Proteomics Variation in Nigella Sativa L. (Rananculaceae) Germplasm

Abstract

Study revealed a first report of proteomics variation in Nigella sativa L. based on analyzing 32 accessions through SDS-PAGE. Three prominent regions along eight subunits were identified. Intra specific variation was observed low whereas the sharpness of bands was high between first and second regions. It was noted that in second region there was no clear evidence of band formation in N. sativa. Prominent and sharp protein peptide bands were recorded in four accessions, namely PK-020561, PK-020609, PK-020620 and PK-020646. Further investigation of single seeds showed almost similar genetic pattern within the single accession. Five clusters were formed on the basis of Euclidean distance. Cluster-I & II contain 1, 1 accession each, likewise Cluster-III and C-IV contain 2, 2 accessions whereas Cluster-V was found diversified as consisted of 26 accessions. Two accessions PK-020878 and PK-020877 were recommended for polymorphism and crop improvement programs. Introduction The Ranunculaceae is a large family containing about 70 genera and at least 3000 species. About 20 annual species are known to be considered for genus Nigella, originated from Mediterranean region spread through West Asia to Northern India (Weiss 2002). In South-East Asia, seeds are used as flavoring dishes, salads, pickles and as medicinal (Benkaci-Ali et al. 2006, Iqbal et al. 2010). Biochemical constituent’s diversity provides wealth of knowledge which would be used efficiently to manage genetic stocks and planning breeding experiments (Iqbal et al. 2017). Plant varieties are usually characterized through agromorphological parameters but are controlled by multiple alleles/genes. To avoid such duplication, biochemical markers like proteins, enzymes, isozymes, and plant hormones, etc. received more attention in recent years for assessment of genetic variability because these are products of genes and their expression is more stable. The seed storage proteins are associated with traits of agricultural significance and are used in legal protection of cultivars (Knoblochova and Galova 2000). These proteins are divided into three groups: The 1 storage proteins-gliadins and glutenins, the 2 proteins with a high metabolic activity albumins and globulins, the 3 protein groups are structural proteins, very difficult to dissolve and fix in a cell structure (Dvoracek and Curn 2003). Among biochemical markers, SDS-PAGE is widely used technique due to its validity and simplicity for describing genetic structure of various plants. Seed storage proteins have been used as genetic markers in four areas: (i) Intra and inter accession variability, (ii) plant adaptation, (iii) whole genome interaction and (iv) in crop improvement (Iqbal et al. 2009b). Considering its importance, 32 accessions were analyzed for banding pattern variation and phylogenetic relationship. *Author for correspondence: <[email protected]>. Bioresource Conservation Institute, National Agricultural Research Centre, Islamabad, Pakistan. DOI: https://doi.org/10.3329/bjb.v50i2.54084 290 IQBAL AND GHAFOOR Materials and Methods Fresh seeds of each of 32 accessions were separately bulked, ground and processed for SDSPAGE seed storage protein analysis (Table 1). Accessions were collected throughout the country including two accessions, one from USA and another from Ukrain. In another experiment, single seed was grounded and protein analysis was performed under the same conditions of the gel electrophoresis. Protein extraction buffers were prepared according to the method provided in Iqbal (2009a). Table 1. List of Nigella sativa L. accessions representing provinces, collection sites and altitudes. Sl. No. Accessions* Province Collecting sites Altitude Sl. No. Accessions* Province Collecting sites Altitude* 1 Pk-020545 KPK Haripur (Hattar) 580 17 Pk-020742 AJK Mirpur 950 2 Pk-020561 Punjab Lahore 290 18 Pk-020749 Punjab Chakwal 525 3 Pk-020567 Faisalabad 230 19 Pk-020766 Multan 125 4 Pk-020576 230 20 Pk-020780 Lahore 290 5 Pk-020585 230 21 Pk-020781 Bahawalpur 190 6 Pk-020592 230 22 Pk-020783 Attock 430 7 Pk-020609 KPK Peshawar 500 23 Pk-020867 Lahore 290 8 Pk-020620 500 24 Pk-020868 290 9 Pk-020631 Punjab Narowal 290 25 Pk-020871 AJK Muzaffarabad 810 10 Pk-020646 KPK Kohat 510 26 Pk-020872 Punjab Mianwali 250 11 Pk-020654 Punjab Gujranwala 270 27 Pk-020873 Rawalpindi (MT) 530 12 Pk-020662 Ukrain 28 Pk-020874 Rawalpindi (Saddar) 521 13 Pk-020663 Pakistan 29 Pk-020875 Capital Islamabad (Aabpara) 550 14 Pk-020699 KPK D. I. Khan 230 30 Pk-020876 Islamabad (Karachi Co.) 550 15 Pk-020720 230 31 Pk-020877 Punjab Rawalpindi (Kohuta) 600 16 Pk-020729 GB Chilas 1450 32 Pk-020878 Lahore 290 Pk-020662 and Pk-020663 were received from United States of America; KPK, Khyber Pakhtun Khawa; D. I. Khan, Dera Ismail Khan; GB, Gilgit Baltistan; AJK, Azad Jummu & Kashmir; MT, Muslim Town. *Accessions enlisted are preserved in National Gene bank of Plant