IJCRR - 3(11), November, 2011
Pages: 128-137
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GENETIC DIVERSITY ANALYSIS IN FIVE ACCESSIONS OF TRIGONELLA USING CYTOLOGICAL
STUDY, PROTEIN ESTIMATION AND SDS-PAGE
Author: Vaseem Raja, Jahangir Ahmad Dar, Rajdeep Kudesia, Manoj Srivastava
Category: General Sciences
Abstract:Genetic variation of cultivars is very interesting in reducing genetic vulnerability as well as
stabilizing production. In this regard, a study was undertaken to evaluate the genetic diversity
among five accessions of Trigonella viz., IC-143851 (A1), IC-144225 (A2), IC-332236 (A3),
IC-371755 (A4) and IC-433589 (A5). For the first assay, mitotic index and protein estimation
were evaluated. A1 accession had the highest mitotic index (15.40%), while A4 accession had
the lowest (6.83%). Highest and lowest protein contents were observed in case of A3
(31.0\?1.16) and A5 (23.2\?0.80) accessions, respectively. Total seed storage protein profiles
were examined using SDS-PAGE. The proteins were resolved in 27 bands with 24 polymorphic
peptides. The similarity coefficient calculated on the basis of presence and absence of bands
ranged from 0.23-0.55. Following the UPGMA algorithm of similarity coefficients, the
accessions could be clustered into two similarity groups. Cluster 1 consisted of two accessions
(A1 and A3) while the cluster 2 grouped rest of the accessions (A2, A4 and A5). Clustering
based on seed storage protein profiles provides information about the phylogenetic relationship
of accessions as all the accessions have at least one or more unique seed storage protein marker
that can separate them from one another.
Keywords: Genetic Diversity , SDS PAGE, Mitotic index, Trigonella Protein profile.Phylogenetic
Full Text:
INTRODUCTION
The family Fabaceae, or bean family, is the second big family of flowering plants in the world with 650 genera and18, 000 species (Rakhee et al., 2004). It includes many crops useful for food, forage, fiber, wood and ornamental purposes. Few members of the family such as chickpea, soybean, fababean, fenugreek, lentil, pea etc. are consumed as grain legumes. The grain legumes are plants used as food in the form of unripe pods, mature seeds or immature dry seeds, directly or indirectly (Rachie and Roberts, 1974). The genus Trigonella is one of the largest genera of the tribe Trifoliatae in the family Fabaceae and sub-family Papilionaceae (Balodi and Rao, 1991), represented by about 110 species in the world (Sechmen et al., 1998). Among Trigonella species, Trigonella foenum graecum (commonly known as fenugreek) is a flowering annual, with autogamous white flowers occasionally visited by insects. Indigenous to countries on the eastern shores of Mediterranean, fenugreek is widely cultivated in India, Egypt, Ethiopia, and Morocco and occasionally in England (Polhil and Raven, 1981). Fenugreek is extensively grown in the tropical and subtropical regions of India during winter season for its seeds, tender shoots and fresh leaves. The current productivity of fenugreek is 1245 kg/ha. The value added products of fenugreek such as fenugreek seeds, fenugreek powder and oleoresins are exported to Europe, North America, South Africa and some Asian countries (Malhotra and Vahishtha, 2008). Traditionally it is consumed as fresh vegetable and as a spice to add flavor to the Indian cuisines. Fenugreek is gaining importance due to its rare medicinal properties (Sharma et al., 1990). According to Ayurveda, fenugreek is herbal drug that is bitter or pungent in taste. It is effective against anorexia and is a gastric stimulant (Rajagopalan, 2001). Diosgenin, a steroidal saponin present in its seeds has been shown to induce apoptosis in a variety of tumor cells (Shishodia and Aggarwal, 2006). Aggarwal and Shishodia (2006) cloned and characterized a small cystine -rich peptide which has antifungal properties in nature (Olli et al., 2007; Olli and Kirti, 2006). Fenugreek seed powder has been shown to demonstrate antidiabetic effect by stabilizing glucose homeostasis and carbohydrate metabolism in type-1 diabetes (Preet et al., 2006).
