International Journal of Current Research and Review
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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.

 

 

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A Study by Purushottam S. G. et al. entitled "Development of Fenofibrate Solid Dispersions for the Plausible Aqueous Solubility Augmentation of this BCS Class-II Drug" is awarded Best article for Vol 13 issue 10
A Study by Kumar S. et al. entitled "A Study on Clinical Spectrum, Laboratory Profile, Complications and Outcome of Pediatric Scrub Typhus Patients Admitted to an Intensive Care Unit from a Tertiary Care Hospital from Eastern India" is awarded Best Article for Vol 13 issue 09
A Study by Mardhiah Kamaruddin et al. entitled "The Pattern of Creatinine Clearance in Gestational and Chronic Hypertension Women from the Third Trimester to 12 Weeks Postpartum" is awarded Best Article for Vol 13 issue 08
A Study by Sarmila G. B. et al. entitled "Study to Compare the Efficacy of Orally Administered Melatonin and Clonidine for Attenuation of Hemodynamic Response During Laryngoscopy and Endotracheal Intubation in Gastrointestinal Surgeries" is awarded Best Article for Vol 13 issue 07
A Study by M. Muthu Uma Maheswari et al. entitled "A Study on C-reactive Protein and Liver Function Tests in Laboratory RT-PCR Positive Covid-19 Patients in a Tertiary Care Centre – A Retrospective Study" is awarded Best Article of Vol 13 issue 06 Special issue Modern approaches for diagnosis of COVID-19 and current status of awareness
A Study by Gainneos PD et al. entitled "A Comparative Evaluation of the Levels of Salivary IgA in HIV Affected Children and the Children of the General Population within the Age Group of 9 – 12 Years – A Cross-Sectional Study" is awarded Best Article of Vol 13 issue 05 Special issue on Recent Advances in Dentistry for better Oral Health
A Study by Alkhansa Mahmoud et al. entitled "mRNA Expression of Somatostatin Receptors (1-5) in MCF7 and MDA-MB231 Breast Cancer Cells" is awarded Best Article of Vol 13 issue 06
A Study by Chen YY and Ghazali SRB entitled "Lifetime Trauma, posttraumatic stress disorder Symptoms and Early Adolescence Risk Factors for Poor Physical Health Outcome Among Malaysian Adolescents" is awarded Best Article of Vol 13 issue 04 Special issue on Current Updates in Plant Biology to Medicine to Healthcare Awareness in Malaysia
A Study by Kumari PM et al. entitled "Study to Evaluate the Adverse Drug Reactions in a Tertiary Care Teaching Hospital in Tamilnadu - A Cross-Sectional Study" is awarded Best Article for Vol 13 issue 05
A Study by Anu et al. entitled "Effectiveness of Cytological Scoring Systems for Evaluation of Breast Lesion Cytology with its Histopathological Correlation" is awarded Best Article of Vol 13 issue 04
A Study by Sharipov R. Kh. et al. entitled "Interaction of Correction of Lipid Peroxidation Disorders with Oxibral" is awarded Best Article of Vol 13 issue 03
A Study by Tarek Elwakil et al. entitled "Led Light Photobiomodulation Effect on Wound Healing Combined with Phenytoin in Mice Model" is awarded Best Article of Vol 13 issue 02
A Study by Mohita Ray et al. entitled "Accuracy of Intra-Operative Frozen Section Consultation of Gastrointestinal Biopsy Samples in Correlation with the Final Histopathological Diagnosis" is awarded Best Article for Vol 13 issue 01
A Study by Badritdinova MN et al. entitled "Peculiarities of a Pain in Patients with Ischemic Heart Disease in the Presence of Individual Combines of the Metabolic Syndrome" is awarded Best Article for Vol 12 issue 24
A Study by Sindhu Priya E S et al. entitled "Neuroprotective activity of Pyrazolone Derivatives Against Paraquat-induced Oxidative Stress and Locomotor Impairment in Drosophila melanogaster" is awarded Best Article for Vol 12 issue 23
A Study by Habiba Suhail et al. entitled "Effect of Majoon Murmakki in Dysmenorrhoea (Usre Tams): A Standard Controlled Clinical Study" is awarded Best Article for Vol 12 issue 22
A Study by Ghaffar UB et al. entitled "Correlation between Height and Foot Length in Saudi Population in Majmaah, Saudi Arabia" is awarded Best Article for Vol 12 issue 21
A Study by Siti Sarah Binti Maidin entitled "Sleep Well: Mobile Application to Address Sleeping Problems" is awarded Best Article for Vol 12 issue 20
A Study by Avijit Singh"Comparison of Post Operative Clinical Outcomes Between “Made in India” TTK Chitra Mechanical Heart Valve Versus St Jude Mechanical Heart Valve in Valve Replacement Surgery" is awarded Best Article for Vol 12 issue 19
A Study by Sonali Banerjee and Mary Mathews N. entitled "Exploring Quality of Life and Perceived Experiences Among Couples Undergoing Fertility Treatment in Western India: A Mixed Methodology" is awarded Best Article for Vol 12 issue 18