Genetic Resources Program, National Agricultural Research Center, Islamabad, Pakistan (Iqbal 2009a). For protein extraction, ten seeds were bulked and ground to fine powder in a mortar and pestle. Sample buffer (400 μl) was added to 0.01 g of seed flour as extraction liquid and mixed thoroughly in eppendorf tube with a small glass rod. The extraction buffer contained the following final concentrations 0.5 M Tris-HCl (pH 6.8), 2.5% SDS, 10% glycerol and 5% 2-mercaptoethanol. Bromophenol Blue (BPB) was added to the sample buffer as tracking dye to watch the movement of proteins in the gel. To purify extraction, the homogenate samples were mixed thoroughly by vortexing and centrifuged at 15,000 rpm for 5 min at room temperature. The extracted crude proteins were recovered as clear supernatant, transferred into new 1.5 ml Eppendorf tubes and stored at ‒20C until electrophoresis. PROTEOMICS VARIATION IN NIGELLA SATIVA L. 291 Total seed protein was analysed through slab type SDS-PAGE using 11.25% polyacrylamide gel following Laemmli (1970). The molecular weights of the dissociated polypeptides were determined by using molecular weight protein standards MW-SDS-70 Kit Sigma Chemical Company, USA following Iqbal et al. (2017). The electrophoresis box used was prepared by Auto Model AL 6500 (donated by JICA), Japan. Depending upon the presence and absence of polypeptide bands, similarity index was calculated for all possible pairs of protein types as 1/0 and data were in a binary data matrix. Based on results of electrophoretic band spectra, Jaccard’s similarity index (S) was calculated for all possible pairs of protein type electrophoregrams using the formula as of Sneath and Sokal (1973); S = W/A + B W, where W is the number of band of common mobility, A the number of bands in protein type A and B is the number of bands in protein type B. The similarity matrix thus generated was converted into a dissimilarity matrix (Dissimilarity = 1-similarity) and used to construct cluster pattern by UPGMA through “Statistica” package. Results and Discussion To get clear banding pattern and reliable results, different concentrations of gel were prepared and tested along with quantity of sample loading. SDS-PAGE of 11.25% polyacrylamide gel with 8 μl loading sample protein yielded best results and gave quality products. Slab type gel containing seed protein banding pattern was divided into three regions where most of the banding patterns were observed between first and second regions. Protein peptide bands with sharpness were present in all regions while third region possessed low intensity bands as compared to first and second region. Fig. 1 represents proteomic profile in 10 accessions at right side. In total 8 subunits were observed and out of them a few were polymorphic. Beside inter-accession, single seeds were also used to extract protein from individual accession which showed extremely low differences (Fig. 1). The visible bands were compared with the standard marker which showed that most of the bands were of between 66.0 to 45.0 Kda and 18.4 to 14.3 Kda of molecular weight. Accessions PK020561 (Lahore, Punjab), PK-020585 (Faisalabad, Punjab), PK-020592 (Faisalabad, Punjab), PK-020609 (Peshawar, Khyber Pakhtun Khawa), PK-020620 (Peshawar, Khyber Pakhtun Khawa) and PK-020646 (Kohat, Khyber Pakhtun Khawa) showed high band intensity and sharpness while there were an evidence of first and second bands of light intensity. In accessions, PK-020609, PK-020620, PK-020631 (Narowal, Punjab) and PK-020646 (Kohat, Khyber Pakhtun Khawa) 1-2 bands were observed clearly as compared to other accessions. On the basis of single seed protein peptide banding pattern very clear bands were observed, in accession number PK-020592, the intensity and sharpness of the bands were very low as compared to other accessions. Cluster pattern formed on the basis of proteomic profile divided into 5 clusters on 75% linkage distances (Fig. 2). Cluster 1 and Cluster-II consisted of one accession in each case, PK020878 (Lahore, Punjab) and PK-020877 (Kohuta, Rawalpindi, Punjab). Cluster-III consisted of two accessions PK-020871 (Muzaffarabad, Azad Jammu and Kashmir) and PK-020868 (Lahore, Punjab). Cluster IV consisted of two accessions PK-020783 (Attock, Punjab) and PK-020592 (Faisalabad, Punjab). Further, Cluster V consisted of 26 accessions of diverse origin, respectively. In the present study, intraspecific variation was limited among Nigella sativa L. accessions. Due to low genetic diversity for proteomic profile, two-dimensional electrophoresis is suggested to separate various proteins observed on gel. SDS-PAGE may be used for interspecific diversity and phylogenetic relationships among various species rather than intraspecific variation. Low genetic diversity may be attributed to the narrow genetic base of the crop, or because one possibility is that Nigella sativa L. spreaded throughout the world from the same origin. Javaid et 292 IQBAL AND GHAFOOR al. (2004) a