Volume 6, Issue 1, February Issue - 2018, Pages:243-248
|Authors: Arna Das, Sarita K. Pandey, Pradipta Bhattacharya, T. Dasgupta|
|Abstract: Seed storage protein markers being less sensitive to environmental fluctuation than phenological traits, has been successfully employed in assessing divergence in many crop plants. The present study was aimed to find out correlation of seed storage protein markers in twenty eight Indian sesame cultivars with their agro-ecological zone of adoption and their seed coat colour. Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS-PAGE) revealed altogether twenty two protein bands of which thirteen were polymorphic with varied molecular weights. Specific bands, relating to specific agro-ecologies were found. Moreover, bands of 93.40 KDa and 68.05 KDa were found associated with production of darker shades of seed coat colour. Clustering pattern based on protein similarity value offered no definite grouping, either to specific agro-ecological zones of adoption or to specific seed coat colour. It is concluded that individual protein banding pattern can be linked to agro-ecological adoption zone and seed coat colour which is helpful in divergence and phylogenetic study in sesame.|
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Sesame (Sesamum indicum L.) as an oilseed is potent to meet the domestic demand of edible oil in India. Though India is one of the major sesame producers in the world (www.faostat.fao.org - 2016), but this crop has been highly neglected and identified as an orphan crop. This resulted into enormous loss of germplasm and drastic reduction in variation. An insight into characterization and preservation of naturally existing variation in sesame, therefore, has become a necessity for further improvement of the crop. Phenological traits, due to pleiotropic effect and polygenic control, exhibit overlapping variation within and between species populations offering taxonomic complexity (Huber-Morath & Phlomis, 1982; Wang et al., 2010; Pabby & Rockman, 2013). Soluble seeds storage protein markers assessed through Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS-PAGE) had been successfully employed to characterize cultivated varieties in several crop plant species, of which Mung (Ghaffor et al., 2002), Pea (Jha & Ohri, 2002), Einkorn wheat (Alvarez et al., 2006), Brassica (Khurshid & Rabbani, 2012) are few to name. Reliability of soluble seed storage protein markers lies in the fact that these are less influenced by environmental fluctuation as compared to phenological markers, therefore stable, uniform and reproducible. Moreover SDS-PAGE takes less time, is simpler to perform and is more economic than nucleic acid markers.
Only a few references are available on soluble seeds storage protein polymorphism study involving Indian sesame germplasm. Akhila & Beevy (2011) reported the presence of fourteen bands in a study of seven sesame genotypes including wild and cultivated species. The protein polymorphism was able to group the genotypes belonging to two different species into separate clusters. Das et al. (2013) reported the presence of twenty two bands in a study with twenty six advanced sesame mutant lines and respective controls. But the study did not offer much specificity in grouping the mutants according to their parental origin. Dar et al., (2014) reported as much as twenty one bands in a study with fifty two Indian sesame germplasms. Similarly, Singharaj & Onsaard (2015) carried out SDS-PAGE in sesame genotypes with varied seed coat colour, for its food value. According to recent study, seed coat colour in sesame is more helpful in phylogenetic study than geographic origin of genotypes (Zhang et al., 2013).
In the present research work an attempt was made to search for correlation between soluble seed storage protein polymorphism of a number of Indian sesame cultivars with the cultivars’ agro-ecological zone of adoption and also with cultivars’ seed coat colour, which would definitely incite more knowledge on divergence and phylogeny in sesame. Correlation of protein markers with seed coat colours would further help in assessing genotypes for those biochemical traits which are linked to particular seed coat colours (Zhang et al., 2013). Such correlation, if exists, can be employed to identify diverse and superior sesame genotypes for future crop improvement programme.
2 Materials and methods
*Source: ICAR-Indian Agricultural Statistics Research Institute;  Mixed colours seeds included pale yellow, brown, deep brown
Twenty eight sesame cultivars from different states in India representing varied agro-ecology had been selected for the study. The detail of the genotypes is presented in Table 1. Seeds of these cultivars were availed from the sesame germplasm collection of the Department of Genetics and Plant Breeding, Institute of Agricultural Science, University of Calcutta, West Bengal, India.
Soluble seed storage protein was extracted and estimated following Lowry‘s method (Lowry et al., 1953). SDS-PAGE (12% separating gel and 4% stacking gel) of those extracted protein was carried out following the method of Laemmli (1970) in a regular mini (10 cm x 10 cm) vertical gel system (Biotech Laboratories, India) applying a current of 30 mA for run through stacking gel and a current of 50 mA for run through separating gel. Standard marker protein, Dalton Mark VI (Sigma, USA) was used as a control. Staining and de-staining of the gel were also carried out following the method of Laemmli (1970). The banding pattern was captured through gel documentation unit (UVP Gel Doc It) and molecular weights (MW) of observed protein bands were then estimated through the Life Sciences Software available with the gel documentation unit. A matrix was prepared by giving a score of ‘1’ for presence and ‘0’ for absence of a particular protein band. This was carried out for all the observed protein bands for all twenty eight genotypes. A dendrogram was prepared based on protein similarity (PS) value of the genotypes with the help of the software NTSYS pc ver 2.20 (Rohlf, 2005). Protein similarity data were generated by the program, SIMQUAL and the dendrogram was generated through SAHN program. Protein dissimilarity (PD) was estimated as 1 - PS.
