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ThesisItem Open Access ASSESSMENT OF GENETIC DIVERSITY AMONG MUSTARD (Brassica juncea (L.) Czern & Coss) GENOTYPES USING PCR BASED DNA MARKERS(jau,junagdh, 2014-07) Gohel Krishna M.; Dr. D. R. MehtaMustard [Brassica juncea (L.), 2n=36] is the world’s third most important source of edible oil. An experiment was conducted to study molecular diversity of mustard Brassica juncea (L.) through RAPD, ISSR and SSR primers. Seven RAPD primers amplified a total of 65 bands/alleles out of which 47 bands were polymorphic (72.31%) with an average of 6.7 bands per primer. Three primers (OPB-07, OPD-02 and OPG-12) demonstrated 100% polymorphism. The phylogenetic tree constructed by UPGMA method generated two main clusters, cluster I and II. A total of 109 bands were obtained from the ten ISSR primers, out of which 78 showed polymorphism of 71.56% with an average of 7.8 bands per primer. The PIC ranged from 0.75 to 0.993 and genetic similarity varied from 0.478 to 1.000. The cluster analysis revealed two main clusters with highest 58% similarity percentage. Ten SSR primers amplified a total of 20 bands and all were polymorphic with an average of 100% polymorphism and two fragments per primer. The PIC value for each primer ranged from zero to 0.666. The present study indicated that SSR markers resulted in the highest percentage of polymorphic loci and greater range of genetic diversity among 20 mustard genotypes. The pooled analysis of all molecular markers revealed that the dendrogram consisted of two main clusters. Cluster I consisted of solitary genotype (Rohini), while cluster-II consisted of remaining nineteen genotypes in different sub-clusters. From the dendrogram depicted, two genotypes viz., Rohini and PBR 97 were found to be more diverse than the other genotypes. The genotype identification through DNA based markers resulted in developing highly diversified dendrogram of 20 mustard genotypes. The data revealed that molecular techniques are more precise and more accurate and can be used for genetic diversity analysis of mustard genotypes. Key Words: RAPD, ISSR, SSRThesisItem Open Access BIOCHEMICAL AND MOLECULAR ANALISYS OF CMS, MAINTANER AND RESTORER LINES IN PEARLMILET (Pennisetum glaucum(L.) R. Br.)(JAU,JUNAGADH, 2013-02) JOGIA ZEEL V.; R.B. MADARIAThesisItem Open Access BIOCHEMICAL AND MOLECULAR ANALYSIS OF CMS, MAINTANER AND RESTORER LINES IN PEARLMILLET (Pennisetum glaucum (L.) R.Br.) 1725(JAU,JUNAGADH, 2013-02) Jogia Zeel Vinodray; Dr. R. B. MadariyaPearlmillet (Pennisatum. glauccum [L.] R.Br.), commonly known as pearl, cat tail, spiked millet is a member of the poaeeae family and has a relatively small diploid genome (2n=2x=14) with a DNA content of lC=2.36pg (Martel et al, 1997). It is the staple food and fodder crop of millions of people living on the most marginal agricultural lands of sub-Saharan Africa and the Indian subcontinent. The present investigation on "Biochemical and molecular analysis of CMS, maintaner and restorer lines in pearlmillet [Pennisetum glaucum (L.) R.Br.)" was planned with three main objectives, (1) To study molecular markers (ISSR and SSR) in different pearl millet CMS, maintaner and restorer lines. (2) To study the protein profiling of different pearl millet CMS, maintainer and restorer lines. (3) To study isoenzymatic patterns in different pearl millet CMS maintainer and restorer lines. Ten ISSR primers produced 31 allelcs with 95.5% polymorphism with an average of 2.8 alleles per primer. 100% polymorphism was m'>'TThesisItem Open Access BIOCHEMICAL AND MOLECULAR CHARACTERIZATION OF BACILLUS SPP. ISOLATED FROM RHIZOSPHERIC SOILS OF FIELD CROPS AND THEIR BIOCONTROL ACTIVITY AGAINST FUSARIUM OXYSPORUM F. SP. CUMINI(jau,junagadh, 2013-08) Ms. Nirvisha R. Thumbar; Dr. S. V. PatelFusarium wilt caused by F. oxysporum f. sp. cumini (FOCu) is the major limiting factor for cumin productivity. Keeping this in view, 27 Bacillus spp. were isolated from 11 crop rhizospheres and screened for their in vitro antagonistic activity. Out of 27 isolates, 11 B. subtilis and 5 B. cereus were subjected to further antagonism which evaluated B. subtilis JND-KHCa-10A as best antagonist with 63.33 % growth inhibition of FOCu at 10 DAI followed by B. subtilis JND-KHCo-11A (56.67%), B. subtilis KSD-RHCu-1A (53.33%). However, B. cereus JND-RHCh-9C gave 35.83% maximum growth inhibition of test pathogen among five B. cereus isolates. The production of defence related substances by antagonists were assayed in synthetic medium containing cell wall of pathogen at 4 DAI. The percent growth inhibition was positively correlated with antibiotics siderophore and lytic enzymes chitinases and β-1,3-glucanase. However, cellulase and IAA production were negatively correlated with percent growth inhibition of test pathogen. Total 16 antagonists and one test pathogen were characterized by whole cell protein profiles using Native and SDS-PAGE. A total 7 bands were generated in range during Native PAGE without any unique bands while SDS-PAGE of whole cell protein extract revealed 17 bands with 94.1% polymorphism and also distinguished B. subtilis from B. cereus by 118.83, 97.58 and 83.64 kDa marker bands. The RAPD profiles of genomic DNA of antagonists and test pathogen demonstrated high level of polymorphism. The calculated PIC values for RAPD markers ranged from 0.152 to o.886 and RAPD primer index (RPI) differed from 0.30 to 12.37 which revealed that OPL-12 is the most inforamtive primer among 20 random primres used. Total 17 primers gave 32 specific unique amplicon for B. subtilis JND-KHCa-10A isolate associated with best antagonism against FOCu. The phylogenetic tree constructed by UPGMA method generated two main clusters and similarity coefficient was ranged from 9.8 to 96 % and it also discriminated best antagonist (B. subtilis JND-KHCa-10A) from other Bacillus spp. with minimum 21.7 % similarity. The discriminative pattern among the Bacillus isolates was validated by the 16s rDNA & 23s ITS analysis which produced the single amplicon of size more than 1kb with a few exceptions. The similarity coefficient was found ranging from 0.433 to 1.000. The Bsub5F & Bsub3R primers showed presence of single amplicon at 713 bp for 11 B. subtilis while it was not found for 5 B. cereus indicating species level identification. On the basis of biochemical as well as molecular results, the isolate B. subtilis JND-KHCa-10A was found to be best biocontrol agent inhibiting the test pathogen FOCu. Biochemical markers chitinase, β-1,3-glucanase, protease, siderophore and salicylic acid are positively correlated with inhibition of test parhogen during in vitro antagonism. Moreover, the molecular characterization of antagonists produced 17 RAPD markers which discriminate best antagonist to control FOCu. Key words: Bacillus spp., Fusarium wilt, Antagonism, Protein profiling, RAPD markers, 16s rDNA amplificationThesisItem Open Access “Biochemical and PCR Based Molecular Marker Systems for Detecting DNA Polymorphism in Pigeonpea [Cajanus cajan (L.) Millsp.]”