Across the world only known and welldefined cultivars are grown in specific areas. Gene banks also harbor scanty germplasm collection of Trigonella species (Hymowitz, 1990). The neglected and the underuse status of these locally important crops indicates a risk of disappearance of important plant material developed over thousands of years. One of the important factors restricting the development of better varieties and their large-scale production is that very little information is available about the genetic diversity, inter- and intraspecific variability and genetic relationship among these species. Therefore, attempts to analyze possible untapped genetic diversity becomes extremely essential for breeding and crop improvement.
Sodium Dodecyl Sulphate - Polyacrylamide Gel Electrophoresis (SDSPAGE) is used for its validity and simplicity to describe genetic structure of crop germplasm, but its implication has been limited mainly to cereals because of less polymorphism in most of the legumes (Ghafoor et al., 2002). Seed protein analysis by SDS-PAGE is a tool to understand the genetic diversity at protein level among the genotypes. It is less expensive, reproducible, reliable and efficient method. Seed protein markers are widely used for the identification of varieties (Cooke 1984) of agricultural and horticultural crops. The validity and simplicity of seed protein profiling has been well documented (Cooke 1988). High stable and reliable seed protein profiling make it a powerful tool in elucidating the taxonomic and evolutionary problems of cultivars (Ladizinsky et al., 1979; Das and Mukarjee, 1995). Researchers have used seed protein profiling by SDS-PAGE as genetic marker system since many years. The use of genetic and seed protein marker can be used to select elite accessions collected from different agro-climatic regions for crop improvement programs. Comparative studies on the proteomic data in leguminous species has been reported (Yasmin et al., 2010). The present study was initiated to study the genetic diversity based on seed protein profiling across the selected accessions.
level (Al-Wadi and Gamal, 2007; Bhat and Kudesia, 2011). Martin et al. (2011) found that some endemic species of Trigonella in Turkey have higher mitotic index by virtue of the fact that they are widespread while other endemic species are restricted to some localities only because of their lower mitotic index. Total protein content in the seeds of Trigonella varied from 23.2-31.0 mg/g with a mean value of (27.04 mg/g.). The lowest protein content was found in A5 accession and the highest protein content was recorded in case of A3. A narrow range of variation was observed in rest of the accessions (Table 1). Our results are in consistency with the findings of Makaii et al. (2004) who revealed 30-32% protein in case of Trigonella. Sammour et al. (2007) also revealed genetic diversity on the basis of protein estimation in Latharus sativa and found the same result. The difference observed in case of the protein content may be attributed to the environmental factors such as geographical area, season of collection, elevation and annual temperature, precipitation and soil fertility (Vargas et al., 2000) and thus expression of different genes. More studies are needed to determine the effects of the environment on the amount of the total seed proteins in seeds of plants.
Electrophoresis of proteins is a powerful tool for population genetics (Parker et al., 1998). The most commonly used proteins are seed storage proteins, which are known to be polymorphic with respect to size, charge, or both these parameters (Cooke, 1984; Martinez et al., 1997). Germplasm characterization based on morphological traits is not up to the mark and requires confirmation at molecular or at least at protein level. Electrophoresis of proteins is a powerful tool for detection of the genetic diversity and the SDS-PAGE of seed protein is particularly considered a reliable technology because seed storage proteins are highly independent of environmental fluctuations (Iqbal et al., 2005; Javid et al., 2004). Genetic diversity of Trigonella germplasm elucidated through SDS-PAGE of proteins from seeds revealed distinct electrophoretic patterns. Twenty seven bands ranging from 50.0 to 97.0 kDa were recognized among five accessions, three were monomorphic (Table 2, Figure 1). The genotypes showed considerable variation in protein band number ranging from 12 to 17. Out of 27 peptide bands 24 bands were polymorphic with 88.88% polymorphism. Our finding reveals that considerable intra-specific variation was available in the analyzed accessions. The variation in the major bands was present in case of A1 and A5, where as the accessions showed variations for the minor bands. Band number 5 (93kDa), 7 (89 kDa) and 27 (50 KDa) were common in all the accessions. Polypeptide band number 8 (87 kDa, A1), 12 (78 kDa, A1) 18 (69 kDa, A2), 21 (65 kDa, A4), 23 (59 kDa, A4), 24 (58 kDa, A3) and 26 (56 kDa, A5) were accession specific. Band number 20 (67 kDa) was absent only in case of A1. Species specific bands may be exploited for hybrid identification in breeding experiments (Maity et al., 2009).
