|Full Text: 1 Introduction 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 |
Table 1 Detail of cultivars under study 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.
Genotypes Seed coat colour State: Agro-ecological zone of adoption* Amrit, Nirmala, Uma Pale yellow Odisha: Sub-humid, Coastal OSC-207, OSC-593 White TKG-352, TKG-22 White Madhya Pradesh: Semi-arid, sub-humid DSS-09 White Karnataka: Arid, Semi-arid TMV-4, TMV-6, VRI-1 Brown Tamil Nadu: Semi-arid, Coastal Gujarat Til-2 (GT-2) White Gujarat: Arid, Semi-arid, Coastal CST-2001-12 White Haryana, Rajasthan: Arid RT-54, RT-348 Brown B-14, Tilottama (B-67), CUMS 3, V-1, V-15, Rama Brown West Bengal: Humid- per humid, Coastal CUMS 9, CUMS 11 Mixed colour  CUMS 17, NIC 8316, Saheb Pale yellow V -10, V-12 Deep Brown *Source: ICAR-Indian Agricultural Statistics Research Institute;  Mixed colours seeds included pale yellow, brown, deep brown
Table 2 Detail of polymorphic bands 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. 3 Results 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.
Band Position Molecular weight (KDa) Band Position Molecular weight (KDa) Band Position Molecular weight (KDa) 1 125.59 8 68.05 17 22.37 2 116.32 11 45.46 19 18.14 3 107.20 12 42.25 21 14.07 4 102.39 13 33.23 5 93.40 16 24.96
Table 3 Distribution of protein polymorphic bands for agro-ecological zone of adoption
Agro-ecological zone of adoption