Biochemical characterization for determination of genetic distances among different indigenous chickpea (Cicer arietinum L.) varieties of North-West Pakistan.

Genetic distances among different chickpea varieties and evaluation of their free amino acid profiles were determined on the basis of Sodium dodecyle sulphate polyacrylamide gels electrophoresis (SDS-PAGE). Total soluble proteins were resolved on 10% SDS Polyacrylamide gel. Low variability in tested varieties was observed. Dendogram based on electrophoretic data clustered the genotypes into 2 groups. The results showed that the average protein content of all the varieties was 26.01% within the range 22.8% for Thal-2006 to 34.06% Sheenghar-2000 of dry seed weight. On the basis of total protein content Bittal-98, Dasht and Sheen Ghar-2000, Karak-3 and CM-98, Paidar -91 and Fakhr-e-Thal, C-44, Balaksar and KK-1showed similar concentrations for protein contents among each other but showed variation from the rest of the varieties. Different proteins were separated on the basis of changes in their molecular weights by means of Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS-PAGE). Dasht, CM-98, and Sheen Ghar showed 100% similarity. Balaksar and Fakhr-e- Thal, KK-2 and Chattan and KC-98, KK-1 and Lawaghar were 100% similar among each other but showed variation from the rest of the accessions. The overall dendrogram showed high and low level of variation among the accessions. The concentration of free amino acids varied among the 16 chickpea varieties. A significant difference of both essential and non-essential amino acids was found among the chickpea cultivars. The total concentration of essential amino acid was recorded 40.81 g/100 g protein while non-essential was recorded 59.18343 g/100 g protein in the given cultivars. The highest concentration of essential amino acids was found in C-44 followed by KK-2, KK-1 and Fakhr E Tal while the lowest concentration was recorded in Cm-98, Paidar-91 and Sheen Ghar-2000 respectively. Cultivars TAL-2006, Chattan and Karak-3 showed maximum concentration of both essential and endogenous amino acids. In conclusion; for broadening the genetic pools in breeding programs or to search for exotic characters, for instance new disease resistance alleles, accession with low similarity coefficients (Lawaghar and Battal-98) may be utilized. Furthermore the information acquired from this study could be used to device a proficient breeding approach intended at improving nutritional as well as broadening the genetic base of this essential food crop of Pakistan.


Introduction
Chickpea (CicerarietinumL.) is an annual grain legume, self-pollinated, diploid (2n = 16) crop. It is comprised of a significant source of dietary proteins. It is the third most important legume cultivated crop of the Indian sub-continent, North Africa and West Asia (Bharadwaj et al., 2010;FAOSTAT, 2011). Chickpea is also the most important nutritive seed crop with high protein content varying between 20 to 26%. It is also valued as a good source of zinc, folate, phosphorus, iron and certain water soluble vitamins. Chickpea, as a high dietary fiber and carbohydrate food contributes a significant food source for persons with insulin sensitivity or diabetes (Singh and Jambunathan (1982); Jukanti et al., 2012). Its seed contains 3% fiber, 4.8 to 5.5% oil, 3% ash, 0.2% calcium, and 0.3% phosphorus, 60 to 66% carbohydrate content, while a small amount of (1 to 6%) lipids., while a small amount of (1 to 6%) lipids depending on different varieties (Hulse, 1991;Huisman and Poel, 1994). Chickpea is a good source of carbohydrates and protein which constitute about 80% of the total dry seed mass (Chibbar et al., 2010;Dhawan et al., 1991;Geervani, 1991). There are two kinds of gram crop, namely Desi and Kabuli in Pakistan. Desi (microsperma) types have a colored and thick seed coat, pink flowers and anthocyanin pigmentation on stems. The kabuli (macrosperma) types have white or beige-colored seeds with a ram's head shape, white flowers and thin seed coat and smooth seed surface. They lack anthocyanin pigmentation on stem (Moreno and Cubero, 1978). The weight of the seeds commonly ranges between 0.1 to 0.3g and 0.2 to 0.6g in desi and kabuli types, respectively (Frimpong et al., 2009). In Africa and Asia the Desi type contributes about 80-85% of the total chickpea area while in Europe and America the Kabuli types are largely grown (Pande and Kishore, 2005). Simon and Muehlbauer (1997) reported that Chickpea (gram) crop is usually cultivated as a single crop or combined with barley, linseed, mustard, pea, sweet potato, wheat, or sorghum, etc. Knowing genetic diversity not only helps in sorting of populations for genome mapping experiments but also accounts an important tool in gene-bank management and breeding experiments like tagging of germplasm, identification and/or elimination of duplicates in the gene stock and establishment of core collections (Kaga et al. 1996). The advancements in germplasm characterization using biochemical fingerprinting has got special attributes due to its increased use in crop improvement and the selection of desirable genotypes for breeding crops. The taxonomic and evolutionary problems of several crop plants have been resolved by using genetic markers and protein profiling (Boutler et al., 1966;Ladizinsky and Hymowitz, 1979;Murphy et al., 1990;Khan, 1990;Nakajima, 1994;Najma et al., 2005;Rao et al., 1992;Das and Mukarjee, 1995;Ghafoor et al., 2002;Javid et al., 2004). Significant studies have been reported regarding the genetic diversity of seed storage proteins for many crops; Lima bean (Lioi et al., 1999), Phaseolus vulgaris (Ferreira et al., 2000) and Cicerarietinum (Ghafoor et al., 2003). Based on SDS-PAGE data, Ahmad and Slinkard (1992) studied phylogenetic relationship among Cicer species and suggested that Cicerreticulatumis would be the wild progenitor of cultivated chickpea. The gene homology being the basic criterion of phylogenetic relationship cannot in many cases be measured directly because of reproductive barriers between species. Therefore seed storage protein analysis is not only regarded as a useful tool in identification and characterization of diversity in crop varieties, cultivars and their wild varieties but also helps to elaborate genetic transgression and phylogenetic relationship of the accessions Hussain et al., 2010). It is also reported that variations in protein bands show the relationship among the collection from various geographical regions (Valizade (2001); Satija et al., 2002;Ghafoor et al., 2003;Asghar et al., 2003). The present study was undertaken to estimate the genetic distances among indigenous chickpea varieties of North-West Pakistan based on their biochemical characteristics, amino acids profiling, protein content and band profiling.