The cluster analysis performed using UPGMA revealed two distinct clusters as evident from dendrogram (Figure 2) constructed from Jaccards similarity matrix. Cluster 1 consisted of two accessions (A3 and A1) while the cluster 2 grouped rest of the accessions (A2, A4 and A5). Clustering based on seed storage protein profiles provides information about the phylogenetic relationship of genotypes as all the genotypes have at least one or more unique seed storage protein marker that can separate them from one another and also from other Trigonella genotypes. The results are in consistence with Hameed et al. (2009).
The SDS-PAGE results revealed that the total amount of polymorphism accounted for principal component was 88.88% which revealed a considerable genetic diversity among the studied accessions. Our results are in consistence with that of Landizinsky (1979), who found genetic diversity among three species of fenugreek based on seed protein profiles. The variability within the investigated accessions agrees with previous biochemical studies (Chowdhury and Slinkard, 2000; Tadesse and Bekele, 2001, 2004). Various reports on the same line are present from the previous investigations. Sammour et al. (2007) used SDS-PAGE technique in Latharus sativa and found 72.72% polymorphism in case of seed proteins .The electrophoretic analysis of seed proteins in the Trigonella accessions revealed a considerable intraspecific variation. This observation is consistent with the electrophoretic data of Latharus sativa (Przybylska et al., 1998). Marked protein polymorphism may be explained by the presence of out crossing in this self-pollinated species as has been augmented by Chowdhury and Slinkard (1997) in case of Latharus sativa. Kaumar and Tata (2010) found 85.57 % polymorphism in case of chilli peppers. Present protein profiles of the selected experimental accessions of Trigonella revealed that accessions A4 and A2 are very close to each other at molecular level. These accessions almost possess same similarity index of about (0.50), (Table 3). Seed protein patterns can be used as a promising tool for distinguishing cultivars of particular crop species (Jha and Ohri, 1996; Mennella et al., 1999). However, only few studies indicated that cultivar identification was not possible with the 133 International Journal of Current Research and Review www.ijcrr.com Vol. 03 issue 11 November 2011 SDS-PAGE method (Ahmad and Slinkard, 1992; De Vries, 1996). The SDS-PAGE is considered to be a practical and reliable method for species identification (Gepts, 1989).
ACKNOWLEDGEMENTS
The authors are very thankful to NBPGR, New Delhi for providing the germplasm of fenugreek. The authors acknowledge the help provided by Dr. Manoj Srivastava, Senior scientist at IGFRI, Jhansi.
References:
1. Aggarwal, B.B. and Shishodia, S. (2006) Molecular targets of dietary agents for prevention and therapy of cancer. Biochem Pharmacol. 71 (10):1397–1421
2. Ahmad, F. and A.E. Slinkard. 1992. Genetic relationships in the genus Cicer L., as revealed b L., by SDSPAGE and AE-HPLC. Seed Science and Technology., 27: 23-35.
3. Akan, H., Ekici, M. and Aytaç, Z. (2005). The endemic species of Trigonella and their conservation In Turkey. XVII. International Botanical Congress, Vienna :( 17-23 July), Abstracts. p. 611.
4. Al.Wadi, H.M.and Gamal, M.A. (2007). Palynological and cytological characters of 3 species of genus solnum (family solaanaceae) from Saudi Arabia.Jour.of.Bio.Sci.4:626-631.
5. Balodi B, Rao RR (1991) The genus Trigonella L. (Fabaceae) in the Northwest Himalaya. J Econ Tax But , 5(1):11-16.
6. Bhat, T.M. and Kudesia, R. (2011). Evaluation of Genetic Diversity in Five Different Species of Family Solanaceae using Cytological Characters and Protein Profiling. GEBJ.20:
7. Chowdhury, M. A. and Slinkard, A.E (1997). Natural outcrossing in grasspea .j. hereditary. 88:154- 156.
8. Chowdhury, M. A. and Slinkard, A.E (2000). Genetics of isozymes in grassb pea .j. hereditary. 91:142- 145.
9. Cooke, R. J. (1984). The characterization and identification of cultivars by electrophoresis. Electrophoresis. 5:59-72.
