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ThesisItem Open Access Polymorphism among thermotolerant and sensitive genotypes of wheat (Triticum aestivum L.) using DNA markers(CCSHAU, 2009) Gupta, Meenu; Dhillon, SantoshWheat is one of the most important staple food crops and is cultivated throughout the world. High-temperature stress is one of the major constrains to wheat production world wide. This study was undertaken with the objective to assess polymorphism among 10 thermosensitive and 10 thermotolerant genotypes of wheat. DNA extracted from young leaves of 20 wheat genotypes was amplified by using 20 ISSR and 25 RAPD primers. Out of these primers, 11 ISSR and 18 RAPD primers were showing amplification and were selected for present investigation. For ISSR and RAPD assays, data was analyzed to calculate various parameters such as the number of total bands, number of polymorphic bands, per cent polymorphism, bands per primer, polymorphic bands per primer, similarity matrices and dendrogram. Both RAPD and ISSR generated a moderate level of average percentage of polymorphism i.e. 60.3% and 48.4% respectively. The ISSR primers yielded average 8.64 bands per primer while RAPD primers amplified average 7 bands per primer. The average number of polymorphic bands was higher in case of RAPDs (4.22) as compared to that in ISSRs (4.18). Overall size of PCR amplified products ranged between 220 bp and 3500 bp for ISSR primers and between 280 bp and 4000 bp for RAPD primers. Based on ISSR similarity matrix data, the value of similarity coefficient ranged from 0.69 to 0.94 with an average genetic similarity of 0.81. RAPD similarity matrices between different genotypes ranged from 0.63 to 0.89 with average similarity coefficient of 0.78. Dendrograms generated using RAPD and ISSR markers separated genotypes into two major clusters which were further divided into sub clusters. However, dendrogram based on RAPD markers was not in accord with dendrogram based on ISSR markers.ISSR-41 primers amplified a ≈2800 bp band which was present in all the 10 thermotolerant genotypes and absent in all thermosensitive genotypes except one (genotype S5). The marker identified using ISSR-41 primer may probably be thermotolerance specific and may have potential for use in marker assisted selection programs for wheat production improvement.ThesisItem Open Access Development of F1 hybrids from crosses between aerobic and high-yielding rice cultivars and their identification via molecular marker analysis(CCSHAU, 2009) Sandhu, Nitika; Jain, R.K.Experiments were conducted to: (i) assess the plant type, yield potential and root traits of some of the aerobic rice genotypes (MAS25, MAS26, MAS109, 3508 and 3512) developed by UAS Bangalore and those selected at rice research station in comparison to the high-yielding indica (PAU201, HKR47) /basmati (HBC19, Pusa Sugandha 4 and Pusa Basmati 1460) rice varieties, (ii) make the crosses between aerobic and high-yielding indica/basmati rice varieties, (iii) assess the genetic diversity in a set of ten rice genotypes comprising of aerobic, indica and basmati rice varieties, and (iv) use microsatellite polymorphism for identification of F1 hybrids. Field evaluation showed that aerobic rice varieties produced optimum yield under aerobic conditions, which declined by 14-24% when cultivated under submerged conditions. On the contrary, indica/basmati had optimum yield under submerged conditions, which declined by 11-25% under aerobic conditions. Aerobic rice varieties have longer and dense root system compared to high-yielding indica/basmati rice varieties. Seedling length and fresh weight decreased under PEG-induced water stress, but the decreasing rate was lesser in aerobic rice varieties. A DNA fingerprinting database of ten parental aerobic, indica and basmati rice varieties have been prepared using 18 microsatellite and a BAD2 aroma gene specific markers. High level of polymorphism was observed among the aerobic, indica and Basmati rice varieties. Number of alleles per locus ranged from 3 to 5 with an average of 3.579 alleles per locus. Size of the PCR products amplified ranged from 80- 585 bp. The molecular size difference between the smallest and largest allele at a locus varied from 15 to 328. No Null alleles were detected. Multiple alleles were also detected at an average frequency of 0.263. Polymorphism information content (PIC), which is an indicative level of polymorphism, varied from 0.330 to 0.780 with an average of 0.620 per locus. The NTSYS-PC UPGMA tree cluster analysis showed the clustering of 10 rice genotypes into two major distinct groups. The group I had three Basmati rice varieties, HBC19, Pusa Sugandha 4 and Pusa Basmati 1460. The other group was further divided into two subgroups with subgroup 1 having indica rice varieties (HKR47 and PAU201) and other subgroup having all the