranged between 5 KDa to 70 KDa. The total number of bands observed was 22, out of which band numbers 10,12, 21 and 22 were common in all the varieties. Band number 2 was present only HC-136, PC-6, PC-1, CSV-15 and MP-Chari. Band number 8 was present in PC-23, PC-121, PC-9B while number 5 was exhibited by PC-1 and band number 4 by MP-Chari and HC-260. Each variety or a group of varieties exhibited characteristic banding pattern on the basis of which they can be identified or classified accordingly.
The objective of the present study was to determine the geographical distribution of genetic variability by protein banding pattern. In order to achieve this objective the following study was performed: 1) electrophoretic detection and characterization of genetic diversity in Cicer arietinum accessions, 2) ascertain inter and intra specific variation in Chickpea germplasm.
MATERIALS AND METHODS
Plant material: The study was conducted at Plant Genetic Resources Institute (PGRI), National Agricultural Research Centre (NARC), Islamabad. Twenty-nine accessions of Chickpea were selected on the basis of geographical distribution. These accessions were collected from the gene bank, PGRI. The species and the origin of the accessions are shown in Table 1. The seed colour, seed shape and texture of the testa are given in Table 2.
Preparation of seed sample: For extraction of protein, individual seeds were ground to fine powder with mortar and pestle. To extract protein in 0.01 g of seed flour, 400 μl of the protein extraction buffer (0.05 M Tris-HCl, 0.2% SDS, 5 M Urea and 1% ß-mercaptethanol) was added to the tube and mixed well by vortex. Then centrifuged at 15,000rpm for 5 min at room temperature. The extracted crude proteins were recovered as clear supernatant and stored at -20°C.
Preparation of gel: Seed proteins were analysed through slab type SDS-PAGE followed by Laemmli (1970) using 11.25% polyacrylamide gel. Electrophoresis was carried out at 100v for two and half hours. In order to check reproducibility of the method two separate gels were run under similar electrophoretic conditions.
| Table 1: | List of accessions used for protein variation |
| Table 2: | Seed characters of chickpea germplasm |
After electrophoresis gels were stained with 0.2% (w/v) Coomassie brilliant blue R250 for about 1 hour than destained over night on a gyratory shaker. After that gel was dried using gel drying processor and analysed.
RESULTS AND DISCUSSION
Twenty-nine samples of Chickpea were evaluated for inter and intra-specific variation in seed storage protein. Results were obtained from the analysis of electrophoregrams and zymograms (Fig. 1, 2, 3). Eighteen bands were observed in total and accessions were classified on the basis of presence/absence and thickness/intensity of specific protein band. All accessions were classified into five clusters (Table 3) on the basis of similarity in banding pattern.
Cluster 1 included nine local wild accessions. These accessions were lacking minor band numbers 1, 2, 3, 4, 6, 7 and 9 and major band numbers 11, 13 and 14. Cluster 2 included only one local accession. This accession had less number of protein bands as compared to other wild accessions. Band numbers 5 and 8 were considerably thin. Accessions in this cluster were lacking those bands, which were absent in cluster 1. In addition to that band numbers 15, 16 and 17 were also absent. Cluster 3 included only two accessions. Both of these belonged to Iran. Band number 15 was absent and all other bands were present in these accessions. The major band numbers 6, 7, 10, 11, 12, 17 and 18 were thick. Cluster 4 included eleven accessions, two from Iran, two local, one from each Italy, India, Jordan, Mexico and Pakistan and two were exotic species. Accessions in this cluster were lacking a major band number 15.
| Fig. 1: | Zymogram (A) and electrophoregrams (B) showing variation of total seed protein in Chickpea (Cicer arietinum) accessions: cluster 1, 2 and 4 |
| Fig. 2: | Zymogram (A) and electrophoregram (B) of total seed protein of chickpea (Cicer arietinum) accessions showing variation in cluster 3 and 4 |
| Fig. 3: | Zymogram (A) and electrophoregram (B) showing variation in accessions of Chickpea germplasm cluster 4 and 5 |
| Table 3: | Grouping of accessions on the basis of similarity in banding patterns |
Cluster 5 included six accessions, two from Morocco, two from Turkey, one from Iran and one accession was exotic. All these accessions had major band number 12 considerably thin and lacking band number 15 (Fig. 1, 2, 3, Table 3).
Germplasm is vital source in generating new plant types having desirable traits that helps in increasing crop quality and production as well, thus improves level of human nutrition. Genetically heterogeneous populations produce more and stable yield than genetically homogenous lines.
Inter and intra-specific variation in SDS-PAGE electrophoregrams of total seed protein in wheat, barley and their wild relatives was detected by Masood et al. (1994). One hundred and eight accessions of Triticum, Aegilops and Hordeum species collected from Central Asia were evaluated for inter and intra-specific variation. A wide range of genetic variation was observed in the number and electrophoretic mobilities of high molecular weight glutenin and hordein subunits. It was suggested that electrophoresis of total seed protein was a useful and effective method to analyse genetic variability present in plant genetic resources. Our findings in Chickpea agreed with the observations of Masood et al. (1994). Similarly Kharkwal conducted another experiment to study intraspecific relationships in Cicer arietinum in 1999. He found that electrophoretic analysis conducted on seed protein extracts of four chickpea varieties indicated that the desi and kabuli types were varieties of the same species.
Keeping in view importance of variation in protein banding pattern our results showed that wild accessions contain less number of protein bands as compared to cultivated accessions. Wild accessions lack major band numbers 11, 13 and 14 and minor band numbers 1, 2, 4, 6, 7 and 9. Where as these bands were observed in all the cultivated accessions belonging to different geographical regions. This may be due to the reason that these bands were either added up due to extensive cross breeding or in the process of evolution. Our findings are similar with the results of Chauhan et al. (2002) where they had identify the different varieties of sorghum on the basis of intensity and presence and absence of specific protein bands.
Accessions, which were placed in one cluster, may or may not be similar. Since the appearance of bands on gel can be influenced by several factors which prevent accurate assessment of band homology among genotypes. Proteins coded by different genes may have similar motilities and produce over lapping bands.
It can be concluded from the results that inter specific variation is more as compared to intra specific variation and genetic variability in chickpea germplasm is not associated with its origin as previously observed by Kharkwal (1999) in his study on desi and kabuli chickpea.