Preparation of protein samples
Total soluble proteins were extracted by grounding the seeds in 50 mM phosphate buffer (pH 7.8) and centrifuged at 14,000 rpm for 10 minutes. The supernatant was separated and used for protein concentration and profiling.

Protein Quantification
CBB-dye binding assay as described by Bradford (1976) was used to measure the protein concentration.

Protein profiling using SDS-PAGE:
Protein profiling of samples was performed using Sodium dodecyle sulphate polyacrylamide gels as described by Laemmli (1970).

Amino Acid profiling
The dried seeds were ground with the help of mortar and pestle. Extraction of amino acids was carried out according to the method described by Mansfield and Baerlocher (2005). A total of 0.3 g of seeds powder was taken in falcon tubes and 5 ml HCl (0.1%) were added. The sample was homogenized through vortexing for 5 minutes. After vortexing the sample was centrifuged at 3500 rpm for 15 minutes and the supernatant was collected in separate tubes and labeled. The amino acid were analyzed using High Performance Liquid Chromatography (HPLC). The system was equipped with UV 338nm detector, column with C 18, 2.5 x 200mm, 5μm column. The mobile phase constituted 1:2:2 (100mM sodium sulphate, pH 7.2; acetonitrile; methanol (v/v/v) with a flow rate of 0.45 ml/minute. The operating temperature was set at 40°C.

Statistical analysis
The data was analyzed using SPSS to the general linear model (GLM) procedure of statistical analysis system. The means were compared by LSD and all pair wise comparison test. Significance was found at (P<0.05).

Results
During the current study, an effort was made to estimate the genetic distances among different indigenous chickpea varieties of North-West Pakistan based on biochemical characteristics, amino acid profiling, protein content and band profiling.

Proteins Quantifications:
Total soluble proteins in different chickpea seeds ranged from 228.2 mg/g (22.8%) to 340.6 mg/g (34.0%) of dry seed weight (Supplementary Material Table 1). The average protein content of all the varieties was 26.01%. Highest percentage for protein content was recorded for Sheen Ghar-2000 while the lowest protein content was observed in Thal-2006. On the basis of total protein content Bittal-98, Dasht and Sheen Ghar-2000, Karak-3 and CM-98, Paidar -91 and Fakhr-e-Thal, C-44, Balaksar and KK-1showed similar concentrations for protein contents among each other but showed variation from the rest of the varieties ( Figure 1).