(jau,junagadh, 2012-07) Ms. Richa Yadav; Dr. D. R. MehtaPigeonpea (Cajanus cajan (L.) Millsp., Fabaceae, 2n=22) is one of the most important pulse crops of the world with genome size 1C= 833.07 Mbp. An experiment was conducted at Biotechnology Laboratory, Department of Genetics and Plant Breeding, J.A.U. Junagadh during 2011-2012 using 15 genotypes of pigeonpea with two main objectives: (i) Biochemical markers analysis through protein profiling using SDS-PAGE and three isoenzymes viz., Peroxidase, Esterase and Polyphenol oxidase and (ii) Molecular markers analysis through RAPD, ISSR and SSR primers. The seed protein profile generated ten bands out of which five bands were shared polymorphic. The cluster analysis revealed two main clusters, cluster I and II with fourteen and one genotype, respectively. The isoenzymes analysis done at 13 DAG revealed that three monomorphic bands of peroxidase were observed with Rm values of 0.218, 0.269 and 0.494, whereas only one shared polymorphic band of esterase was detected (Rm = 0.041). Polyphenol oxidase (PPO) generated three monomorphic bands with Rm values of 0.143, 0.206 and 0.453. Isozymes studies showed very less polymorphism which cannot be used to distinguish the 15 genotypes of pigeonpea. Twelve RAPD primers amplified a total of 70 bands/alleles in which 66 bands/alleles were polymorphic (94.28%) with average 5.5 bands per primer. Twelve unique polymorphic bands were observed by six RAPD primers viz., OPP 6, 13ES10AC24, 14ES10A25, 17ES10C28, 21ES10A32 and 20ES10A31. The PIC ranged from zero to 0.914, whereas RAPD primer index (RPI) ranged from zero to 11.88. Nine ISSR primers amplified a total of 60 bands ranged from 158 bp to 1584 bp and all the bands were polymorphic (100%) with an average of 6.67 bands per primer. Three unique polymorphic bands were observed by UBC 808, UBC 854 and UBC 840 primers. The PIC varied from 0.742 to 0.932 and ISSR primer index (IPI) ranged from 2.97 to 9.96 with an average of 5.65 per primer index. Cluster analysis of both RAPD and ISSR revealed two main clusters, cluster I and cluster II with eleven and four genotypes, respectively. Ten SSR primers generated total 16 bands out of which 15 bands were polymorphic (93.75%) with fragments ranged from 60 bp to 328 bp. The PIC value ranged from zero to 0.5 with SSR primer index (SPI) varied from zero to one. Fifteen pigeonpea genotypes were grouped into two main clusters, cluster I and cluster II with twelve and three genotypes, respectively. All the three molecular markers produced higher percentage of polymorphic loci in which ISSR showed higher polymorphism (100%) among 15 genotypes as compared to RAPD (94.28%) and SSR (93.75%). There was a significant and positive correlation of combined data with similarity coefficient of RAPD, ISSR and SSR data. The dendrogram constructed by combined data of RAPD, ISSR and SSR showed 12.6% to 89.2% similarity between pair of genotypes. The cluster analysis revealed the two main clusters I and II with eleven and four genotypes, respectively. The data revealed that molecular techniques are more accurate than biochemical markers, and can be used for genetic diversity analysis of pigeonpea genotypes.ThesisItem Open Access BIOCHEMICAL AND PHYSIOLOGICAL ANALYSIS AND PROTEIN PROFILING IN WHEAT {Triticum aestivum L.) GENOTYPES UNDER HEAT STRESS 1939(JAU, JUNAGADH, 2014-10) Hitesh Ranchhodbhai Raman!; Dr. M. K. MandaviaThe present experiment on "Biochemical and Physiological Analysis and Protein Profiling in Wheat {Triticum aestivum L.) Genotypes under Heat Stress" was conducted at Department of Biotechnology, Junagadh Agricultural University, Junagadh. The experiment-1 was carried out using fourteen wheat genotypes and four heat treatments using Factorial CRD design, where seeds were grown in germination bag filled with soil for 10 days. The seedlings were subjected to control and heat treatments at 35°C, 40°C and 45°C for four hour and samples were analysed for relative water content, membrane stability and injury, lipid peroxidation, hydrogen peroxide content and chlorophyll stability index. Heat tolerant genotype GW-190 genotype showed highest membrane stability and relative water content and lowest membrane injury, lipid peroxidation and hydrogen peroxide compared to other genotype so it was selected as best heat tolerant genotype. Heat susceptible genotype J-2010-11 showed lowest membrane stability (MS) and relative water content and highest membrane injury, lipid peroxidation and hydrogen peroxide content so it was selected as highly susceptible genotype. Above physiological and biochemical parameters may be used for screening the susceptible and tolerant wheat genotypes against heat stress. H2O2 and MS are more effective indicators for screening heat tolerant genotypes under stress condition. From results of the experiment-1, one heat tolerant (GW-190) and heat susceptible (J-2010-11) wheat genotypes were selected and the plants were subjected to two groups; control and heat treatments where 40°C and 45°C heat treatments given for 2 h and 4 h of duration and physiological, biochemical, antioxidant enzyme activities, Isoenzymes and Protein profiling by 2D electrophoresis analysis were performed. Relative water content and membrane stability were found to be higher in heat tolerant genotype GW-190 compared to heat susceptible genotype J-2010-11 at tillering and grain filling stages. As heat stress and duration of heat stress increased the relative water content and membrane stability of heat tolerant and heat susceptible genotypes were decreased at both the stages of wheat development. Compared to tillering stage, relative water content and membrane stability were found lower in grain filling stage because of increased temperature. Protein, Proline and glycine betaine, Glutathione reductase, Peroxidase and Superoxide dismutase acitivities were found to be higher in heat tolerant genotype GW-190 compared to heat susceptible genotype J-2010-11 at tillering and grain filling stages. As heat stress and duration of heat stress increased, the biochemical constitutes and antioxidant enzymes activities of heat tolerant and heat susceptible genotypes also increased at both the stages of wheat development. As well all the biochemical constitutes and all three antioxidant enzymes activities were found to be higher in tillering stage compared to grain filling stage. At tillering stage, in case of Peroxidase, band No. 5 (Rm= 0.289) and band No. 6 (Rm=0.495) were present only in heat tolerant genotype while it was absent in heat susceptible genotype. At grain filling stage, band No. 3 (Rm=0.160) was present only in heat tolerant genotype. In case of Superoxide dismutase at tillering stage all the bands were present in heat tolerant as well in heat susceptible genotypes. At graing filling stage, band No. 2 (Rm—0.072) was present only in heat tolerant genotype. So isoenzymes may be useful for screening the heat tolerant and heat susceptible genotypes. At tillering stage, more total protein spots (1207) were recorded compared to that of (972) spots at grain filling stage. Compared to control, in heat stress condition expression of spots were increased. This was not true for grain filling stage. At tillering stage, highest numbers of protein spots (207) were found at 45°C for 4h duration in heat tolerant genotype GW-190 while it was true (148) spots at 40°C for 2h duration in heat tolerant genotype GW-190 at grain filling stage. The protein spots showed the differential expression pattern in treated heat tolerant genotype might be responsible for the stronger heat tolerance. Scanning electron microscopy of wheat leaves showed that analysis of variance indicated significant differences for stomatal length existed among heat tolerant and susceptible genotype as well significant differences were found for stomatal width among heat tolerant and susceptible genotype. Total 12 Operon series RAPD primers were amplified to generate the 105 fragments. The percent polymorphism obtained for RAPD primers were ranged from 71.4% to 100% with an average value of 92.33% per primer. Subcluster A1 (b) of cluster-1 consisted of only one heat tolerant genotype J-2010-06 with more than 66% of similarity. Subcluster A2 of cluster-I consisted of only one heat susceptible genotype J-2010-13 similarity of more than 85%. Cluster-II consisted of two genotypes J-2010-05 and GW-190 sh " similarity of more than 85% that belongs to heat tolerant groups. These primers be used to screen the genotypes against heat stress.ThesisItem Open Access “CHARACTERISATION AND GENETIC DIVERSITY OF COWPEA (VIGNA UNGUICULATA L.) GENOTYPES RESISTANT TO YELLOW MOSAIC VIRUS USING BIOCHEMICAL AND MOLECULAR MARKERS”(jau,junagadh, 2012-08) Mr. Dhananjay kumar; Dr. A.M ParakhiaCow 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.ThesisItem Open Access “CHARACTERISATION OF DOWNY MILDEW RESISTANT AND SUSCEPTIBLE PEARL MILLET (Pennisetum glaucum (L.) R.Br.) GENOTYPES USING BIOCHEMICAL AND MOLECULAR MARKERS”(jau,junagadh, 2014-05) Dharmesh Balvantbhai Bavadiya; Dr. M. K. MandaviaThe present experiment on “CHARACTERISATION OF DOWNY MILDEW RESISTANT AND SUSCEPTIBLE PEARL MILLET (Pennisetum glaucum (L.) R.Br.) GENOTYPES USING BIOCHEMICAL AND MOLECULAR MARKERS” was conducted at Department of Biotechnology, Junagadh Agricultural University, Junagadh with objectives, to detect the polymorphism among different pearlmillet genotypes using biochemical techniques viz., Isoenzymes and to screen the disease susceptibility and suceptibilty analysis among pearlmillet genotypes using various molecular markers viz. Random Amplified Polymorphic DNA (RAPD), Inter Simple Sequence Repeats (ISSR) and Simple Sequence Repeats (SSR). The protein profile (Native-PAGE) produced thirteen bands with Rm values ranging from 0.176 to 0.950 while SDS-PAGE of pearlmillet seed produced total of seventeen bands with Rm values ranging from 0.041 to 0.899. The isoenzymic study of peroxidase revealed seven bands of Rm values were ranging from 0.262 to 0.840 while isoenzymic study of super oxide dismutase revealed three bands of Rm values ranging from 0.256 to 0.622,while isoenzymic study of poly phenol oxidase revealed five bands of Rm values ranging from 0.120 to 0.430. The combined study of protein profiling and isoenzymes revealed that the phylogenetic tree consisted of two main clusters with similarity coefficient range 78 to 97%. Total 10 RAPD primers generated 118 bands in which 105 bands were polymorphic with an average of 10.5 bands per primer. The 9 ISSR primers were screened to generate 120 bands in which 79 bands were polymorphic with an average of 7.90 bands per primer. Total 80 SSR primers generated the 244 fragments in which 169 bands were polymorphic with an average of 2.1 bands per primer. The similarity coefficient ranged from 44 to 79% for RAPD, 62 to 64% for ISSR and 58 to 72% for SSR. The pooled study of RAPD, ISSR and SSR generated clustering pattern which seem to be similar as SSR clustering pattern. No RAPD primers was able to produce genotype specific band, while ISSR primers (ISSR-V, ISSR-VIII and ISSR-IX) and SSR primers (PSMP-2030, PSMP-2073, PSMP-2072, PSMP-2077, PSMP-2225, PSMP-2070, PSMP-2084, PSMP-2202, PSMP-2208, PSMP-2274, PSMP-2018, PSMP-2224, ICMP-3013, ICMP-3017, ICMP-3024, ICMP-3057, Xicmp-3088, ICMP-4014, Xpsmp-2255, Xpsmp-2248, Xpsmp-2249, Xpsmp-2270, Xpsmp-2227, m 13 Xpsmp-2237, Xpsmp-2273, Xpsmp-2269) produced genotype specific bands and discriminated different genotypes among all 10 pearlmillet genotypes respectively. Among all molecular and biochemical study techniques, SSR markers ans SDS-PAGE seem to be more informative than RAPD, ISSR and other isoenzyme assay to access distinguish disease resistant and suceptible pearlmillet genotypes. Thus, molecular markers were proved to be more accurate and effective than biochemical markers exept SDS-PAGE as some of RAPD, ISSR and SSR primers found to be genotype specific markers in the present study. Key words: Isoenzyme, RAPD, ISSR, SSR, Pennisetum glaucum.ThesisItem Open Access “Characterization of Brinjal (Solanum melongena L.) Genotypes through Biochemical and Molecular Markers”(jau,junagadh, 2011-05) Ms. Thakkar Jalpa R.; Dr. B. A. GolakiyaBrinjal (Solanum melongena L.) is an important crop and has a growing reputation and is now cultivated globally. It is a valuable member of the human diet in Asia, especially in India, which is a primary diversity center of the species. India is the second in Asia in terms of brinjal production. This crop showed a high level of morphological diversity which results in confusion about its systematic and this diversity is at the level of genera, species and cultivars. The aims of the studies reported in this thesis were to characterize brinjal genotypes with different morphological biochemical and molecular tools. To reveal characterization among ten brinjal genotypes, different morphological characters were recorded like 6 quantitative characters and 15 qualitative characters. In that, the highest plant height was observed in GOB-1, the highest number of branches was observed in KS-331, while lowest was observed in JBR-3-16. The highest fruit length was observed in Pb-Sadabahar, while genotype JBOB-04-04 was found with lowest fruit girth, lowest plant height, highest plant spread and acute leaf blade tip angle, while genotypes JBR-2-11, Pb-Sadabahar and KS-331 were observed with violet corolla. In case of growth habit, JBCOB-06-08 and GOB-1 were having semi spreading, while JBR-3-16 was found with long petiole length and oval fruits. Genotype GBL-1 was found with petiole spikes and ABR-02-23 was found with very weak leaf blade lobing. In case of JBGR-1, it was found with highest fruit girth, lowest fruit length, oval shaped, green fruits and having the spikes in its fruit cap. For the biochemical markers, the isoenzymes like peroxidase, esterase, polyphenol oxidase and superoxide dismutase and protein profiling were used to know the electrophoretic banding patterns of ten different brinjal genotypes by NATIVE-PAGE. A total of 8 alleles were generated by peroxidase isozymes at 