aerobic rice varieties (MAS25, MAS26, MAS109, 3508 and 3512). Polymorphism for four (RM440, RM162, RM144, RM240) of the 18 SSR markers with a base difference of >30 bp in the amplified products, could be clearly visualized on 2.5% w/v agarose gels. A total of 22 crosses were made between aerobic (MAS25, MAS26, MAS109) and high yielding indica (HKR47, PAU201)/Basmati (HBC19, Pusa Sugandha 4 and Pusa Basmati 1460) varieties. The crossed seeds were recovered from 14 crosses. Two plants obtained from the Pusa Sugandha 4 x MAS25 and HBC19 x MAS26 crosses were confirmed as F1 hybrids by molecular marker analysis.ThesisItem Open Access Molecular characterization of cold tolerant and susceptible genotypes of chickpea (Cicer arietinum L.)(CCSHAU, 2009) Mahavir; Boora, Khazan SinghChickpea (Cicer arietinum L.) is an annual grain legume cultivated in many parts of world and constitutes an important source of protein in human diet. Production of chickpea is affected by many biotic and abiotic factors. So, present investigation was undertaken to assess genetic differences between cold tolerant and susceptible genotypes of chickpea (Cicer arietinum L.) using SSR markers. Forty one SSR primers were used to assess molecular polymorphism in forty one chickpea genotypes. A total of 72 amplified products were obtained out of which 67 were polymorphic and 5 were monomorphic. Average polymorphism across forty genotypes was found to be 93.05%. For the genotypes tested, 1 to 7 bands were obtained, with an average of 1.75 bands per primer. The size of amplified fragments ranged from100-1300 bp. One primer also produced unique alleles in specific xx genotype E 100Ym which could be used to distinguish it. Analysis of this polymorphism profile, generated using suitable statistical programmes, grouped the forty one genotypes into two major clusters at a similarity coefficient of 0.60. These groups were further divided into sub groups and sub sub groups. The maximum similarity value of 0.94 obtained between Pusa 267 on the other hand PG 96006 & H04-11 and H04-11 & Pusa 244 found to be the most divergent with similarity value of 0.41. Genetic map was constructed and compared using Kosambi function. The marker regression function calculated by interval mapping the highest likelihood ratio statistic 3.668, 1.498, 1.429, 1.284, 0.848, 0.727, 0.693, 0.4 for each of the eight chromosomes at H1O09 (loc930), H2L102 (loc158), H1H24 (loc98), H1E06 (loc1), H1P17 (loc30), H4H05 (loc8), H1C19 (loc60) and H3H022 (loc21).These markers can be used in MAS for improvement of chickpea for cold tolerance. Fine mapping of the QTLs may be done to isolate the genes for cold tolerance using map based cloning.ThesisItem Open Access “Drudgery reduction in use of improved chullah : an ergonomic approach(CCSHAU, 2009) Goyal, Neha; Mehta, ManjuThe present study was undertaken in Hisar district of Haryana state to determine feedback of improved chullah by the users and the physiological and environmental parameters while using improved chullah. A sample of 147 respondents from three villages was selected randomly for studying the acceptability of improved chullah installed through RHWE. Out of 147 respondents, 10 willing respondent were taken for second and experimental phase to determine the drudgery through physiological and environmental parameter. The survey of first phase revealed that majority of respondent were in between 38-50 years of age, illiterate, belongs to farming families, had nuclear families with 3-6 members, and belonging to high cast with monthly income Rs.1000-4000. Data showed that most of the respondent discontinued the improved chullah mainly because of economical restrains with total acceptability of 29.93%.However, the time expenditure was 2hrs. 23 min. on improved chullah in comparison to 3-4 hrs. on traditional chullah. Respondent selected for experimental evaluation and second phase were belonging to age group of 22-38 and 39-54 years. Majority of the respondents were in to mesomorph category.The assessment of drudgery revealed that increased PEFR and reduced heart rate in terms of physiological parameters with significant amount of drudgery reduction and low percentage of SPM, SO2, NO2/NO3 in smoke of improved chullah which decreases the risk of adverse effect on respiratory tract, lungs and defense mechanism. Use of improved chullah leads to cooking comfort, saving of time, less health hazards, smoke free kitchen and convenience resulting, in a better work environment.ThesisItem Open Access Micronutrient and molecular diversity analysis in mungbean [Vigna radiata (L.) wilczek] genotypes(CCSHAU, 2010) Aneja, Bharti; Yadav, Neelam R.Vigna radiata (L.) Wilczek, commonly called mungbean or greengram is the third most important pulse crop of India occupying nearly 3 million hectare. It is an excellent source of inexpensive and easily digestible