Studies on Genetic diversity in chilli (Capsicum annuum L.) through SDS-PAGE protein profiling

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1 2017; 5(6): P-ISSN: E-ISSN: IJCS 2017; 5(6): IJCS Received: Accepted: Athikho Kayia Alice Rakesh Kr Dubey Indian Institute of Vegetable Research, Varanasi, Uttar AK Pandey Vikas Singh Regional Research Station, IIVR, Sargatia, Seorahi, Kushinagar, Uttar Pradesh, India Siddhartha Singh Correspondence Siddhartha Singh Studies on Genetic diversity in chilli (Capsicum annuum L.) through SDS-PAGE protein profiling Athikho Kayia Alice, Rakesh Kr Dubey, AK Pandey, Vikas Singh and Siddhartha Singh Abstract Study on genetic diversity was with 31 chilli (Capsicum annuum L.) genotypes collected from different regions of North-East India was carried out at College of Horticulture and. Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS-PAGE) was carried out to determine the protein banding patterns of 31 chilli genotypes. The genotypes showed considerable variation in band number of protein in present investigation which ranged from A total of 70 protein bands as per Rm values were identified by silver staining. Keywords: Genetic diversity; chilli; SDS PAGE, dendrogram; seed protein Introduction Chilli is grown for its pungent fruits, which are used both green and ripe/dry form. Chilli peppers cultivated in India are considered to be worlds famous for two important commercial qualities: pungency (hotness) and colour. Rich variability in morphological traits in hot pepper occurs throughout India particularly in south peninsular region, North Eastern foot hills of Himalayas and Gangetic plains (Pradheep and Veeraragavathatham, 2006) [19]. Although chillies are cultivated throughout the country, North-eastern states are home to the genetic variability where several interspecific hybrids/derivatives were originated, among which Naga king chilli is one of the world s hottest pepper (Dhaliwal et al., 2014) [5]. The North-eastern region comprising the states of Arunachal Pradesh, Assam, Manipur, Meghalaya, Mizoram, Nagaland, Sikkim and Tripura which is located between N and E. Chilli is grown in almost all the parts of NEH region and most of them are local cultivars or landraces. Landraces are variable plant populations adapted to local agro climatic conditions, which are locally named, selected and maintained by the traditional farmers to meet their social, economic, cultural and ecological needs (Teshome et al., 1997) [22]. The chilli landraces have been selected by farmers for agronomic and horticultural traits important to them (e.g., fruit size, heat level, colour and early maturity) and as a result of natural selection, are well adapted to the specific environment to this region (Yatung et al., 2014) [23]. Selection of parents depends on specific objective of the research programme and their performance. In crops improvement programme, genetic diversity is one of the important tools to quantity genetic variability in both cross and self-pollinated crops (Gaur et al., 1978) [7]. In order to benefit transgressive segregation, the knowledge of genetic distance between parents is necessary (Joshi et al., 2004; Khodadadi et al., 2011) [10]. Electrophoresis of seed proteins is based on the concept that each genotype/cultivar is distinct and relatively homogenous at the genetic level. Seed protein electrophoresis is frequently employed as an additional approach for species identification and as a useful tool for back-tracking the evolution of various groups of plants (Ladizinski and Hymowitz, 1979) [13]. Seed protein profiling is generally used for studying genetic diversity and phylogenetic relationship among the chilli varieties. As, seed proteins have the advantage of being scorable from unviable organs or tissues and the electrophoretic protocol for bulk protein is generally simpler than that for isozymes (Cook, 1984) [2]. Among the biochemical techniques, Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) is economical, simple and provides the best resolution in the identification of germplasm by protein patterns and extensively used technique for describing ~ 465 ~

