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ThesisItem Open Access Biofortification of hexaploid wheat with iron and zinc through classical and molecular approaches(G.B. Pant University of Agriculture and Technology, Pantnagar - 263145 (Uttarakhand), 2016-01) Mathpal, Priyanka; Sundip KumarThesisItem Open Access Transcriptomic approach for understanding the role of calcium signaling and transport genes in relation to grain calcium content and calcium responsiveness in finger millet(G.B. Pant University of Agriculture and Technology, Pantnagar - 263145 (Uttarakhand), 2013-08) Singh, Uma Maheshwar; Anil KumarFinger millet is a plant with exceptionally high seed Ca content. Little is known about the genetic and epigenetic basis of calcium accumulation in seed. In order to understand the mechanisms of high seed Ca accumulation, two genotypes of finger millet differing in seed Ca content i.e. GPHCPB-1 (low Ca) and GPHCPB-45 (high Ca) were selected in present study. Through PCR based approach a total of 8 genes of Calcium regulated protein kinases were isolated, but these were quite few to be able to explain the exact mechanism of high seed Ca accumulation. Therefore, to cover all Ca signalling and transport genes expressed during seed development, the transcriptome of developing spike of both GPHCPB-1 and GPHCPB-45 genotypes were sequenced. A total of 55 and 52 Ca signaling and transport genes were identified in GPHCPB-1 and GPHCPB-45 genotype respectively. On the basis of phylogeny, per cent sequence identity, and conserved sequence analysis all the genes were classified into Ca sensor and transporter gene families. Differential expression analysis showed that 15 genes were highly expressed in GPHCPB-45, 7 were highly expressed in GPHCPB-1 and remaining 32 had very less (FPKM<1) expression value. Structural and functional analysis of these genes were also done and high level of sequence similarity among Ca sensor genes and low level of sequence similarity among Ca transporter genes of finger millet with their rice orthologues was found. Simultaneously effect of different levels of exogenously supplied Ca on both GPHCPB-1 and GPHCPB-45 genotypes were also studied. The responsiveness of both the genotypes towards increasing exogenous application of Ca was determined in terms of changes in Ca accumulation in different tissues and different morpho-physio-agro-biochemical parameters. During the spike development (S1, S2, S3 and S4) the effect of exogenous Ca (Ca deficient-0.1mM, Ca sufficient-5.0 mM, Ca excess-10 mM and Ca toxic- 20 mM) on Ca accumulation in root, stem, leaf and spike and also on root length, root dry matter accumulation, plant height, RWC, chlorophyll content, SPAD value, oxalate and phytate content was recorded. During Ca deficient conditions, genotype GPHCPB-45 performed better than GPHCPB-1; while at toxic level of Ca genotype GPHCPB-1 withstand that condition better than GPHCPB-45 in terms of Ca accumulation as well as different agro-morphophysio-biochemical parameters. Based on such studies it was revealed that threshold potential of Ca accumulation was high for GPHCPB-1 in even Ca toxic conditions, however GPHCPB-45 was efficient accumulator under Ca deficient conditions. The results of present study clearly suggest the differential responsiveness of genotypes towards exogenous application of Ca in different tissues, which is suggestive of genetic and epigenetic basis of seed Ca accumulation trait and induction of differential calcium signaling and transport machinery. The responsiveness of differentially expressed genes (highly expressed in GPHCPB-45; FPKM>4) were studied towards exogenous application of Ca in both GPHCPB-1 and GPHCPB-45 genotypes. The overall high expression of most of the Ca sensor genes i.e., EcCDPK16, EcCDPK19, EcCIPK2, EcCIPK9, and EcCIPK11 were observed in GPHCPB-45 genotype in later stages of spike development, in a concentration dependent manner. Similarly, the overall high expression of Ca transporter genes i.e., EcPM3ATPase2, EcPM4ATPase8, EcPM12ATPase4, EcER3ATPase3 and EcCaX3 were observed in GPHCPB-45 