proteins of low flatulence and is available in several edible forms. Iron and Zinc are important micronutrients for human health whose deficiency causes anemia and malfunctioning of immune system. Commercial cultivars contain low levels of iron and zinc and it is important to assess genetic variability in available germplasm for improving micronutrient content in commercial cultivars. The present study was undertaken to study SRAP polymorphism among 21 mungbean genotypes using 29 SRAP primers. A total of 121 amplified bands were produced which were polymorphic with an average of 4.65 bands per primer. The size of amplified bands ranged from 70-3000 bp. Unique bands were displayed by 8 mungbean genotypes using SRAP analysis. Six out of 29 SRAP primers were most useful in fingerprinting mungbean genotypes under study. The similarity coefficients between different genotypes ranged from 0.45-0.96 with an average similarity value of 0.71. At an arbitrary cut-off at 60 per cent similarity level on a dendrogram, the mungbean accessions were categorized into two major clusters. ML1108 and 2KM115 were found to genetically similar. SMH99-1A and ML776 showed high iron and zinc content while Satya was poor in iron as well as zinc content. Recombinant inbred lines involving ML776 and Satya could be used for tagging gene for micronutrient content. The results indicated that SRAP markers were efficient for identification of Vigna radiata genotypes and for determination of the genetic relationships among them.ThesisItem Open Access Cloning and characterisation of sex-specific DNA marker in jojoba (Simmondsia chinensis L.)(CCSHAU, 2010) Surender; Kharb, PushpaJojoba (Simmondsia chinensis L. Schneider) is a slow growing, dioecious shrub of arid zones and belongs to family Simmondsiaceace. It is commercially valued for multipurpose uses of its oil present in seeds. Sex of the young jojoba plant cannot be judged until the first flower buds appear which may take up to 3-4 years. Bhardwaj (2008) identified a male specific ISSR marker in jojoba, amplifying a unique allele of 1100 bp in male genotypes only. This study was undertaken to clone and characterize this sex-specific marker and to convert this marker into more specific SCAR marker. PCR amplification of DNA, from ten male and ten females plants of jojoba, was carried out using primer IS52. The amplified products were separated by agarose gel (1.5%) electrophoresis. A fragment of size 1100bp size was found to be present only in male plants. This specific band was eluted from the agarose gel using Qiagen gel extraction kit and cloned in to pJET1.2/blunt Cloning Vector using CloneJETTM PCR cloning kit supplied by Fermentas USA. The ligation mixture was transformed into freshly prepared competent cells of E.coli JM107 strain. The transformed colonies were allowed to grow on LB (Luria Bertani) plates containing ampicillin (100μg/ml).Only recombinant colonies appeared on the LB plates. The plasmid DNA from one colony was isolated using modified alkaline lysis method and then checked on 0.7% agarose gel. The male specific cloned DNA fragment was got sequenced using automated DNA sequencer ABI 3130XL Genetic Analyzer (Applied Biosystem, USA) located at Department of Animal Biotechnology, COVS, CCS HAU, Hisar. The sequence generated was compared with the sequences available in NCBI database and the maximum homology search was done using BLASTn. The sequence did not have any homology with any known nucleotide sequences in the available databases. Three open reading frames (108, 40 and 36 amino acids) were revealed in the sequences. Out of these, two ORF (40 and 36 amino acids) did not show any homology in available databases. One ORF of 108 amino acids showed 32% similarity with monothiol glutaredoxin-5 and mitochondrial precursor of Aspergillus terrus. SCAR scF (Simmondsia chinensis forward) and SCAR scR (Simmondsia chinensis reverse) primers were designed based on the sequence of male specific fragment. This primer pair amplified a unique allele of 1000 bp in male genotypes only (Patent filed).ThesisItem Open Access Study on personality of adolescent boys from rural and urban disorganized families(CCSHAU, 2010) Rathour, Savita; Duhan, KrishnaThe present study was undertaken in Hisar district of Haryana state. Five senior secondary schools of Hisar city and three higher/senior secondary schools of Kaimari, Mangali and Gangwa villages of Hisar I block were selected as per the demand of study. A sample of 45 rural and 45 urban respondents of 13-18 year age were taken, thus to make a total sample of 90 adolescents. Their parents also participated in study. Multi dimensional Assessment of Personality form for adults and teenagers, developed by Vohra, S. in 1993, 1996 was used to assess the personality of parents and adolescents. The finding revealed significant differences in