The SDS-PAGE banding pattern for soluble seed storage protein revealed a total of twenty two bands covering all the twenty eight genotypes as reported earlier by the authors (Das et al., 2013) in their previous study on seed storage protein polymorphism involving sesame mutants and their parents. The heaviest band at the top of the gel with molecular weight of 125.59 KDa was marked as one (1), the lightest polypeptide was the 22nd band of 12.19 KDa found at the bottom. All the other polypeptides found in between these two bands had molecular weights ranging in between 125.59 KDa and 12.19 KDa. Sigma, Dalton Mark VI gave six marker bands of molecular weights 66 KDa, 45 KDa, 34.7 KDa, 24 KDa, 18.4 KDa and 14.4 KDa respectively, distributed from top to bottom of the gel. Marker band of 45 KDa divided all the bands almost at the mid-way. Ten bands appeared above 45 KDa band and twelve below. Besides, variation in total number of bands was observed in different genotypes offering seed storage protein polymorphism of 59.1%. A zymogram for ten genotypes is given in Figure 1, along with marker bands. Seven protein bands, namely, band no. 6, 7, 9, 10, 14, 15, 18, 20 and 22, out of these twenty two bands were monomorphic. The other thirteen bands, namely, band no. 1, 2, 3, 4, 5, 8, 11, 12, 13, 16, 17, 19 and 21 exhibited polymorphism. The detail of the polymorphic bands is given in Table 2.
Akbar F, Yousaf N, Ashiq Rabbani M, Khan Shinwari Z, Shahid Masood M (2012) Study of total seed proteins pattern of sesame (Sesamum Indicum L.) landraces via sodium dodecyl sulfate polyacrylamide gel elecrophoresis (SDS-PAGE). Pakistan Journal of Botany 44:2009-2014.
Akhila H, Beevy S (2011) Morphological and seed protein characterization of the cultivated and the wild taxa of Sesamum L. (pedaliaceae). Plant Systematics and Evolution 293: 65-70.
Alvarez JB, Moral A, Martin LM (2006) Polymorphism and genetic diversity for the seed storage proteins in Spanish cultivated einkorn wheat (Triticum monococcum L. ssp. monococcum). Genetic Resources and Crop Evolution 53 :1061-67.
Dar AA, Roy Choudhury A, Arumugam N (2014) A study on seed protein profile of Indian cultivars of Sesamum indicum L. International Journal of Current Biotechnology 2:10-17.
Das A, Pandey S, Dasgupta T (2013) Genotype-environment interaction in sesame (Sesamum indicum L.) genotypes and identification of stable types. In: Proceedings of International conference on Agri & Animal Sciences, 8-9 July 2013; Colombo, Sri Lanka. International Center for Research and Development, Pp. 43 – 72.
FAOSTAT (2016) Food and Agriculture Organization of the United Nations [online] Available at: http://faostat.fao.org/site/339/default.aspx, accessed on December 12, 2016.
Ghafoor A, Ahmad Z, Qureshi A, Bashir M (2002) Genetic relationship in Vigna mungo (L.) Hepper and V. radiata (L.) R. Wilczek based on morphological traits and SDS-PAGE. Euphytica 123:367-78.
http://vikaspedia.in/InDG Available at: http://vikaspedia.in/agriculture/crop-production/weather-information/agro-climatic-zones-in-india, accessed on December 12, 2016
Huber-Morath A, Phlomis L (1982) Flora of Turkey and East Aegean Island. In: Davis PH (Ed.), Edinburgh University Press, Edinburgh, Pp.102-126.
Jha SS, Ohri D (2002) Comparative study of seed protein profiles in the genus Pisum. Biologia Plantarum 45:529-32.
Kanu PJ (2011) Biochemical analysis of black and white sesame seeds from China. American Journal of biochemistry and Molecular Biology 1:145-57.
Khurshid H, Rabbani MA (2012) Comparison of electrophoretic protein profiles from seed of different oilseed Brassica cultivars. Journal of Public Health and Biological Sciences 1:36-42.
Laemmli UK (1970) Cleavage of structural proteins during the
Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1953) Protein measurement with the folin-phenols reagent. Journal of Biological Chemistry 193:265-75.
Pabby AB, Rockman MV (2013) The many faces of pleiotropy. Trends in Genetics 29: 67-73.
Pham TD, Bui TM, Werlemark G, Bui TC, Merker A, Carlsson AS (2009) A study of genetic diversity of sesame (Sesamum indicum L.) in Vietnam and Cambodia estimated by RAPD markers. Genetic Resources and Crop Evolution 56:679–690.
Rohlf FJ (2005) Numerical Taxonomy and Multivariate Analysis System, Version 2.20, Applied Biostatistics, New York.
Singharaj S, Onsaard E (2015) Production and characteristic of sesame proteins. Journal of Food Science and Agricultural Technology 1:188-192.
Stankiewicz M, Gadanski G, Gawronski SW (2001) Genetic variation and phylogenetic relationships of triazine resistant and triazine susceptible biotypes of Solanum nigrum analysis using RAPD markers. Weed Research 41: 287–300.
Wang Z, Liao BY, Zhang J (2010) Genomic patterns of pleiotropy and the evolution of complexity. Proceedings of The National academy of Sciences 107:18034-18039.
Wei LB, Zhang HY, Zheng YZ, Guo WZ, Zhang TZ (2008) Development and utilization of EST-derived microsatellites in sesame (Sesamum indicum L.). Acta Agronomica Sinica 34: 2077−2084.
Zhang H, Hongmei M, Libin W, chun L, Zhao R, Wang (2013) Genetic analysis and qtl mapping of seed coat color in sesame (Sesamum indicum L.). PLoS ONE 8: e63898. doi:10.1371/journal.pone.0063898.