9 DAG. Relative mobility was varied between 0.052-0.44 with 37.5% polymorphism. The total 6 bands of esterase isozymes were observed having 0.208-0.406 relative mobility with 13.71% polymorphism. In case of polyphenol oxidase isozymes, total 9 bands were observed having relative mobility of 0.182-0.913 with 66.6% polymorphism. Total 6 bands of superoxide dismutase isozymes were observed having relative mobility between 0.176-0.891 with 66% polymorphism, while protein profiling showed relative mobility of total 7 bands in the range of 0.272 to 0.965 with 71% polymorphism. Combined analysis of isoenzymes and protein profiling were performed. It revealed that the genotype JBR-2-11 showed highest variability with other genotypes. This was also obtained by peroxidase isoenzyme and in combined isoenzymes studies. The RAPD, ISSR and SSR marker system were applied to the brinjal genotypes. For the RAPD data, an r value of 0.868 was obtained which was in the better scale. Dendrogram constructed using the RAPD data clearly distinguished all genotypes. It revealed that JBOB-04-04 and Pb-Sadabahar found in one cluster and shared maximum 77% similarity; however, genotype GOB-1 have out grouped from other 9 genotypes and shared minimum 56% similarity. In case of ISSR data, an r value of 0.871 was obtained which was in the good scale. Jaccard’s similarity coefficient ranged from 0.680 and 0.930. The ISSR results indicated that maximum similarity 92.8% was found between Pb-Sadabahar and JBGR-1, while minimum similarity 56.5% was obtained between GOB-1 and GBL-1. For the SSR data, r value of 0.911was obtained which was in the best scale and Jaccard’s similarity coefficient ranged from 0.636 to 1.000. So SSR data revealed that JBOB-04-04 showed maximum variability compared to other nine genotypes. The combined RAPD, ISSR and SSR analysis revealed that out of ten genotypes JBOB-04-04 and GOB-1 were showed maximum (83.3%) similarity. The lowest similarity of 63% was found between GBL-1 and GOB-1. The GOB-1 genotype has oblong fruit shape with blackish purple colour and also found highest in plant height. This genotype was found alone in most clustering pattern with other 9 genotypes. It was evident from present study that isoenzymes and molecular markers were proficient to distinguish ten brinjal genotypes.ThesisItem Open Access Characterization of Castor (Ricinus communis L.) Cultivars Through DNA Fingerprinting, Isozymes and Protein Profiling.(jau,junagadh, 2010-04) Lakhani Hardik Natvarlal.; Dr. B. A. GolakiyaCastor (Ricinus communis L. 2n=20, family : Euphorbiaceae) is an industrially important crop for production of non-edible oil. Three DNA based molecular marker techniques, viz. Random Amplified Polymorphism DNA (RAPD), Inter Simple Sequence Repeat (ISSR) Simple Sequence Repeat (SSR), and were used to study the genetic diversity in castor cultivars. Peroxidase, Esterase and Polyphenol oxidase isozyme patterns and protein profiling were used for biochemical characterization of castor cultivars. Out of the 28 RAPD, 10 ISSR and 10 SSR primers screened, a total of 30 polymorphic primers (16 RAPD, 7 ISSR and 7 SSR) were used for further study. Amplification of genomic DNA of 13 cultivars, using RAPD analysis, yielded 99 polymorphic fragments with 100 % polymorphism. Number of amplified fragments with RAPD primers ranged from 6 to 12 and varied in size from 60 to 1814 bp. A dendrogram based on UPGMA analysis grouped the 13 castor cultivars into two main clusters named A and B, with Jaccard’s similarity coefficient ranging from 0.494 to 0.929. The 7 ISSR primers produced 38 bands across 13 cultivars, which were polymorphic. The size of amplified bands varied from 240 to 2700 bp. A dendrogram based on UPGMA analysis of 13 castor genotypes generated by pooled ISSR molecular data formed two main clusters, named A and B and Jaccard’s similarity coefficient ranged from 0.421 to 1. The 7 SSR primers produced 15 bands across 13 cultivars, which were polymorphic. A dendrogram based on UPGMA analysis of 13 castor cultivars generated by pooled ISSR molecular data formed two main clusters, named A and B and Jaccard’s similarity coefficient ranged from 0.424 to 1.00. Isozymes patterns were carried out to assess the biochemical character in castor cultivars. Three enzyme systems viz., Peroxidase, Esterase and Polyphenol oxidase were studied at different days after germination. Maximum banding pattern was obtained at 10 days after germination (DAG) than 15 and 5 DAG. Total 9 bands of peroxidase isozymes were observed at 5, 10 and 15 DAG. Relative mobility of 5 DAG varied between 0.170-0.250. At 10 DAG it varied from 0.390-0.579, while at 15 DAG it varied from 0.390-0.519. Polymorphism, observed were 100%, 33% and 66% at 5, 10 and 15 DAG respectively. Total 14 bands of esterase isozymes were observed at 5, 10 and 15 DAG. Relative mobility at 5 DAG varied between 0.310-0.370, at 10 DAG it varied from 0.144-0.871, while at 15 DAG it varied from 0.571-0.822. Polymorphism observed were 50%, 85% and 40% at 5, 10 and 15 DAG respectively. Total 9 bands of polyphenol oxidase isozymes were observed at 5, 10 and 15 DAG. Relative mobility of 5 DAG varied between 0.146-0.369, at 10 DAG it varied from 0.470-0.700, while at 15 DAG varied from 0.562-0.847. Polymorphism observed 66%, 100% and 33% at 5, 10 and 15 DAG, respectively. Total of 5 bands were generated by protein profiling using polyacrylamide gel electrophoresis in which 3 were polymorphic and showing 60% polymorphism. PIC value for all thirteen cultivars reveled by protein profiling was 0.786. The dendrogram constructed based on genetic distance revealed that thirteen castor cultivars fell into two main clusters, named A and B.ThesisItem Open Access “Characterization of Cotton (Gossypium Spp.) Cultivars through DNA Fingerprinting, Isozymes and Protein Profiling”(jau,junagadh, 2010-06) Mr. Kahodariya J. H.; Dr. D. N. VakhariaThe present investigation on “Characterization of Cotton (Gossypium Spp.) Cultivars through DNA Fingerprinting, Isozymes and Protein Profiling” was planned to conduct with two main objectives, (1) DNA fingerprinting through RAPD, ISSR, SSR, STMS primers and development of locus specific SCAR markers, (2) Biochemical analysis through four isoenzymes Viz. catalase, esterase, peroxidase and polyphenol oxidase, and protein profiling using Native-PAGE. Twenty one RAPD primers generated a total of 245 bands/alleles with the 97.88 % polymorphism with an average of 11.66 bands per primer. The dendrogram generated two main clusters that consists all the cultivars of three species grouped together in their respective sub-cluster. Among the screened primers OPA-05, OPA-10, OPA-15, and OPB-11 showed cultivar specific markers while OPA-09, OPA-15, OPA-16, OPA-19 and OPB-11 produced species specific DNA fragments. Nine ISSR primers engendered 85 bands/alleles with 95.69 % polymorphism with an average of 9.44 bands per primer. The cluster analysis revealed the two main clusters. The clustering of G. hirsutum cultivars found to be the same as RAPD. Cultivar and