10. Cooke, R.J.(1988).Electrophoresis in plants testing and breeding.Advances in Electrophoresis. 2: 171-261.
11. Das S, Mukarjee KK,1995. Comparative study on seed proteins of Ipomoea. Seed Science and Technology,23: 501–509.
12. Davis, P.H., Mill, R.R. and Tan, K. (1988). Flora of Turkey and east Aegean islands (Supplement 1). Edinberg.Edinbergh university press. 220-223.
13. De Vries, I.M. (1996). Characterisation and identification of Lactuca sativa cultivars and wild relatives with SDSelectrophoresis (Lactuca sect. Lactuca, Compositae). GRACE., 43: 193-202.
14. Gepts P. 1989. Genetic diversity of seed storage proteins in plants. In: Brown AHD, Clegg MT, Kahler AL, and Weir BS [eds.], Plant population genetics, breeding and genetic resources, 64–82. Sinauer Associates Inc., Sunderland, Massachusetts.
15. Ghafoor A, Ahmad Z, Qureshi AS, 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: 367–378.
16. Hameed, A., Shah, T.M., Atta, B.M., Iqbal, N., Haq, M.A. and Ali, H.(2009). Comparative seed storage protein profiling of Kabuli chickpea genotypes, Pakistan Journal of Botany. 41(2):703- 710.
17. Huber-Morath, A. (1970). In Davis, P.H. (eds.), Flora of Turkey and the East Aegean Islands. Edinburgh Univ. Press, Edinburgh. 3: 452-482.
18. Hymowitz, T. (1990). Grain Legumes. In In Advances in new crop.Edited by Janick J, Simon JE. Timber Press, Portland, OR :54-57.
19. Iqbal, S.H., A. Ghafoor and N. Ayub. (2005). Relationship between SDSPAGE markers and Ascochyta blight in chickpea. Pak. J. Bot., 37: 87-96. 136 International Journal of Current Research and Review www.ijcrr.com Vol. 03 issue 11 November 2011
20. Javaid, A., A. Ghafoor and R. Anwar. 2004. Seed storage protein electrophoresis in groundnut for evaluating genetic diversity. Pakistan Journal of Botany, 36: 25-29.
21. Jha, S.S. and D. Ohri. 1996. Phylogenetic relationships of Cajanus cajan (L.) Millsp. (pigeonpea) and its wild relatives based on seed protein profiles. GRACE, 43: 275-281.
22. Ladizinsky G, Hymowitz T,1979 Seed protein electrophoresis in taxonomic and evolutionary studies. Theoretical and Applied Genetics 54: 145-151.
23. Ladizinsky, G. (1979). Seed protein electrophoresis in section Trigonella Foenum-graecum of (Fabaceae). Plant Systematics and Evolution. 133(1- 2):87-94.
24. Laemmli, U.K. (1970).Cleavage of structural proteins during the assembly of the head of bacteriophage T4 . Nature. 227:680–685
25. Maity,S., Datta,A.K. and Chattopadhyay,A.(2009).Seed protein polymorphism in nine species of Jute (Corchorus, Family :Tiliaceae).Indian journal of Science and Technology. 2(1): 34-36.
26. Makai,P.S., Makai,S.and András,K. (2004).Comparative test of fenugreek / Trigonella foenum-graecum L. / varieties. J. Cent. Eur. Agric. 5(4): 259-262.
27. Malhotra, S.K. and Vashishtha, B.B. (2008) Organic production of seed spices. National Research Centre for Seed Spices, Ajmer, India, p 90
28. Martin1,E., Akan, H. , Ekici, M. and Aytaç, Z.(2011). New chromosome numbers in the genus Trigonella L. (Fabaceae) from Turkey. African Journal of Biotechnology .10 (2): 116- 125.
29. Martinez, E. N., Castellani, O. F. and Anon, M. C. (1997).Common molecular features among amaranth storage proteins.J Agric food chem.45:3832-3839.
