Assessment of Genetic Diversity in Bambara Groundnut (Vigna subterranea) Landraces Based on Seed Storage Proteins Electrophoretic Pattern

Bambara groundnut (Vigna subterranea) is a leguminous crop that is considered underutilized and has previously received little research attention. Variability in a number of physiological factors, including germination rate, widely affects its production. Seed storage protein, its fractions and protein profile of six Bambara groundnut local landraces were studied to assess their genetic relatedness. Total seed storage protein and its fractions were estimated by Bradford’s method. SDS-PAGE analysis was used to evaluate storage protein profile. The results showed significant differences (p<0.05) in protein contents among the landraces. The major seed storage proteins were found to be globulins (0.048 to 0.088mg/mL ), albumins (0.023 to 0.038mg/mL ), glutelins (0.007 to 0.013mg/mL ) and prolamins (0.002 to 0.004mg/mL ). Five peptide bands were detected with molecular weights corresponding to 97.4 kDa, 45 kDa, 29 kDa, 20.1 kDa and 18 kDa, respectively. Three peptide bands corresponding to 97.4 kDa, 45 kDa and 18 kDa were detected in all the landraces and two peptide bands between 29 kDa and 20.1 kDa were detected in five landraces. Dendrogram generated by UPGMA grouped the six landraces into one major cluster with two sub-clusters. The observed diversity in storage protein pattern of the landraces indicated their potential as materials for crop improvement.


Verdc.) is a leguminous crop that is native to Africa
; it is commonly cultivated for its seeds by subsistence farmers (Linnemann and Azam-Ali, 1993;Massawe et al., 2005) and has the prospect of contributing to climate change ready agriculture (Mayes et al., 2019). Among the legumes in Africa, Bambara groundnut has been reported to be the most important after groundnut and cowpea with which it shares its origin of genetic diversity. Though extensively grown in Nigeria, it has been reported to be one of the crops less investigated scientifically (Bamshaiye et al., 2011). Genetic diversity is of great importance in implementing successful breeding programs in both vegetatively and sexually propagated crops, the variation can occur naturally or artificially (Agbolade et al., 2016). One of the techniques employed for assessing the genetic variability and relatedness among crop germplasms is the analysis of seed storage protein profile, and it stands as an alternative and a valid method for varietal or species identification (Menella et al., 1999;Naworu, 2020). Proteins are dynamic in nature, hence the differences in their characteristics defines one from another. Studies on storage protein, its fractions and peptide pattern of some Bambara groundnut landraces indicated differences among landraces in terms of their protein content and molecular weights (Paulter, 1981;Odeigah and Osanyinpeju, 1998;Oludare et al., 2017). The biosynthesis of seed storage proteins takes place in the developing embryo when cell division is complete. These proteins which accumulate in protein bodies in the cotyledonary parenchyma cells are distinguished from other proteins by their lack of any functional activity besides storage and are utilized during germination and seedling establishment (Surekha and Mandal, 2000;Li et al., 2006). Seed storage proteins have varied solubility, and this serves as the basis for their classification.
Albumins are water soluble, globulins are salt soluble, glutelins are alcohol soluble while the glutelins are alkali soluble (Surekha and Mandal, 2000). Seed storage proteins are genetic markers that have found utility in intra-and inter-species genetic diversity analyses, conservation of genetic resources and as a molecular tool in crop improvement (Ghafoor et al., 2002). The genetic diversity of storage proteins can be very useful for breed lines analysis and enhanced nutritional quality of crop varieties by indicating lines having variably expressed storage proteins (Shende et al., 2019). As proteins are direct products of gene expression, the electrophoretic banding of proteins from any species can be used to evaluate the genetic variability within and with other species (Agbolade et al., 2017).