species specific markers were also observed with the ISSR primers Viz. IS-4, IS-7, ISSR-6, ISSR-7, ISSR-10 and ISSR-12. Ten SSR primers out of fifteen and four STMS primers generated total 19 and 14 bands respectively out of which 12 and 14 bands were polymorphic. In case of SSR, 58.33 % polymorphism was recorded while STMS markers were 100 % polymorphic. The character specific band was obtained with STMS primer CM43 that was linked to the leaf red colour of cotton leaves present only in two cultivars Viz. G.Cot-19 and G.Cot MDH-11. The microsatellite primers Viz. BNL1053, BNL3408 and CM43 also produced cultivar and species specific markers in studied cotton cultivars. Fifteen cotton cultivars were grouped into two main clusters and the grouping of cultivars were somewhat similar to the RAPD analysis. Among the studied technique SSR and STMS seems to be more effective than other techniques. The cluster analysis revealed grouping of cultivars according to species. The average similarity of 15 cotton cultivars was 25 % in RAPD and ISSR that is not possible in nature among the cultivars of the same genus, while was 47 % in SSR and STMS. The locus specific SCAR marker was developed by cloning and sequencing the band of around 900 bp present in all G. arboreum cultivars. The sequenced fragment generated a novel sequence of 914 bp. The 20-mer primer was designed from the sequence to develop 297 bp SCAR and was submitted in NCBI genebank with Accession No. 490146. Thus by developing one SCAR marker specific to each cultivars could eliminate the use of all other technique of DNA fingerprinting i.e RAPD, ISSR, SSR and STMS and biochemical markers. Isoenzymes and protein were used for the characterization of cotton cultivars. The isoenzyme analysis was carried out at 4, 8 and 12 DAG and protein analysis was done from the seeds as well as from the seedlings of 4, 8 and 12 DAG. There was no isoenzyme variation found in catalase. The maximum numbers of 7 bands at 8 DAG, 5 bands at 4 DAG and 8 bands at 4 DAG were visualized on Native-PAGE by esterase, peroxidase and polyphenol oxidase isoenzymes respectively. The esterase, peroxidase and polyphenol oxidase isoenzymes generated dissimilar clusters of 15 cotton cultivars from each other. However somewhat similarity was observed in cluster-I of all the dendrogram generated from these three isoenzymes i.e. consists only G. hirsutum cultivars. The polyphenol oxidase isoenzyme was found to be superior in clustering of cotton cultivars according to species. The average similarity was 86 %, 21 %, 38% and 24 % for catalase, esterase, peroxidase and polyphenol oxidase isoenzymes respectively. The protein profile generated the highest number of 10 bands at 12 DAG. First time in the present investigation the cultivars of G. arboreum Viz. G.cot-15, G.cot-19 and G.Cot MDH-11 was found in a separate cluster than G. herbaceum. The pooled study of all molecular and biochemical markers revealed a dendrogram consisted of two clusters. First cluster consisted only of G. hirsutum cultivars while cluster-II consisted of G. herbaceum and G. arboreum cultivars both in different sub-clusters. The cultivars G.Cot hyb-8 and G. cot hyb-10, and G.cot-15 G.cot-19 were found to be similar. The cultivars of G. herbaceum Viz. G.Cot-21, G.Cot-23 and V797 shared a same position in most of the dendrogram generated using various techniques. The results pointed out that biochemical techniques were not able to distinguish cotton cultivars precisely as compared to the DNA fingerprinting techniques. The cultivar identification through molecular and biochemical markers resulted in developing highly diversified map of 15 cotton cultivars belonging to three different species of Gossypium. The results revealed that molecular techniques are more accurate than biochemical markers, and can be used for characterization of cotton cultivars and seed purity study.ThesisItem Open Access CHARACTERIZATION OF COWPEA (Vigna Unguiculata L.) GENOTYPES THROUGH MOLECULAR AND BIOCHEMICAL MARKERS(jau,junagadh, 2012-04) Mr. Anatala Tushar J.; Dr. B. A. GolakiyaCowpea (Vigna unguiculata L.) vegetable plays a vital role in the health and nutritional security of human being and it belongs to the family of fabaceae, is a diploid with chromosome number 2n=22 and genome size is 600 Mb. Cowpea is one of the oldest source of human food. It is grown in tropic and sub tropic regions of Asia, Africa, Central and Southern America, parts of southern Europe and USA, India and Ethiopia as primary center of origin of cowpea and China as a secondary center of origin. Though India constitutes one of the main centers of diversity, the historical as most probable place of origin of cowpea’s non-specific wild forms are found in Africa. Morphological, cowpea seeds colours are very different. This crop showed a high level of morphological diversity which results in confusion about its systematics and this diversity is at the level of genera, species and cultivars. The aims of the studies reported in this thesis were to characterize cowpea genotypes with different morphological, molecular and biochemical tools. To reveal characterization among ten cowpea genotypes, three different morphological characters were recorded [100 seeds weight, seeds size and seeds colour]. In that, the maximum seed weight was observed in JCPL-99 (14.65g) with large seed size and reddish brown colour, while minimum seed weight was observed in JCP-96-24-2(8.41g) with small seed size and white colour with brown spots. The RAPD, ISSR and SSR marker systems were applied to the cowpea genotypes. For the RAPD data, based on PIC value, it can be said that primer OPD-08 was the best primer resulting in good amplification with maximum PIC value (0.923). Dendrogram constructed using the RAPD data clearly distinguished all genotypes. It revealed that JCPL-99 and JCPL-116 found in one cluster and shared maximum 81.00% similarity; however, genotype JCPL-2 have out grouped from other 9 genotypes and shared minimum 60.00% similarity. In case of ISSR data, based on PIC value it can be said that the primer 834 was the best primer resulting in good amplification with maximum PIC value (0.890). Jaccard’s similarity coefficient ranged from 0.612 and 0.890. The ISSR results indicated that maximum similarity of 92.90% was found between JCPL-116 and JCPL-118, while minimum similarity of 68.40% was obtained between JCPL-2 and JCP-96-3-2-1. For the SSR data, based on PIC value it can be said that the primer VM-11 was the best primer resulting in good amplification with maximum PIC value (0.905). Jaccard’s similarity coefficient ranged from 0.333 to 0.627. SSR data revealed that JCPL-116 showed maximum variability compared to other nine genotypes. The combined RAPD, ISSR and SSR analysis revealed that out of ten genotypes JCPL-48 and JCPL-52 showed maximum (78.10%) similarity. The lowest similarity of 61.20% was found between JCPL-2 and JCP-96-3-2-1. The JCPL-2 genotype was medium in size i.e. 14.19 g of 100 seeds weight with yellowish white colour. This genotype was found