2 the seed protein diversity of crop germplasm (Cook, 1995; Das and Mukherjee, 1995; Fufa et al., 2005; Iqbal et al., 2005) [3, 4, 6, 8]. In the present study 31 chilli genotypes from different regions of NEH were collected and cultivated with standard package of practices at College of Horticulture and Pradesh and the diversity analysis was carried out using seed protein profiling through SDS-PAGE. Materials and Methods Plant Materials Thirty one chilli (Capsicum annuum L.) genotypes (Table 1) were collected from different North-East region of India and cultivated in the research farm of College of Horticulture and. Table 1: List of chilli genotypes with their sources of collection. Genotype Source CHFC-31 A landrace of Pasighat (Arunachal Pradesh) CHFC-32 A landrace of Pasighat (Arunachal Pradesh) CHFC-33 A landrace of Pasighat (Arunachal Pradesh) CHFC-34 A landrace of Pasighat (Arunachal Pradesh) CHFC-35 A landrace of Mao-gate (Manipur) CHFC-36 A promising genotype of Guwahati (Assam) CHFC-37 A landrace of Pasighat (Arunachal Pradesh) CHFC-38 A landrace of Pasighat (Arunachal Pradesh) CHFC-39 A landrace of Guwahati market (Assam) CHFC-40 A landrace of Mao-gate (Manipur) CHFC-41 A landrace of Choinu village (Manipur) CHFC-42 A landrace of Choinu village (Manipur) CHFC-43 A landrace of Chandel District (Manipur) CHFC-44 A promising genotype of Local (Sikkim) CHFC-45 A landrace of Mayangkhang village (Manipur) CHFC-46 A promising genotype of local (Tripura) CHFC-47 A landrace of Pasighat (Arunachal Pradesh) CHFC-48 A landrace of Pasighat (Arunachal Pradesh) CHFC-49 A landrace of Pasighat (Arunachal Pradesh) CHFC-50 A landrace of Pasighat (Arunachal Pradesh) CHFC-51 A landrace of Senapati (Manipur) CHFC-52 A landrace of Maram (Manipur) CHFC-53 A landrace of Mao-gate (Manipur) CHFC-54 A landrace of Senapati (Manipur) CHFC-55 A landrace of Pasighat (Arunachal Pradesh) CHFC-56 A promising genotype of Guwahati (Assam) CHFC-57 A promising genotype of Imphal (Manipur) CHFC-58 A promising genotype of Senapati (Manipur) CHFC-59 A landrace of Pasighat (Arunachal Pradesh) CHFC-60 A landrace of Local (Tripura) CHFC-61 A promising genotype of Itanagar (Arunachal Pradesh) Extraction of total seed protein Seeds of thirty one chilli genotypes evaluated in the field were collected and 0.1 g seed was taken in pestle and mortar and added 25μL of buffer (0.06 M Tris -HCl, 2.5% Glycerol, 0.5% SDS, 1.25% β-mercaptoethanol, 0.1% TCA, 10 mm urea, 1 mm EDTA). The sample was homogenized and heated in a boiling water bath for 5 min at 100 C for denaturation of proteins. The protocol followed in the present study was in accordance with Kumar and Tata (2010) [11] with some modifications. Protein Estimation Estimation of protein was done as Lowry s method (1951). Seeds of thirty-one germplasm lines were collected and subjected to gel electrophoresis. A standard curve of absorbance at 660 nm versus µg of BSA was drawn and from this standard curve, the amount of protein in the sample tube was determined as protein per gram of the sample. Sodium Dodecyl Polyacrylamide Gel Electrophoresis (SDS-PAGE) Polyacrylamide gel electrophoresis in presence of denaturing agent (SDS) was carried out as per procedure described by Laemmli (1970) [14] with some modifications. The soluble seed proteins were subjected to SDS-PAGE in gel slabs of 1mm thickness (5% stacking and 10% separating gels). Electrophoresis was performed with a discontinuous buffer system in a vertical electrophoresis unit. The gel was run at 25 ma until the tracking dye approached the bottom of the gel. The system was disconnected and the gel was taken out from the slab. The gel was then subjected to silver staining as per procedure described by Mortz et al. (2001) [16] through sensitizing with 0.02% sodium thiosulphate solution for 5 minutes and then washing twice with double distilled water for 1 minute. It was then transferred to staining solution and kept on gel rocker for 20 minutes in dark. The gel was then washed twice with distilled water for 45 seconds, transferred to developing solution and finally the reaction