SDS-PAGE based Genetic Diversity
SDS-PAGE was utilized to estimate the extent of genetic diversity existing among the germplasm of chickpea. Different proteins were separated on the basis of change in their molecular weights of all the tested varieties by means of SDS-PAGE. The binary data matrix of all the genotypes was computed for the formation of phylogenetic tree (dendrogram) using UPGMA. Total of 13 bands were observed. The cluster analysis was performed for total banding pattern.

Cluster analysis on the basis of SDS-PAGE
Cluster analysis of chickpea seed storage proteins was performed on the results of SDS-PAGE (Figure 2 and 3). The software "popgene" was used to calculate the genetic diversity among the given varieties. The results of cluster analysis are presented as phylogenetic tree (dendogram) in Figure 4 on the basis of linkage distance by using "Un-weighted pair group method with arithmetic means" (UPGMA) (Nei and Li, 1979).
In cluster analysis for whole polypeptide bands, the whole samples were divided into two groups or lineages, lineage 1 (L1) and lineage 2 (L2), at a linage distance of 0.25 ( Figure 4). These lineages were further divided into clusters at linkage distance 0.15, forming five clusters (C1 -C5). Lineage 1 consists of two clusters while lineage 2 consists of 3 clusters. Among the linage 1, cluster 1 (C1) consists of a single accession, while cluster 2 (C2) contains two accessions. Similarly in linage 2, cluster 3 (C3) consists of a single accession while cluster 4 (C4) and clusters 5 (C5) consists of two accessions each. Cluster 2 and 4 were further divided into sub-clusters at linkage distance of 0.06. The percentage of each cluster in total population (16 genotypes) is given in Table 1.   In lineage 1 Bittal-98 showed highest variations at LD = 0.06, In lineage 2 Balaksar and Fakhr-e-Thal showed variation from the rest of the accessions in the same clusters at LD= 0.16 while in cluster 4 Karrak-3 showed variation at LD = 0.06. In lineage 1 accession Dasht, CM-98, and Sheen Ghar showed 100% similarity at LD = 0.03 and could not be differentiated among each other on the basis of total protein bands.
In lineage 2 at LD = 0.16 accession Balaksar and Fakhr-e-Thal at cluster 3, at LD = 0.08 accession C-44, PB-91 and Thal-2006 at cluster 4 and at LD= 0.08 accession KK-2 and Chattan at cluster 5 and KC-98, KK-1 and Lawaghar at cluster 5 were 100% similar among each other but showed variation from the rest of the accessions. Therefore, they were grouped in each cluster. The overall dendogram showed high and low level of variation among the accessions (Figure 4). The average variation observed was 30%. Accession line; Balaksar showed maximum variation whereas Karak-3 showed the lowest variation. Sheen Ghar-2000, Thal-2006 and CM-98 did not show any variation ( Table 2).

Amino Acids profiling of Chickpea
Amino acids are significant component of the food and play an important role to indicate its nutritional value.  (Table 3) . Furthermore the total concentration of essential amino acid was recorded 40.81 g/100 g protein while non-essential was recorded 59.18343 g/100 g protein in the given cultivars. A significant difference of both essential and non-essential amino acids was found among the chickpea cultivars. The highest concentration of essential amino acids was found in C-44 followed