alone in most clustering pattern with other 9 genotypes. For the biochemical markers, the isoenzymes; peroxidase, esterase, polyphenol oxidase and superoxide dismutase and protein profiling were used to know the electrophoretic banding patterns of ten different cowpea genotypes by NATIVE-PAGE. A total of six alleles were generated by peroxidase isozymes at eight DAG. Relative mobility was varied between 0.160-0.735 with 17.67% polymorphism. The total nine bands of esterase isozymes were observed having 0.169-0.797 relative mobility with 33.33% polymorphism. In case of polyphenol oxidase isozymes, total seven bands were observed having relative mobility of 0.165-0.756 with 28.57% polymorphism. Total four bands of superoxide dismutase isozymes were observed having relative mobility between 0.202-0.414, while protein profiling showed relative mobility of total 10 bands in the range of 0.366 to 0.920 with 10% polymorphism. Combined analysis of isoenzymes and protein profiling revealed that the genotype JCPl-2 and JCPL-52 had minimum similarity [82.80%]. Combined analysis of isoenzymes revealed that the genotype JCPl-52 and JCPL-118 had minimum similarity of [76.90%]. It was evident from present study that isoenzymes and molecular markers were proficient to distinguish ten cowpea genotypes.ThesisItem Open Access “CHARACTERIZATION OF DROUGHT RESISTANT AND SUSCEPTIBLE PEARL MILLET (Pennisetumglaucum(L.) R.Br.) GENOTYPES USING BIOCHEMICAL AND MOLECULAR MARKERS”(jau,junagdh, 2015-09) Parmar Suhani Bhanubhai; Dr. M. K. MandaviaThe present experiment on “CHARACTERIZATION OF DROUGHT RESISTANT AND SUSCEPTIBLE PEARL MILLET (Pennisetumglaucum(L.)R.Br.) GENOTYPES USING BIOCHEMICAL AND MOLECULAR MARKERS”was conducted at Department of Biotechnology, Junagadh Agricultural University, Junagadh with objectives,to detect the polymorphism among differentpearlmillet genotypes using biochemical techniques viz., Isoenzymes and to screen the drought susceptibility and suceptibilty analysis among pearlmillet genotypes using various molecular markers viz. RandomAmplified Polymorphic DNA (RAPD), Inter Simple Sequence Repeats (ISSR) and Simple Sequence Repeats (SSR). The protein profile (Native-PAGE) produced eight bands with Rm values ranging from 0.031 to 0.659 while SDS-PAGE of pearlmilletseed produced total of twenty bands with Rm values ranging from 0.043 to 0.882. SDS-PAGE in present study showed specific bands linked with drought reaction trait in resistant genotype J-2454. The isoenzymic study of peroxidase revealed fourteen bands ofRm values were ranging from 0.029 to 0.721 whileisoenzymic study of super oxide dismutase revealed three bands ofRm values ranging from 0.253 to 0.619,whileisoenzymic study of poly phenol oxidase revealed eight bands ofRm values ranging from 0.123 to 0.742.The combined study of protein profiling and isoenzymes revealed that the phylogenetic tree consisted of two main clusters with similarity coefficient range 48 to 83%. Total 20 RAPD primers generated 378 bands in which 273 bands were polymorphic with an average of 13.65bands per primer. The 7 ISSR primers were screened to generate 133bands in which 120 bands were polymorphic with an average of 17.14bands per primer.Total 56 SSR primers generated the 487 fragments in which 419 bands were polymorphic with an average of 7.48 bands per primer. Thesimilarity coefficientranged from 60 to 81% for RAPD, 34 to 68% for ISSR and 45 to 66% for SSR.The pooled study of RAPD, ISSR and SSR generated clustering pattern which seem to be similar as SSR clustering pattern.RAPD primers (OPA-18, OPC-05, OPD-03, OPD-05, OPM-14, OPN-13, OPO-09, OPO-15, OPQ-12, OPQ-14, OPR-14 and OPZ-10) was able to produce genotype specific band, while ISSR primers (ISSR-I, ISSR-II, ISSR-IV, ISSR-X and ISSR-25) and SSR primers (PSMP-2018, PSMP-2072, PSMP-2074, PSMP-2077, PSMP-2080, PSMP-2084, PSMP-2089, PSMP-2201, PSMP-2203, PSMP-2208, PSMP-2220, PSMP-2232, PSMP-2267, PSMP-2273, PSMP-2276, PSMP-2277, ICMP-3002, ICMP-3024, ICMP-3050, ICMP-3066, ICMP-3080, ICMP-3081, ICMP-3086, ICMP-3088, ICMP-4010, Xpsmp-2069, Xpsmp-2089, Xpsmp-2208, Xpsmp-2219, Xpsmp-2255, Xpsmp-2266, Xpsmp-2270, Xpsmp-2273, Xpsmp-2275, Xicmp-3027, Xicmp-3088 and Xctm-12) produced genotype specific bands and discriminated different genotypes among all 10 pearlmillet genotypes respectively.Among all molecular and biochemical study techniques, ISSR and SSR markers and SDS-PAGE seem to be more informative than RAPD, and other isoenzymeassay to access distinguish drought resistant and susceptible pearlmillet genotypes. RAPD, ISSR and SSR study indicated that resistant genotype J-2467 is most diverse among 10 pearlmillet genotypes used in this study. Thus, molecular markers were proved to be more accurate and effective than biochemical markers except SDS-PAGE and ISSR as some of RAPD and SSR primers found to be genotype specific markers in the present study. Key words: Isoenzyme, RAPD, ISSR, SSR, Pennisetumglaucum.ThesisItem Open Access Characterization of Groundnut (Arachis hypogaea L.) varieties through DNA Fingerprinting, Isozymes and Protein Profiling.(jau,junagadh, 2011-05) Patel Sahil Vallabhbhai; Dr. B. A. GolakiyaGroundnut (Arachis hypogaea L., 2n=40, Family: Fabaceae) is an important crop for the production of oil. Three DNA based molecular marker techniquesviz.Random Amplified Polymorphism DNA (RAPD), Inter Simple Sequence Repeat (ISSR) and Simple Sequence Repeat (SSR), were used to study the genetic diversity in groundnut varieties.Peroxidase, Esterase, Polyphenol oxidase and Superoxide dismutase Isozymepatterns and protein profiling were used for biochemical characterization of groundnut varieties. Out of the 30RAPD,10 ISSR and 10 SSR primers screened, a total of 39 polymorphic primers (22 RAPD, 9 ISSR and 8 SSR) were used for further study. Amplification of genomic DNA of 12varieties, using RAPD analysis, yielded 96 polymorphic fragments with 100 % polymorphism. Number of amplified fragments with RAPD primers ranged from 3 to 10 and varied in size from 144 to 3315bp. A dendrogram based on UPGMA analysis grouped the 12 groundnut varieties into two main clusters named A and B, with Jaccard’s similarity coefficient ranging from 0.274 to 0.726. The 9 ISSR primers produced 58 bands across 12groundnut varieties, which were polymorphic. The size of amplified bands varied from 153 to 915bp. A dendrogram based on UPGMA analysis of 12 groundnut varieties generated by pooled ISSR molecular data formed two main clusters, named A and B and Jaccard’s similarity coefficient ranged from 0.414 to0.789. The 8 SSR primers produced 15 bands across 12groundnut varieties, which were polymorphic. A dendrogram based on UPGMA analysis of 12 groundnut varieties generated by pooled SSR molecular data formed two main clusters, named A and B and Jaccard’s similarity coefficient ranged from 0.142 to 0.717. Isozymes patterns werecarried out to assess the biochemical character in groundnut varieties. Four enzyme systems viz., esterase, peroxidase, polyphenol oxidase and superoxide dismutase were studied at different