was stopped with 12% acetic solution. Gel was washed thoroughly but gently with double distilled water until protein bands became clearly visible for bands scoring. The electrophorograms were prepared on the basis of protein mobility and the density expressed in Rm values. Statistical analysis The gels were scored as presence (+) or absence (-) of protein polypeptide bands. Depending upon the presence or absence of polypeptide bands, similarity index (SI) (Nei and Li, 1979) [17] between the genotypes was calculated by the following formula: SI= ( 2Z ) X 100 X+Y Where, Z= Number of similar bands between the genotypes, and X+Y =Total number of bands in the two genotypes compared. Cluster analysis UPGMA (Unweighted pair group method with arithmetic mean analysis) by using statistical software SPSS for windows package (Version 14). Results Cluster analysis of banding pattern of thirty-one genotypes based on similarity index and UPGMA resulted in distinct clusters (Fig.1). Protein distribution patterns in thirty-one genotypes of chilli were studied and summarized in table 2. Based on the dendrogram, all the genotypes could be grouped into 2 major clusters. The first cluster contained the genotypes CHFC-31, CHFC-32, CHFC-33, CHFC-34, CHFC-35, CHFC-36, CHFC-37, CHFC-38, CHFC-39, CHFC-40, CHFC-51, CHFC-52, CHFC-53, CHFC-54, CHFC-55, CHCF-56, CHFC-57, CHFC-58, CHFC-59, CHFC-60 and CHFC-61. Second cluster was found to be incorporating 10 genotypes viz., CHFC-41, CHFC-42, CHFC-43, CHFC-44, CHFC-45, CHFC-46, CHFC-47, CHFC-48, CHFC-49 and CHFC-50. ~ 466 ~

3 Fig 1: UPGMA of thirty-one chilli genotypes based on total Seed protein profiles obtained by SDS-PAGE A total of 70 protein bands as per R m values were identified by silver staining (Table 3). The genotypes showed considerable variation in protein band number ranging from Among the genotypes CHFC-51 showed maximum number (18) of protein bands while the minimum number (8) of bands was present in genotypes CHFC-37. Band number 1 (R m =0.30) was present in genotype CHFC-41, CHFC-42, CHFC-43, CHFC-44, CHFC-45, CHFC-46, CHFC-47, CHFC-49 and CHFC-50 only. Band number 4 (R m =0.121), 7 (R m =0.161), 21 (R m =0.395), 27 (R m =0.444), 30 (R m =0.484), 35 (R m =0.556), 38 (R m =0.589), 40 (R m =0.605), 47 (R m =0.685), 50 (R m =0.726), 52 (R m =0.750), 54 (0.766), 59 (R m =0.823) and 61 (R m =0.847) were present in both the genotype CHFC-56 and CHFC-57. Band number 16 (R m =0.343) and band number 66 (R m =0.939) were exclusively present in genotype CHFC-48. Band number 17 (R m =0.347) and band number 24 (R m =0.427) were exclusively present in genotype CHFC 51. Band number 26 (R m =0.442) in genotype CHFC-40, band number 45 (R m =0.646) in genotype CHFC- 42 and band number 70 (R m =0.968) in genotype CHFC-61 were solely present. Band number 59 (R m =0.823) and band number 64 (R m =0.895) was present in maximum (11) number of genotypes. Table 2: Major cluster produced by SDS-PAGE analysis in thirty-one chilli genotypes Cluster Sub cluster Sub-sub cluster I A 1 I A I A 2 I I B 1 I B I B 2 II A II II B Genotypes CHFC-51, CHFC-52, CHFC-53, CHFC-54, CHFC-55, CHCF-56 and CHFC-57 CHFC-58, CHFC-59, CHFC-60 and CHFC-61 CHFC-36 and CHFC-37 CHFC-31, CHFC-32, CHFC-33, CHFC-34, CHFC-35, CHFC-38, CHFC-39 and CHFC-40 CHFC-47, CHFC-48, CHFC-49 and CHFC-50 CHFC-41, CHFC-42, CHFC-43, CHFC-44, CHFC-45 and CHFC-46 Table 3: Comparision of scorable protein bands among 31 chilli genotypes S. Rm C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C No value ~ 467 ~

4 Total The genetic distance coefficient matrix utilizing SDS-PAGE analysis for the thirty one genotypes of chilli under study has been presented in table 4 and the dendrogram generated with matrix value has been presented in Fig.1. Genetic distance generated by SDS-PAGE analysis in the germplasm under study ranged from to Values of genetic distance coefficient matrix suggested least genetic distance of genotype CHFC-56 with CHFC-57 (0.000) while genotype CHFC-50 was found to have maximum genetic distance with genotype CHFC-52 as evident by coefficient value Table 4: Genetic distance estimates among 31 genotypes of chilli using SDS-PAGE analysis. Genotypes CHFC CHFC CHFC CHFC ~ 468 ~

