Biochemical and cytological analysis of five cultivars of Cicer ( chickpea )

During the present study, protein estimation and protein profiling of five cultivars of Cicer namely Dhawal, ujjwal, Shubhra, DCP-92-3 and IPC-04-20 have been carried out. The protein content on per gram fresh weight basis was found highest in the Dhawal measuring 35.2± 3.83 mg followed by DCP-923 and lowest was found in the IPC-04-20 cultivar. The protein profiling of seed proteins using sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) revealed a high polymorphism between the five cultivars. The total number of polypeptide bands recorded was 36, the 4 bands were monomorphic and the rest32 were polymorphic with an average polymorphism of 88.88%. The Jacards similarity ranged from 0.25000 to 0.56000. The similarity index calculated was highest between Ujjwal and IPC-04-20 (37.5%) where as lowest was calculated between Shubhra and IPC-04-20 (22.22%) variety. The unweighted pair group method with arithmetic mean (UPGMA) clustering method revealed two major clusters in the dendrogram that is, cluster 1 and 2, comprising two varieties each. The Shubhra occupies a distinct place as depicted in the dendrogram. Moreover, cytological studies of the five cultivars by calculating the mitotic index were carried out. The mitotic index varied between 4.82 to 10.83% and was found highest in DCP-92-3 while as minimum value was observed in Ujjwal.


INTRODUCTION
The genus Cicer include 33 perennial, eight annual, one unspecified wild species as well as the cultivated ones ( Van der Maesen, 1987).Chickpea is the second most important cool season pulse crop in the world and is grown in at least 33 countries including central and west Asia, South Europe, Ethiopia, North Africa, North and South America and Australia (Ladizinsky and Adler, 1976;Singh and Ocampo, 1997).It is native to South Europe and is the most important pulse crop of India, commonly grown in Uttar Pradesh, Panjab, Maharashtra, Rajasthan, Bihar and Madhya Pradesh accounting for more than 90% of the total area under it.India is the largest producer of chickpea, accounting for 66% of the world production (FAO, 2004).The average annual yield world wide (0.78 ton/ha) is considered to be somewhat lower than its potential yield (Singh et al., 1994;Sudupak et al., 2002).In India, gram is sown as Rabi crop at the end of the rainy season.Sowing takes place from September to November, and harvesting from February to April.Genotyping of different species is necessary for characterization of different accessions of crop gemplasm, testing varietal purity and registration of newly *Corresponding author.E-mail: Shahzada194@gmail.com.
Abbreviations: UPGMA, Unweighted pair group method with arithmetic mean; SDS-PAGE; sodium dodecyl sulphate polyacrylamide gel electrophoresis.developed cultivars (Chowdhury et al., 2002).Among numerous techniques available for assessing the genetic variability and relatedness, seed storage protein analysis represents a valid alternative to varietal identification (Manella et al., 1999).Seed storage protein profiling based on SDS-PAGE can be employed for various purposes, such as characterization of germplasm (Javid et al., 2004;Iqbal et al., 2005), varietal identfication, biosystematic analysis, determination of phylogenetic relationship between different species (Sammour, 1991;Isemura et al., 2001;Ghafoor et al., 2002).It is a useful tool for studying genetic diversity via sodium dodecyl sulphate polyacrylamide gel electrophoreseis (SDS-PAGE) (Sadia et al., 2009).
Seed storage protein markers are highly polymorphic and environmental influence on their electrophoretic pattern is limited (Gepts et al., 1986;Sadia et al., 2009).Genetic diversity of seed storage proteins has been reported for lima bean (Lioi et al., 1999), Phaseolus vulgare (Ferreira et al., 2000) and chickpea (Ghafoor et al., 2003).Phylogenetic relationship among Cicer species based on SDS-PAGE data has suggested that Cicer reticulatum is the wild progenitor of cultivated chickpea (Ahmad and Slinkard, 1992).

MATERIALS AND METHODS
The germplasm of five accessions of Cicer were obtained from Indian Institute of Pulse Research, Kanpur (U.P.) India.During the present study, five accessions were used for biochemical and cytological analysis.The details of five accessions are given in Table 1.

Protein estimation
Cicer being one of the highly proteinaceous crops was subjected to protein estimation in order to know the total protein content.The protein content was measured by using the method of Bradford et al. (1976) and was calculated by using the formula: Where, O. D. is Optical Density, D. F. is dilution factor and F. W. is fresh weight.

Protein extraction
For total seed protein extraction from individual seed samples, 1 g seed of each accession was taken and ground into fine powder using pestle and mortar.500 ml of protein extraction buffer was added to 0.01 g of seed flour and vortexed thoroughly to homogenize.The homogenate samples were centrifuged at 6,000 rpm for 10 min at room temperature.The extracted crude proteins were recovered as clear supernatant and were transferred to a new 1.5 ml eppendorf tubes and stored at 4°C until they were run on the polyacrylamide gel.

Protein profiling
Protein profiling of extracted samples was carried out through SDS-PAGE using 12% polyacrylamide gel.Electrophoresis was carried out at 75 V for 3 h.A protein marker was loaded as standard along with the samples with equal quantities of protein (4 ml) into each well of the gel.The gels were then fixed in solution (10% acetic acid and 40% ethanol) for 15 min with constant shaking and then stained with 0.2% (W/V) Commassie brilliant blue R 250 overnight on an electric shaker using Double Shaker Mixer Model DH-10.Destaining was carried out for a couple of hours followed by gel preservation, scanning and photography.