Sample collection and preparation
Seeds for six landraces of Bambara groundnut were locally obtained from Niger , Yobe and Kano States of Nigeria (Figure 1). The seeds were identified in the Department of Plant Biology, Bayero University, Kano, with a voucher number '0509'. The seeds were named Niger cream, Yobe cream, Yobe black, Kano mottled, Kano maroon and Kano brown based on their places of collection and colours. Only seeds with normal shapes were used, while those displaying any visible damage or abnormal shape were discarded. The seeds were cleaned by washing with distilled water. Twenty (20) randomly selected seeds from each landrace were weigh ed and crushed using a mortar and pestle into flour, sieved and stored in airtight plastic containers. Extraction of total storage protein Seeds flour was transferred into a 250 mL beaker and defatted with n-hexane (1:3, w/v) at room temperature by continuous shaking for thirty minutes and then allowed to settle. The hexane was removed by decantation and the flour left to dry at room temperature. The extraction of total proteins was done according to Hameed et al. (2009). Seed flour (1 g) was dissolved in a 10 mL extraction buffer (50 mM phosphate buffer, pH 7.8). The homogenate was vortexed and centrifuged at 14,000 rpm for 10 minutes at 4 °C. The supernatant was used for total protein estimation and sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) analysis.

Extraction of storage protein fractions
Storage protein fractions were sequentially extracted based on their solubility (Silva et al., 2016). Seeds flour (1g) was dissolved in 10 mL distilled water, the homogenate was incubated for 30 minutes at 30 °C and subsequently centrifuged at 3000 x g for 20 minutes. The

Niger cream Kano mottled Kano maroon
Kano brown supernatant was recovered and the pellet resuspended in 5 mL distilled water, incubated for 30 minutes at 30 °C and centrifuged at 3000 x g for 20 minutes. The supernatants were combined as albumin fraction. Globulins fraction was extracted in a similar manner using 1% NaCl as the solvent; glutelins fraction was extracted with 0.1 M NaOH while prolamins fraction was extracted with 70% ethanol.

Estimation of protein
Protein quantification was carried out according to Bradford's method using bovine serum albumin (BSA) as the standard (Bradford, 1976).

Determination of storage protein banding pattern
SDS-PAGE analysis of total storage proteins from the six Bambara groundnut landraces was carried out in 12% polyacrylamide slab gels according to the method of Laemmli (1970). Ten microliters (10µl) each of the protein ladder (acculadder TM Protein size marker, 6.5 kDa to 116 kDa) and samples were loaded into the wells of stacking gel. Electrophoresis was done at 80 V for 1 hour until the bromophenol blue ran to the bottom of the gel. After electrophoresis the gels were stained with 2% coomassie blue for 1 hour and destained by solution containing 5% (v/v) acetic acid, 20% (v/v) methanol and distilled water in the ratio of 5:20:75 (v/v) for 2 hours.

Assessment of seed storage protein diversity
The diversity of storage protein profiles was assessed using Jaccard's similarity matrix based on the presence or absence of peptide bands in the electrophoretic pattern of total seed storage proteins. Depending on the presence or absence of a band, peptide was scored 1 for presence and 0 for absence of band and the score was used to generate a similarity matrix. The similarity index was converted into a dissimilarity index using the relationship: dissimilarity = 1 -similarity. Following an unweighted pair-group method with arithmetic averages (UPGMA), the dissimilarity values were used to generate a dendrogram.

Statistical analysis
All data are presented as the mean ± standard deviation of three replicate analyses. For comparison, data were analyzed by one-way ANOVA. Tu key's test was used to determine the differences between protein content among Bambara groundnut landraces. All statistical analysis was based on a significant level of 0.05. Analyses were done with SPSS Statistics version 16.0.

Results
The concentrations of total seed storage protein and protein fractions of the Bambara groundnut landraces are presented in  SDS-PAGE analysis of the total seed storage protein ( Figure 2) revealed similarities in peptide pattern between the studied Bambara groundnut landraces even though there were some observable differences in terms of number of peptide bands detected. Niger cream and Yobe cream landraces have the highest number of bands with five bands each, followed by Yobe black, Kano maroon and Kano brown with four bands each and then Kano mottled with 3 bands. All the six landraces have 3 bands in common, corresponding to 97.4 kDa, 45 kDa and 18 kDa. Two bands between 20.1 kDa and 29 kDa were detected except in the Kano mottled landrace.