5 CHFC CHFC CHFC CHFC CHFC CHFC CHFC CHFC CHFC CHFC CHFC CHFC CHFC CHFC CHFC CHFC CHFC CHFC CHFC CHFC CHFC CHFC CHFC CHFC CHFC CHFC CHFC Discussion Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS-PAGE) is an economical, simple and extensively used biochemical technique for describing the seed protein diversity of crop germplasm (Cook, 1995; Das [3, 4, and Mukarjee, 1995; Fufa et al., 2005; Iqbal et al., 2005) 6, 8]. Seed protein variants have been observed to be the most widely used biochemical genetic markers during the last quarter century. Its success depends upon the polymorphism of seed and seedling proteins and the fact that these proteins represent primary gene products and are largely unaffected by the environmental interactions (Smith and Smith, 1992) [21]. Caution should be taken in interpreting darkness and thickness as the kind of variation may be due to the lack of separation on the gels of several proteins having similar migration rates and studies are required to estimate the number of genes causing quantitative variation in seed protein bands (Ladizinsky and Haymowitz, 1979) [13]. The Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS-PAGE) was carried out to determine the protein banding patterns of 31 chilli genotypes. The genotypes showed considerable variation in band number of protein in present investigation which ranged from 8-18 which is in concordance with the reports of Anu and Peter (2003) [1], Singh et al. (2009) [20], Kumar and Tata (2010) [11], Kumar et al. (2010) [12], Yatung et al. (2014) [23] and Peddakasim et al. (2015) [18]. Among the genotypes CHFC-51 showed maximum number (18) of protein bands while the minimum numbers (8) of bands were present in genotype CHFC-37. The genetic distance estimates among 31 genotypes of chilli using SDS-PAGE analysis showed a maximum degree of genetic distance exhibiting value of between genotype CHFC-50 (A landrace of Pasighat, Arunachal Pradesh) and CHFC-52 (A landrace of Maram, Manipur). Further, the genotype CHFC-56 (A promising genotype of Guwahati, Assam) showed minimum genetic distance with genotype CHFC-57 (A promising genotype of Imphal, Manipur) as evident by lowest value of genetic distance i.e Present findings can be supported by the results of Anu and Peter (2003) [1], Fufa et al. (2005) [6], Kumar and Tata (2010) [11], Kumar et al. (2010) [12] and Yatung et al. (2014) [23]. Conclusion The thirty one genotypes of chilli (Capsicum annuum L.) under study were grouped into six clusters irrespective of their origin. Based on the present study the genotypes CHFC- 50 (A landrace of Pasighat, Arunachal Pradesh) was most distantly related to CHFC-52 (A landrace of Maram, Manipur). Hence, it was recommended that these two genotypes (CHFC-50 and CHFC-52) could be utilized for crossing programme to create more genetic diversity or segregants of desired characteristics through chilli breeding programmes. Although SDS-PAGE analysis could show discrete variation among few genotypes of chilli under study, this protein marker should be applied in future to more number of genotypes to arrive at a reasonable conclusion. Therefore, seed protein electrophoresis could be proved to be a successful technique in certain cases to distinguish morphologically indistinguishable genotypes. SDS-PAGE is a predominant tool to study molecular systematic for identification of genotypes based on proteins and a promising method to discriminate between morphologically similar chilli genotypes in a limited way. Acknowledgements The authors are thankful to Department of Biotechnology, Govt. of India for providing facilities under the project IBT Hub at Forestry, Central Agricultural Pradesh. References 1. Anu A, Peter KV. Analysis of seed protein of 29 lines of Capsicum annuum L. by polyacrylamide gel electrophoresis. Genetic Resources and Crop Evolution. 2003; 50(3): Cook RJ. The characterization and identification of crop cultivars by electrophoresis. Electrophoresis. 1984; 5: ~ 469 ~

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