Protein imaging and data analysis
Gel photographing and documentation were carried out.With regard to variation in protein banding pattern, electrophorogram of each accession was scored for the presence or absence of bands and used to construct a dendrogram by the unweighted pair group mean and arithmetic average method (UPGMA).

Mitotic index
During the present study, the mitotic index was examined.Root tips of each accession were harvested at about 6 a.m., fixed in 3:1 ratio of ethyl alcohol and glacial acetic acid solution for about 6 h and then preserved in 70% alcohol for analysis.The mitotic index was calculated by using the formula:

Protein estimation
Cicer being a proteinacious crop was subjected to protein estimation.The protein content was found highest in Dhawal (35.2 mg/g F. W.) followed by DCP-92-3 (34.3 mg/g F. W.), Ujjwal (32.9 mg/g F. W.) and Shubhra (31.0  mg/g F. W.); while as the lowest protein content was observed in IPC-04-20 (28.7 mg/g F. W.) (Table 2).The variation might be due to different geographical regions.

Protein profiling
The SDS-PAGE of seed proteins of five genotypes was carried out to investigate the genetic diversity at molecular level.Seed storage protein profiling showed distinct polymorphism in electrophoretic banding patterns and led to detection of 36 polypeptide bands in total.Out of 36 bands, only four were monomorphic and the rest 32 were polymorphic.The average polymorphism was 88.88%.Similar results were also reported by Nisar et al. (2007) in Chickpea.Polymorphism was evident in all seed proteins of diverse molecular weights among all accessions but major diversity was found in low molecular weight region.The molecular weights of peptides ranged from 99 to 40 KD with the presence or absence of particular band.The maximum protein bands generated in case of Dhawal ( 21) and the minimum (16) in Shubhra.The protein band for highest molecular weight (that is, 99 KD) was generated in all the five accessions while that of lowest molecular weight (that is, 40 KD) was generated in IPC-04-20 (Table 3 and Figure 2).The similarity index calculated was found highest between Ujjwal and IPC-04-20 (that is, 37.5%), and between Dhawal and DCP-92-3 (30.76%) while as minimum similarity was found between Shubhra and IPC-04-20 (22.22%) (Table 4).
The data obtained from SDS-PAGE analysis was used for construction of dendrogram using unweighted pair group mean and arithmetic average (UPGMA) procedure is presented in Figure 1.Five accessions were grouped in two clusters.Cluster I and cluster II, comprising of two accessions each.The cluster analysis revealed that Ujjwal and IPC-04-20 are very close to each other.Accession Shubhra occupies a distinct place as revealed in the dendrogram.

Cytological study
The cytological study was done to find out the mitotic index.The mitotic index varied from 4.82 to 10.83% (Table 5).It was found highest in DCP-92-3 (10.83%) and in Dhawal (8.98%) which revealed that these accessions have highest power of division, while as the minimum mitotic index was found in Ujjwal (4.82%) and Shubhra (7%) which indicates that these have low rate of division.Similarity index of different protein bands was calculated by the formula:

Jaccard's similarity index
Jaccard's similarity index is given in Table 6.

DISCUSSION
Previously morphological and cytological assay procedures were used to estimate existing genetic variability in the crops of commercial importance including legumes (Islam and Shepherd, 1991).These essay procedures though were successful in many cases but were not considered suitable for large scale screening mainly because of limited number of markers and time consuming for the essay procedures.Most recently, protein markers (especially seed storage proteins) are being used for better and more reliable estimation of genetic distances among species/lines/populations (Weber et al., 2005).Comparison of seed storage proteins have been found to provide no biological basis for separating closely related small and large seeded lentils (Ladizinsky, 1979).Same is the case with chickpea genotypes evaluated in the present study as no difference in seed storage proteins of bold and small seeded genotypes was observed.The protein profiling of seed proteins using SDS-PAGE revealed a high polymorphism between the five cultivars.The total number of polypeptide bands recorded was 36, the 4 bands were monomorphic and the rest 32 were polymorphic with an average polymorphism of 88.88%.The Jacards similarity ranged from 0.25000 to 0.56000.The similarity index calculated was highest between Ujjwal and IPC-04-20 (37.5%); whereas lowest was Total number of similar bands S. I. = × 100 Total number of bands The Shubhra occupies a distinct place as depicted in the dendrogram.Similar results were also reported by Nisar et al. (2007) in Chickpea.The results of present studies are further strengthened by previous finding of Ghafoor et al. (2003), Yasmin et al. (2010), Asghar et al. (2003), Ferreira et al. (2000) and Dasgupta and Singh (2003) who reported high genetic diversity in various legume species using protein profiling.

Conclusion
Genetic diversity is important for improving any crop species.An understanding of the magnitude and patterns of genetic diversity in crop plants has important implications in breeding programs and for conservation of genetic resources.From the result of the study, it is clear that higher amount of genetic diversity of seed storage proteins is present in chickpea genotypes which can be utilized in breeding programs aimed at increasing level of genetic diversity which ultimately will be useful for the development of new improved genotypes of chickpea.Evaluation of genetic diversity and identification of chickpea accessions by SDS-PAGE is easy and early approach and it is also useful for molecular weight analysis of chickpea seed storage proteins.It is concluded from the result that Shubhra and IPC-04-20 are genetically dissimilar; hence, it is recommended that these two accessions should be used for future breeding programs.

Table 1 .
Five experimental accessions of Cicer.

Table 2 .
Protein content of five accessions of Cicer.

Table 3 .
Presence and absence of bands of different molecular weights in different samples.

Table 5 .
Mitotic index of five Cicer accessions.