30. Mennella, G., S.V. Onofaro, A. Tonini and V. Magnifico. 1999. Seed storage protein characterization of Solanum species and of cultivars and androgenetic lines of S. melongena L., by SDS-PAGE and AE-HPLC. Seed Science and Technology., 27: 23-35.
31. Olli, S. and Kirti, P.B. (2006) Cloning, characterization and antifungal activity of defensin Tfgd I from Trigonella foenum-graecum L. J Biochem Mol Biol. 39(3):278–283.
32. Olli, S., Guruprasad, L. and Kirti, P.B. (2007) Characterization of defensin (TFGD 2) from Trigonella foenumgraecum. Current Sci. 93 (3):365–369.
33. Parker, P. G., Snow, A. A., Schug, M. D. Booton, G. C. and Fuerst, P. A.(1998).What molecules can tell us abot populations;choosing and using molecular markers.Ecology.79:361- 382.
34. Polhil RM, Raven PH (1981) Advances in legume systematic. Royal Botanical Gardens, Kew, England .,2:
35. Preet, A., Siddiqui, M.R., Taha, A., Badhai, J., Hussain, M.E., Yadava, P.K. and Baquer,N.Z.(2006). Longterm effect of Trigonella foenumgraecum and its combination with
36. sodium orthovanadate in preventing histopathological and biochemical abnormalities in diabetic rat ocular tissues. J Mol Cellular Biochem..289(1/2):137–147.
37. Przybylska,J.,Zimnik- Przybylska,Z. and Krajewiki,P.(1998).Diversity of seed albumins in the grasspea (Latharus sativus L.):an electrophoretic 137 International Journal of Current Research and Review www.ijcrr.com Vol. 03 issue 11 November 2011 study.Genet.Resour.Crop.Evol.45:423- 431.
38. Rachie KO, Roberts LM. (1974) Grain legumes of the lowland tropics. Advances in Agronomy,26:1-32.
39. Rajagopalan MS .( 2001) Fenugreek a savory Medicinal. Supplement industry Executive 5(6):43- 44.
40. Rakhee, S.D., Meena, D.L., Lal, B.C., Prabhakar, K.R. and Vidya, S.G.(2004). Assessment of genetic diversity in trigonella foenum graecum and Trigonella caerulea using ISSR and RAPD markers,BMC Plant Biology. 4:13
41. Sammour, R., Mustafa, A. Z., Badar, S. and Tahr, W. (2007).Genetic variation in accessions of Latharus sativus L.Acta.Bot.Croat.66:1-13.
42. Sammour,R.,Mustafa,A.Z.,Badar,S. and Tahr,W.(2007).Genetic variation in accessions of Latharus sativus L.Acta.Bot.Croat.66:1-13.
43. Secmen, O., Gemici, Y., Gork, G., Bekat, L. and Leblebict, E.(1998). Tohumlu bitkiler sistematigi, BornovaIzmir. 236-240.
44. Shishodia, S. and Aggarwal, B.B. (2006). Diosgenin inhibits osteoclastogenesis, invasion and proliferation through the downregulation of Akt, I Kappa B kinase activation and NF- Kappa Bregulated gene expression. Oncogene. 25(10):1463–1473.
45. Tadesse,W.,Bekele,E.(2001).Factor analysis of components of yield in grass pea (Lathyrus sativus L.). Lathyrus lathyrism Newsletter.2: 43- 46.
46. Tadesse,W.,Bekele,E.(2004). Isozymes, Protein and ODAP variability of grass pea (Lathyrus sativus L.) in Ethopia. Ethopian .J.Sci. 27.
47. Vargas, E. M., Macaya, G., Bauboin, J. P. and Rocha, O. J. (2000).Variation in the content of phaseolin in wild populations of lima beans Phaseolus lunatus L.In the central valley of Costa Rica.PGR Newsletter. 121: 53-58.
48. Yasmin, B., Habib, A., Sazia, S., Sajid, G. and Imtiyaz A.K. 2010. Comparative proteomic studies in leguminous species. Asian Journal of Agricultural Sciences. 2(3):89-93.
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