Discussion
Some of the significant factors that determine the level of genetic variability are extent of distribution; areas sampled and plant characteristics such as mode of reproduction, breeding behavior and generation time. Total soluble proteins of 16 different chickpea varieties extracted and quantified showed that the average protein content of all the varieties was 26.01% within the range of 22.8% for Thal-2006 to 34.06% Sheenghar-2000 of dry seed weight. Our findings are complementary with the results of Milan-Carillo et al. (2000) who have recorded mean value of 22.5% protein for desi chickpea genotypes. Protein content are also in concordance with Jambunathan and Singh (1981) who analyzed 8 desi and 7 kabuli chickpea cultivars and observed higher crude protein for kabuli types (241 g/kg) than desi type (217g/kg). Difference in crude protein content has been reported to depend on geographical origin of seed in addition to genetic differences but the role of location and season in the genotypic expression of protein content is low. Indian subcontinent has a very diverse Germplasm and its diversity includes the total diversity expressed by the exotic accession. One of the most extensively used and reliable techniques to separate and differentiate proteins is SDS-PAGE (Javed et al., 2004;Iqbal et al., 2005). It is used to separate different proteins on the basis of changes in their molecular weights. A powerful tool for population genetics is protein electrophoresis (Parker et al., 1998). In the present study the 16 different chickpea varieties showed both high and low levels of genetic variations during cluster analysis which is in agreement with Hameed et al. (2009), who reported the same results for seed storage protein of Kabuli Chickpea genotypes. The whole polypeptide bands were divided into two groups or lineages, Lineage 1 and lineage 2, at a linkage distance of 0.25. These lineages were further divided into clusters at lineage distance of 0.15, forming five clusters (C1-C5). Lineage 1 contains 2 clusters while lineage 2 contains 3 clusters. Among the linage 1, cluster 1 (C1) consists of a single accession, while cluster 2 (C2) contains two accessions. Similarly in linage 2, cluster 3 (C3) consists of a single accession while cluster 4 (C4) and clusters 5 (C5) consists of two accessions each. Cluster 2 and 4 were further divided into sub-clusters at linkage distance of 0.06. The percentage of each cluster in total population (16 genotypes) is given in Table 1. In lineage 1 Bittal-98 showed highest variations at LD = 0.06. In lineage 2 Balaksar and Fakhr-e-Thal showed variation from the rest of the accessions in the same clusters at LD= 0.16 while in cluster 4 Karrak-3 showed variation at LD = 0.06 In lineage 1 accession Dasht, CM-98, and Sheen Ghar showed 100% similarity at LD = 0.03 and could not be differentiated among each other on the basis of total protein bands. In lineage 2 at LD = 0.16 accession Balaksar and Fakhr-e-Thal at cluster 3, at LD = 0.08 accession C-44, PB-91 and Thal-2006 at cluster 4 and at LD= 0.08 accession KK-2 and Chattan at cluster 5 and KC-98, KK-1 and Lawaghar at cluster 5 were 100% similar among each other but showed variation from the rest of the accessions. Therefore, they were grouped in each cluster. The overall dendogram showed high and low level of variation among the accessions (Figure 4). The average variation observed was 30% (Table 2) showing a significant variation earlier examined in various studies. Hameed et al., 2009 studied eight Kabuli Chickpea genotypes and observed total variation of 20%. Similarly Ahmad et al. (2012) found 37% variation based on molecular characterization of Chickpea germplasm. A low level of intra specific variation was studied among the chickpea accessions, which confirms the reports of Thakare et al. (1987), Mehrani (2002) and Ghafoor et al. (2003). This low level of variation signifies a risk to the genotypes examined in the study therefore variation must be high for excellent yield, disease resistance and other environmental susceptibility, reducing the chances of harmful effect of environment and other factors. SDS-PAGE results confirmed that the method provides a tool for reliable germplasm characterization based on genetic dissimilarities in seed storage proteins composition in chickpea. Our results did not confirm the findings of Nisar et al. (2007) who studied considerable intra specific variation in local and exotic chickpea germplasm. Large amounts of proteins are stored in Legume seeds during their development. They do not play any role in the development of cotyledonary tissue therefore they are accumulated in storage vacuoles or protein bodies. During seed maturation, the cotyledonary parenchyma cells survive desiccation and go through proteolysis at germination, thus supply free amino acids, as well as ammonia and carbon skeletons to the developing seedlings. These seed proteins are called storage proteins (Casey et al., 1986). Several comparatively minor proteins, including protease and amylase inhibitors, lectins, lipoxygenase and defense proteins are found in Legume seeds which are important to the nutritional/functional quality of the seed (Murray, 1979). The potential quality of a protein food is presented by its Amino acid composition therefore their bioavailability is critical for the supply of amino acids in the diet (Sarwar and Peace, 1986). A higher amount of essential amino acid is found in all chickpea varieties (WHO, 1973;FAO, 1973). Amino acid content normally shows the nutritive value of a protein source (Bodwell et al., 1980) and is broadly used for screening prospective protein foods

Conclusion
To broaden the genetic pools in breeding programs or to search for exotic characters, for instance new disease resistance alleles, accession with low similarity coefficients (Lawaghar and Battal-98) may be utilized. Conversely it is very complicated to establish the genetic similarities based on seed storage protein profile to reveal genetic diversity, therefore DNA based genetic markers such as SSR, RAPD should be used to further investigate related varieties of chickpea to determine its genetic identity. The information acquired from this study could be used to device a proficient breeding approach intended at improving nutritional as well as broadening the genetic base of this essential food crop of Pakistan. Since the information attained reveals the potential worth of Chickpea germplasm collections therefore efforts are being made to expand the data base by characterizing the remaining germplasm.