Discussion
Seeds of plants contain one or more groups of proteins that supply amino acids important during germination and seedling establishment. These storage proteins are important because they determine both the quantity and quality of seed proteins for various metabolic uses (Alwhibi, 2017). In the present study, total seed storage proteins and its four fractions differed significantly among the Bambara groundnut landraces. Odeigah and Osanyinpeju (1998) and Oludare et al., (2017) reported seed storage protein content of Bambara groundnuts within the same range (0.053 to 0.066 mg/ml). However, Poulter (1981) reported albumin fractions having the highest content (20.34 to 74.46%) in Bambara groundnut cultivars, followed by globulin fractions (2.52 to 46.30%) while Yagoub and Abdalla (2007) reported albumin fractions (72.64%) as the major storage protein, followed by glutelin (8.60%), globulin (8.20) and then prolamin fractions (0.84%). The differences in protein content can be a reflection of genetic variation existing in the crop since proteins are the primary products of gene expression. Also nutritionally, the Bambara groundnut seed contains all the four types of storage proteins making it an idle crop that can be used to supplement protein in cereal crops diet.
SDS-PAGE analysis of the seed storage protein shows that the Bambara groundnut landraces studied have similarity in electrophoretic band patterns irrespective of their seed colour, but slightly differ in terms of number of bands and band intensity. Niger cream (NC) (Oludare et al., 2017). Odeigah and Osanyinpeju (1998) reported that SDS-PAGE of Bambara groundnut storage protein did not show any definite correlation between any polypeptide and seed phenotypic characteristics. Smartt (1990) and Odeigah and Osanyinpeju (1998) suggested that differences may be present between Bambara groundnut storage proteins at finer level which SDS-PAGE could not detect. Similar results on storage protein electrophoresis showing some slight variations in seed storage protein electrophoregrams were reported in Bambara groundnut Oludare et al., 2017), sesame (Alege, 2015) and groundnut (Muniappan et al., 2016). Dudwadkar et al. (2015) reported that the diversity and variation in seed storage proteins have potential for species delimitation and can also serve as markers for intra-and inter-specific hybridization. Landraces are an important source of genetic divergence and the greater the level of variation, the greater the chances of a landrace possessing gene combinations of interest in plant breeding (Sadia et al., 2009). There is a lack of unique bands peculiar to a specific landrace. Despite morphological diversity possessed by landraces, it seems there exist genotype duplicates among Bambara groundnut as revealed by the similar peptides pattern of some landraces. These similarities may represent the characters shared by the landraces . The presence of bands of similar molecular weight across varieties signifies, among others, that the genes encoding the proteins are ubiquitous in the species (Akinwusi and Illoh, 1995). Inability of accessions or cultivars to express a peculiar band is an indication of common gene expression among the crop genotypes while possession of common bands in all studied accessions or cultivars strongly indicates that the gene coding for the peptide bands is fixed (Alege, 2015). Cluster analysis put the six Bambara groundnut landraces into one major cluster (A) with two subclusters A1 (consisting of four landraces) and A2 (consisting of two landraces). The grouping of all six landraces into one single cluster reveals low genetic diversity among the studied landraces. The occurrence of low genetic diversity could be attributed to genotype homogeneity or purity (Peddakasim et al., 2015). Since Bambara groundnut seeds samples used in the present study were sourced from markets, the clustering pattern cannot be solely based on the environment where the seeds samples were purchased since seeds samples can be brought to market from different locations across the country. Tabatabei et al. (2011) reported that human factors are very important for the association between accessions from different locations. Results within close range of similarity coefficient were similarly reported in many other species; for example, 0.82-0.24 in cowpea cultivars and 0.96-0.47 in sesame accessions Alege, 2015).

Conclusion
The findings from the present study indicated some variation in seed storage protein content and profile of the Bambara groundnut landraces. The observed variations indicate substantial genetic variability in storage proteins among the studied Bambara groundnut landraces indicating their potentials as materials for crop breeding and improvement. Furthermore the presence or absence of peptide bands can be useful for identification of landrace among others.