days after germination. Diverse banding pattern was obtained at 6 days after germination (DAG) and9 DAG. Total 6 bands of esterase isozyme were observed at 6 and 9 DAG. Relative mobility of 6 DAG varied between 0.146-0.283 while at 9 DAG it varied from 0.580-0.648.Polymorphism, observed was 100%, and 66.67 % at 6 and 9 DAG, respectively. Total 14 bands of peroxidase isozyme were observed at 6 and 9 DAG. Relative mobility at6 DAG varied between 0.196-0.570; at9 DAG it varied from 0.084-0.508. Polymorphism observed was62.50 %and33.33 % at 6 and 9 DAG, respectively. Total 8 bands of polyphenol oxidaseisozymes were observed at 6 and 9 DAG. Relative mobility of 6 DAG varied between 0.209-0.497 while at 9DAGvaried from 0.261-0.684. Polymorphism observed was 66.67 %and 60 % at 6 and 9 DAG, respectively. Total 8 bands of superoxide dismutaseisozymes were observed at 6 and 9 DAG. Relative mobility of 6 DAG varied between 0.451-0.570 while at 9DAG varied from 0.490-0.735. Polymorphism observed was 0 %and 50 % at 6 and 9 DAG, respectively. Total of 14 bands were generated by protein profiling using polyacrylamide gel electrophoresis inwhich 4 were polymorphic and showing 66.67 % polymorphism. Overall PIC value for all groundnut varieties reveled by protein profiling was 0.821.The dendrogram constructed based on genetic distance revealed that twelve groundnut varietiesfell into two main clusters,named A and B.ThesisItem Open Access “CHARACTERIZATION OF MUNG BEAN (Vigna radiata L.) GENOTYPES THROUGH BIOCHEMICAL AND MOLECULAR MARKERS”(jau,junagadh, 2012-07) Mr. Sanghani Jayeshkumar M.; Dr. B. A. GolakiyaMung bean (Vigna radiata L.) plays a vital role in the health and nutritional security of human beings. It belongs to leguminosae family and diploid chromosome number (2n=22) with 600 Mb genome size. Mung bean is one of the oldest source of human food. It is grown in tropic and sub tropic regions of Asia, Africa, Central and Southern America, parts of Southern Europe, USA and India. This crop showed a high level of morphological diversity which results in confusion about its systematic and this diversity is at the level of genera, species and cultivars. Mungbean seeds also showed variation in colour, size and shape. The aims of the studies reported in this thesis were to characterize mung bean genotypes with different biochemical and molecular tools. For the biochemical markers, the isoenzymes like peroxidase, polyphenol oxidase, esterase, superoxide dismutase and Native protein profiling were studied to differentiate eleven mungbean genotype. A total of 6 alleles were generated by peroxidase isozymes at 10 days after germination (DAG). Relative mobility was varied between 0.034-0.555 with 50% polymorphism. The total 8 bands of polyphenol oxidase isozymes were observed having 0.170-0.852 relative mobility with 62.5% polymorphism. In case of esterase isozymes, total 8 bands were observed having relative mobility of 0.091-0.587 with 37.5% polymorphism. Total 4 bands of superoxide dismutase isozymes were observed having relative mobility between 0.263-0.780 with 25% polymorphism. While protein profiling showed relative mobility of total 8 bands in the range of 0.118 to 0.597 with 62.5% polymorphism. Combined analysis of isoenzymes and protein profiling were performed. It revealed that the genotype PUSHA VISHAL and PUSA-9537 showed minimum similarity of 59.4%. The RAPD, ISSR and SSR marker system was applied to the mung bean genotypes. The RAPD data showed that primer OPC-11 was the best primer resulting good amplification with maximum PIC value (0.893). Dendrogram constructed using the RAPD data clearly distinguished all genotypes. It revealed that MEHA and HUM-1 found in one cluster and shared maximum 75.00% similarity; however, genotype PUSHA VISHAL have out grouped from other 10 genotypes and shared minimum 45.5% similarity. In case of ISSR data showed that primer 810 was the best primer resulting good amplification with maximum PIC value (0.907). Jaccard’s similarity coefficient ranged from 0.229 to 0.886. The ISSR results indicated that maximum similarity 88.6% was found between IPM-2-3 and GM-4, while minimum similarity 22.9% was obtained between PUSHA VISHAL and K-851. For the SSR data was find that the primer VR-304 was the best primer resulting good amplification with maximum PIC value (0.746). Jaccard’s similarity coefficient ranged from 0.607 to 0.952. So SSR data revealed that MEHA showed maximum variability compared to other ten genotypes. The combined RAPD, ISSR and SSR analysis revealed that out of eleven genotypes IPM-2-3 and HUM-1 were showed maximum 80.9% similarity. The lowest similarity of 40.2% was found between PUSHA VISHAL and K-851. It was evident from present study that isoenzymes and molecular markers were proficient to distinguish eleven mung bean genotypes. The molecular markers are more reliable than biochemical markers to distinguish the genotype. Among the molecular markers, co dominant SSR marker gave precise results to discriminate genotype specific traits.ThesisItem Open Access “CHARACTERIZATION OF PHYTOPHTHORA BLIGHT RESISTANT AND SUSCEPTIBLE SESAME (sesamum indicum L.) GENOTYPES USING MOLECULAR MARKERS”(jau,junagdh, 2015-06) Mr. Karad Kiritkumar Dhanjibhai; Dr. S. V. PatelThe present investigation on “CHARACTERIZATION OF PHYTOPHTHORA BLIGHT RESISTANT AND SUSCEPTIBLE SESAME (sesamum indicum L.) GENOTYPES USING MOLECULAR MARKERS” was conducted at Department of Biotechnology, Junagadh Agricultural University, Junagadh with objectives, to screen sesame genotypes for Phytophthora blight resistance and susceptible based on molecular markers viz. Random Amplified Polymorphic DNA (RAPD), Inter Simple Sequence Repeats (ISSR) and Simple Sequence Repeats (SSR). Total 20 RAPD primers generated 299 bands in which 279 were polymorphic with an average of 13.95 bands and 94.2% polymorphism per primer. The RAPD primers augmented fragment size was ranged from 132 bp-3600 bp. The 12 ISSR primers engendered 133 bands in which 126 bands were polymorphic that was equal to 95.60% polymorphism with an average of 10.5 bands per primer. The amplified fragments were in range of 130-2538 bp. Total 19 SSR primers generated the 117 fragments in which 89 bands were polymorphic with 70.25% polymorphism and average of 4.68 bands per primer. The similarity coefficient of clusters analysis was ranged from 31 to 79% for RAPD, 32 to 75% for ISSR and 53 to 79% for SSR. The pooled study of RAPD, ISSR and SSR generated clustering pattern which seem to be similar as ISSR clustering pattern. The 10 RAPD primers (B8, OPA-01, OPA-02, OPA-03, OPA-11, OPA-19, OPC-13, OPD-12, OPE-08 and OPM-13), 6 ISSR primers (ISSR 1, ISSR 6, ISSR 10, ISSR 16, ISSR 17 and ISSR 18) and 7 SSR primers (AY838909, AY838916, SI-ssr11, SI-ssr29, SI-ssr30, SEM-12-27 and SEM-12-147) produced genotype specific bands and discriminated six, eight and nine genotypes across the 16 sesame genotypes, respectively. Among all molecular marker studied, ISSR markers seem to be more informative than RAPD and SSR to distinguish Phytophthora