A Study by Jabbar Desai et al. entitled "Prevalence of Obstructive Airway Disease in Patients with Ischemic Heart Disease and Hypertension" is awarded Best Article for Vol 12 issue 17
A Study by Juna Byun et al. entitled "Study on Difference in Coronavirus-19 Related Anxiety between Face-to-face and Non-face-to-face Classes among University Students in South Korea" is awarded Best Article for Vol 12 issue 16
A Study by Sudha Ramachandra & Vinay Chavan entitled "Enhanced-Hybrid-Age Layered Population Structure (E-Hybrid-ALPS): A Genetic Algorithm with Adaptive Crossover for Molecular Docking Studies of Drug Discovery Process" is awarded Best article for Vol 12 issue 15
A Study by Varsha M. Shindhe et al. entitled "A Study on Effect of Smokeless Tobacco on Pulmonary Function Tests in Class IV Workers of USM-KLE (Universiti Sains Malaysia-Karnataka Lingayat Education Society) International Medical Programme, Belagavi" is awarded Best article of Vol 12 issue 14, July 2020
A study by Amruta Choudhary et al. entitled "Family Planning Knowledge, Attitude and Practice Among Women of Reproductive Age from Rural Area of Central India" is awarded Best Article for special issue "Modern Therapeutics Applications"
A study by Raunak Das entitled "Study of Cardiovascular Dysfunctions in Interstitial Lung Diseas epatients by Correlating the Levels of Serum NT PRO BNP and Microalbuminuria (Biomarkers of Cardiovascular Dysfunction) with Echocardiographic, Bronchoscopic and HighResolution Computed Tomography Findings of These ILD Patients" is awarded Best Article of Vol 12 issue 13 
A Study by Kannamani Ramasamy et al. entitled "COVID-19 Situation at Chennai City – Forecasting for the Better Pandemic Management" is awarded best article for  Vol 12 issue 12
A Study by Muhammet Lutfi SELCUK and Fatma entitled "Distinction of Gray and White Matter for Some Histological Staining Methods in New Zealand Rabbit's Brain" is awarded best article for  Vol 12 issue 11
A Study by Anamul Haq et al. entitled "Etiology of Abnormal Uterine Bleeding in Adolescents – Emphasis Upon Polycystic Ovarian Syndrome" is awarded best article for  Vol 12 issue 10
A Study by entitled "Estimation of Reference Interval of Serum Progesterone During Three Trimesters of Normal Pregnancy in a Tertiary Care Hospital of Kolkata" is awarded best article for  Vol 12 issue 09
A Study by Ilona Gracie De Souza & Pavan Kumar G. entitled "Effect of Releasing Myofascial Chain in Patients with Patellofemoral Pain Syndrome - A Randomized Clinical Trial" is awarded best article for  Vol 12 issue 08
A Study by Virendra Atam et. al. entitled "Clinical Profile and Short - Term Mortality Predictors in Acute Stroke with Emphasis on Stress Hyperglycemia and THRIVE Score : An Observational Study" is awarded best article for  Vol 12 issue 07
A Study by K. Krupashree et. al. entitled "Protective Effects of Picrorhizakurroa Against Fumonisin B1 Induced Hepatotoxicity in Mice" is awarded best article for issue Vol 10 issue 20
A study by Mithun K.P. et al "Larvicidal Activity of Crude Solanum Nigrum Leaf and Berries Extract Against Dengue Vector-Aedesaegypti" is awarded Best Article for Vol 10 issue 14 of IJCRR
A study by Asha Menon "Women in Child Care and Early Education: Truly Nontraditional Work" is awarded Best Article for Vol 10 issue 13
A study by Deep J. M. "Prevalence of Molar-Incisor Hypomineralization in 7-13 Years Old Children of Biratnagar, Nepal: A Cross Sectional Study" is awarded Best Article for Vol 10 issue 11 of IJCRR
A review by Chitra et al to analyse relation between Obesity and Type 2 diabetes is awarded 'Best Article' for Vol 10 issue 10 by IJCRR. 
A study by Karanpreet et al "Pregnancy Induced Hypertension: A Study on Its Multisystem Involvement" is given Best Paper Award for Vol 10 issue 09

List of Awardees

A Study by Ese Anibor et al. "Evaluation of Temporomandibular Joint Disorders Among Delta State University Students in Abraka, Nigeria" from Vol 13 issue 16 received Emerging Researcher Award


A Study by Alkhansa Mahmoud et al. entitled "mRNA Expression of Somatostatin Receptors (1-5) in MCF7 and MDA-MB231 Breast Cancer Cells" from Vol 13 issue 06 received Emerging Researcher Award


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International Journal of Current Research and Review (IJCRR) provides platform for researchers to publish and discuss their original research and review work. IJCRR can not be held responsible for views, opinions and written statements of researchers published in this journal

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