“CHARACTERISATION AND GENETIC DIVERSITY OF COWPEA (VIGNA UNGUICULATA L.) GENOTYPES RESISTANT TO YELLOW MOSAIC VIRUS USING BIOCHEMICAL AND MOLECULAR MARKERS”

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Date
2012-08
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jau,junagadh
Abstract
Cow pea (Vigna unguiculata L. Fabaceae, 2n=22) is one of the most important vegetables play a vital role in the health and nutritional security of human beings with genome size 1C= 600.00 Mbp. An experiment was conducted at Department of Biotechnology, Junagadh Agriculture University, Junagadh during 2011-2012 using 10 genotypes of cowpea with three main objectives: (i) To screen and select cowpea genotypes susceptible or resistant against cowpea yellow mosaic virus. (ii) Molecular diagnosis of different selected cowpea genotypes using various molecular markers viz. Random Amplified Polymorphic DNA (RAPD), Inter Simple Sequence Repeats (ISSR) and Simple Sequence Repeats (SSR) (iii) To examine the isoenzymatic pattern of different selected cowpea genotypes by Polyacrylamide Gel Electrophoresis (PAGE). DAS-ELISA (Double Antibody Sandwich- Enzyme Linked Immunosorbent Assay) is a serological technique based on quantative analysis. It is very useful to detect viruses in plant. The fifteen cowpea genotypes were selected to detect the presence of virus and also for screening among these resistant or susceptible. The positive control having absorbance at 405 nm is 6.000 and negative control having absorbance at 405nm is 0.296385. Variability in virus infection was obtained in cowpea genotypes. JCPL-2 had maximum absorbance at 405nm (2.17292) is highly susceptible and JCPL-11 have minimum absorbance at 405nm (0.167178) was highly tolerant. A combined study in which fifteen cowpea seeds, fifteen mechanically infected leaves and fifteen naturally infected leaves is taken to study virus infection. The absorbance at 405 nm of most susceptible genotypes (JCPL-2) in seed was 0.66188, in mechanically infected leaves was 2.19292 and naturally infected leaves was 0.739349. The average of these three absorbance is 1.198049 and the absorbance at 405 nm of most tolerant genotypes (JCPL-11) in seed was 0.205838, in mechanically infected leaves was 0.261121 and naturally infected leaves was 0.252483 the average of these three absorbance is 0.239814. The RAPD, ISSR and SSR marker system were applied to the cowpea genotypes. For the RAPD data, based on PIC value, it can be said that primer OPAI-15 was the best primer resulting good amplification with maximum PIC value (0.764). Dendrogram constructed using the RAPD data clearly distinguished all the genotypes. It revealed that JCPL-44 and JCPL-45 found in one cluster and shared maximum 96.00% similarity; however, genotype JCPL-2 (highly susceptible) have out grouped from other 9 genotypes and shared minimum 41.00% similarity with other genotypes. In case of ISSR data, based on PIC value it can be said that the primer 835 was the best primer resulting good amplification with maximum PIC value (0.837). Jaccard’s similarity coefficient ranged from 0.625 and 0.906. The ISSR results indicated that maximum similarity (90.60%) was found between JCPL-2 and JCPL-18, while minimum similarity (62.50%) was obtained between JCPL-2 and JCP- 87. For the SSR data, based on PIC value it can be said that the primer VM-13 was the best primer resulting good amplification with maximum PIC value (0.668). Jaccard’s similarity coefficient ranged from 0.500 to 1.000. So SSR data revealed that JCPL-2 and JCPL-18 showed maximum variability compared to other eight genotypes. The combined RAPD, ISSR and SSR analysis revealed that out of ten genotypes JCPL-44 and JCPL-45 showed maximum (91.0%) similarity. The lowest similarity of 56.00% was found between JCPL-2 and JCP-87. Isoenzyme studies were under taken for diversity analysis at 8 DAG. Peroxidase, esterase and PPO profile showed the significant differences and provided useful information of cowpea genotypes identification. A total of 3 alleles were generated by peroxidase isozymes at 8 DAG in cowpea genotypes. Relative mobility varied between 0.396-0.868 for peroxidase isozymes. For esterase isozymes total 2 bands were observed ranging the relative mobility from 0.598 to 0.833. The banding pattern showed 50.00% polymorphism. In PPO isozymes, total 3 bands were observed having relative mobility of 0.237- 0.661 with 66.67% polymorphism. Combined analysis of peroxidase, esterase and PPO isoenzymes were performed. In that, similarity ranged from minimum 62.50% to maximum 100.0%. The clustering analysis indicated that genotype JCPL-44 and other varieties had maximum variability when compared to other genotypes. The data revealed that molecular techniques are more accurate than biochemical markers, and can be used for genetic diversity analysis of cowpea genotypes with respect to resistant to yellow mosaic virus in cowpea.
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biotechnology
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