blight resistant and susceptible sesame genotypes. Key words: Sesame, Phytophthora blight, RAPD, ISSR, SSR, sesamum indicum L.ThesisItem Open Access “CHARACTERIZATION OF PYRICULARIYA LEAF SPOT RESISTANT AND SUSCEPTIBLE PEARL MILLET GENOTYPES USING BIOCHEMICAL AND MOLECULAR MARKERS”(jau,junagdh, 2015-06) Ms. Vidushi N. Vaja; Dr. H. P. GajeraThe present experiment on “CHARACTERIZATION OF PYRICULARIYA LEAF SPOT RESISTANT AND SUSCEPTIBLE PEARL MILLET GENOTYPES USING BIOCHEMICAL AND MOLECULAR MARKERS”was conducted at Department of Biotechnology, Junagadh Agricultural University, Junagadh with objectives, (i) to detect the polymorphism among different pearl millet genotypes using Isoenzymes and (ii) to screen the disease susceptibility and suceptibilty analysis among pearlmillet genotypes using various molecular markers viz. Random Amplified Polymorphic DNA (RAPD) and Simple Sequence Repeats (SSR). The protein profile by SDS PAGE of pearl millet genotypes produced total 16 bands with Rm values ranging from 0.407 to 0.847. Band with Rm value 0.757 showed only in susceptible genotypes J-2526, 100-SB-13, 159-SM-13,144-SM-13 and ICMB-95444. The profile generated by Super oxide dismutase (SOD), Guaiacol peroxidase (GPX), Ascorbate peroxidase (APX), Polyphenol oxidase (PPO) and Catalase (CAT) isoenzymes produced respectively three, six, eight, six and three bands of different intensities. The Rm value of SOD ranged from 0.400 to 0.648, GPX ranged from 0.331 to 0.762, APX ranged from 0.310 to 0.815, PPO ranged from 0.356 to 0.613 and in CAT it was ranged from 0.303 to 0.375. Combined Analysis of all five isoenzymes ranged from 61% to 91%. The genotypes were grouped into two main clusters viz. cluster-A and cluster-B and shared 61% similarity. Jaccard’s similarity coefficient ranged from 73% to 94% for combined analysis of isoenzyme and protein data. The 32 RAPD primers generated 481 bands across fifteen pearl millet genotypes in which 342 bands were polymorphic with an average of 10.68 bands per primer and 139 bands were monomorphic. The 80 SSR primers generated total 768 bands in which 624 bands were polymorphic with an average of 7.8 bands per primer, while remaining 144 bands were monomorphic. The similarity coefficient ranged from 63% to 79% for RAPD and 54% to 71% for SSR. The pooled study of RAPD and SSR generated clustering pattern which seem to be similar as SSR clustering pattern. Out of 32 RAPD primers, fifteen were able to produce 18 unique specific bands, of which 10 bands were found in five resistant genotypes by 9 RAPD primers (OPA-16, OPC-05, OPD-02, OPD-03, OPE-09, OPN-10, OPO-09, OPO-13 and OPZ-15). However, 45 SSR primers produced 77 genotype specific bands, of which 35 bands were found in discrimination of five pyricularia leaf spot resistant pearl millet genotypes using 25 SSR primers (ICMP-3004, ICMP-3005, ICMP-3013, ICMP-3024, ICMP-3027, ICMP-3043, ICMP-3077, ICMP-3080, ICMP-3092, ICMP-3096, ICMP-4014, PSMP-2018, PSMP-2074, PSMP-2203, PSMP-2220, PSMP-2237, PSMP-2261, Xicmp-3027, Xpsmp-2089, Xpsmp-2225, Xpsmp-2227, XpsmP-2236, Xpsmp-2248, Xpsmp-2251, Xpsmp-2255). Among all molecular and biochemical techniques, SSR markers and SDS-PAGE seem to be more informative than RAPD and other isoenzyme assay to discriminate disease resistant and susceptible pearl millet genotypes. Molecular markers were proved to be more accurate and effective than biochemical markers except SDS-PAGE. The RAPD and SSR primers found to be specific for pyricularia leaf spot resistant genotypes.ThesisItem Open Access “Characterization of Sesame (Sesamum indicum L.) Genotypes through Biochemical and Molecular markers”(jau,junagadh, 2011-05) Mr. K. J. Vadher; Dr. D. N. VakhariaThe present investigation on “Characterization of sesame (Sesamum indicum L.) Genotypes through Biochemical and Molecular markers” was planned with two main objectives, (1) Biochemical analysis through protein profiling and isoenzymes Viz. amylase, esterase, peroxidase and polyphenol oxidase using Native-Polyacrylamide Gel Electrophoresis (Native-PAGE), (2) DNA fingerprinting through RAPD, ISSR and SSR primers. Protein analysis was done from the seeds and the isoenzymes study was conducted at 6, 9 and 12 Days After Germination for the characterization of sesame genotypes. Sesame seeds of 16 genotypes showed about 12 bands of protein with Rm values ranged from 0.41 to 0.98. The cluster analysis of protein profile formed two major clusters. The sesame genotypes possessed characteristics protein profile which can be used for the discrimination of those genotypes. Native-PAGE of esterase, peroxidase, polyphenol oxidase and amylase isoenzymes produced maximum numbers of 7 (6 DAG), 6 (12 DAG), 8 (9 DAG) and 3 bands (9 DAG) respectively. These isoenzymes generated dissimilar enzymatic lineage of 16 sesame genotypes with an average value of 97, 87, 86 and 84 % similarity respectively. This resulted 88 % similarity in pooled analysis. The polyphenol oxidase isoenzyme was found to be superior in clustering the sesame genotypes. The dendrogram constructed in the pooled study of isoenzymes and protein was somewhat identical only with minor changes to that of observed in pooled isozymatic study. Sixteen RAPD primers generated total of 126 alleles in which 121 bands were polymorphic with an average of 14.82 bands and 96.6% polymorphism per primer. RAPD primers augmented fragment size ranged from 161 bp-2962 bp. The dendrogram constructed by UPGMA method generated two main clusters. The primers OPA-02, OPA-03, OPA-05, OPA-11, OPC-13, OPD-12, OPE-08, OPM-06, OPM-13, B7 and B15 amplified some of the unique and genotype specific alleles. Nine ISSR primers engendered 70 bands/alleles in which 56 bands were polymorphic that was equal to 80.6% polymorphism with an average of 7.77 bands per primer. The amplified fragments were in range of 126-3919 bp. Dendrogram constructed using UPGMA based on Jaccard’s similarity coefficient for 16 sesame genotypes split into two main clusters-I and II with the similarity of 63 %. Some of the ISSR primers also amplified unique and genotype specific alleles. Seven SSR primers out of ten primers generated total 12 alleles. The largest fragment of 366 bp was amplified by AT838916 and smallest fragment of 163 bp was found in AY838904. Among the screened SSR primers AY838913, AY838915, AY838916 and AY838921 amplified some of the unique and genotype specific bands. The collective study of all molecular and biochemical markers revealed a dendrogram consisted of two main clusters were near about similar to pooled study of molecular. The combined study of biochemical markers resulted in poor lineage between genotypes than pooled study of molecular marker. The biochemical techniques poorly distinguished the sesame genotype as compared to the techniques of DNA fingerprinting. For genetic diversity analysis molecular marker study proved to be better than the biochemical tools since they were less polymorphic than molecular markers as well as fluctuated by the environmental conditions and can be used for genotype identification.