boldness(Z=5.46*), general ability(Z=2.57*), guilt proneness(Z=2.52*), leadership(Z=5.90*), mental health(Z=1.81*), sensitivity(Z=2.50*),self sufficiency(Z=4.74*), social warmth(Z=2.46*) and tension level(Z=2.55*) of rural and urban respondents. Significant correlation existed between parental personality aspects and adolescent’s personality aspects. Adolescent’s adaptability, boldness, general ability, guilt proneness, innovation, leadership, maturity, self control, self sufficiency and tension had significant positive correlation with adaptability, boldness, general ability, guilt proneness, innovation, leadership, maturity, self control, self sufficiency and tension level of their parents. Whereas there was a significant negative correlation was observed between adolescent’s enthusiasm and parent’s tension, adolescent’s innovation and parent’s excitability, adolescent’s maturity and parent’s tension. Results further revealed significant association between age and enthusiasm level of the respondent. Economic status of family had significant effect on adolescent’s general ability, guilt proneness, leadership, mental health, self control and social warmth. Social variables viz. caste, family type, family size, parental sex and parental education were significantly associated with enthusiasm, general ability, guilt proneness, individualism, leadership, maturity, mental health, self control, self sufficiency and tension level of adolescents.ThesisItem Open Access Molecular analysis of thermotolerant and sensitive genotypes of wheat (Triticum aestivum L.em.Thell) using ISSR markers(CCSHAU, 2010) Richa; Dhillon, SantoshWheat is one of the most important staple food crops and is cultivated throughout the world. High temperature stress is one of the major constrains to wheat production world wide. This study was undertaken with the objective to assess polymorphism among 7 thermosensitive and 7 thermotolerant genotypes of wheat. DNA extracted from young leaves of 14 wheat genotypes was amplified by using 40 ISSR primers. Out of 40 primers, 26 ISSR primers showed amplification and were selected for allele scoring. ISSR data was analyzed to calculate various parameters such as the number of total bands, number of polymorphic bands, per cent polymorphism, bands per primer, polymorphic bands per primer, similarity matrices and dendrogram. ISSR generated a moderate level of average polymorphism i.e. 49.2%. An average of 7.19 bands per primer were generated. Overall size of PCR amplified products ranged between 220 bp and 3000 bp. Based on similarity matrix data, the value of similarity coefficient ranged from 0.73 to 0.87 with an average genetic similarity of 0.8. Dendrogram separated genotypes into two major clusters, one consisted of thermosensitive genotypes except S1(WH157) and other had all thermotolerant genotypes. Among all the 40 ISSR primers screened, primer ISSR-24 showed≈2200bp band which was present in all 7 thermosensitive genotypes but absent in all thermotolerant genotypes. Another differentiating band of ≈1750bp was observed with primer ISSR-29.This band was present in all thermotolerant genotypes and absent in all thermosensitive genotypes except S1. Primer ISSR-24 and ISSR-29 were found to be putative markers linked to thermotolernace in wheat crop and hence find application in wheat improvement program.ThesisItem Open Access Molecular characterization of poly hydroxy butyrate producing bacteria(CCSHAU, 2010) Shalini, Swati; Sikka, Virendra K.Polyhydroxybutyrates are the biopolymers and have a potential to be used as biodegradable plastics to replace synthetic plastics. The present investigation has been undertaken to isolate polyhydroxybutyrate producing bacteria and analyze the diversity among them related with sequence of PHB synthase gene. The soil and water sample for isolation of bacteria were collected from fourteen diverse sources. Out of hundred bacterial isolates forty were found PHB producing after initial fluorescence based screening on Nile blue A containing medium. By trying different combinations of carbon and nitrogen sources 2g glucose as carbon source and 0.65g potassium nitrate was found as most suitable for intracellular PHB accumulation. Six best PHB accumulating strains were selected and characterized for optimization of PHB production media. The selected strains were producing 59-29% PHB of dry weight of cell pellet. Different combinations of Agri-byproducts were tried as carbon source for cost effective PHB production and 3g molasses was found as best carbon source for PHB production (60% of cell dry weight).PCR amplification was performed by PHB synthase gene specific primers with genomic DNA of selected strains. Diverse pattern of amplification was observed which indicate towards diversity of phbC gene sequence. The gene specific band was eluted and cloned in TA sequencing vector.