in later stages of spike development, in a concentration dependent manner. The expression of two genes EcCIPK19 and EcPM8ATPase was comparatively higher in GPHCPB-1 as compared to GPHCPB-45 genotype. The exceptionally higher expression of EcCaX3 in response to exogenous Ca and at later stages of spike development in high Ca containing genotype establishes this gene as a strong candidate for higher seed Ca accumulation and its further use for Ca biofortification. However, exploration of its spatial distribution within seed, over expression and knockout studies will help in understanding the exact role of this gene in high seed calcium accumulation. Finally, transcriptome based functional annotation provides a greater insight into the mechanisms related to seed calcium accumulation and would help to accelerate future research in this very important area of seed biology.ThesisItem Open Access Marker assisted conversion of normal maize into quality protein maize(G.B. Pant University of Agriculture and Technology, Pantnagar - 263145 (Uttarakhand), 2014-01) Tufchi, Mahak; Singh, N.K.The present investigation on “Marker assisted conversion of normal maize into Quality Protein Maize” was carried out with the objective to convert normal maize lines into quality protein maize (QPM). Polymorphisms among normal maize lines namely Pant 10k 1375, Pant 11R-53, Pant 11R-126 and Pant 11R-128 and QPM donor lines namely CML161, CML162, CML187 and CML189 were determined using both molecular and phenotypic markers. The polymorphic parents were crossed to generate F1s. Nine selected F1s were used to generate BC1F1s and BC2F1s populations by subsequent crossing with their respective recurrent parents. Individual plants from BC1F1s and BC2F1s populations heterozygous for o2 allele were selected using SSR marker phi057 whereas phenotypic criteria in both generations followed by marker assisted selection of loci other than o2 locus in BC2F1s were used to select plants similar to recurrent parents. The number of plants selected in BC1F1s and BC2F1s populations varied from 12 to 24 and from 10 to 28, respectively. All the BC2F2 individuals were screened using light box and plants with kernels less than 25% opaqueness were selected which varied from 2 plants in Pant11R-128xCML189 to 10 plants in Pant11R-126xCML162. Selected progenies were advanced to BC2F3 by self pollination and self seeds of each plants were used to determine protein, tryptophan, lysine, quality index (QI) and carotenoids. The number of lines across the nine population with tryptophan higher than 0.075% were 29 whereas the number of lines identified to be QPM based on protein, tryptophan and QI were 5, 3, 2, 2, 1, 1, 2, 2, 1 in Pant10k1375xCML161, Pant10k1375xCML187, Pant11R128xCML187, Pant11R53x CML189, Pant11R128xCML189, Pant11R126xCML189, Pant11R53xCML162, Pant11R126xCML162 and Pant11R128xCML162, respectively. These QPM lines could be a potential source of germplasm in development of single cross QPM hybrids in different combination among themselves as well as in combination with other diverse QPM lines. Analysis of carotenoids revealed that four lines namely BC2F3:17, BC2F3:8, BC2F3:10, BC2F3:14 derived from the cross between Pant10k1375xCML161 had carotenoids higher than the higher parent and therefore indicates that carotenoids can be enhanced using classical breeding approach. The relation between kernel colour and carotenoids content was found to be significant positive. However, regression analysis revealed low coefficient of determination of kernel colour on carotenoids as indicated by R2 value of 0.204. Pair-wise correlation between protein, tryptophan, lysine, QI and carotenoids were positive however low coefficient of determination indicated that one parameter cannot be used in determination of othersThesisItem Open Access Synthesis of antigenic peptides of rabies virus and development of anti-peptide antibodies for diagnostic uses(G.B. Pant University of Agriculture and Technology, Pantnagar - 263145 (Uttarakhand), 2010-01) Pandey, Vinod Chandra; Rajesh ChandraRabies, a unique viral disease of warm blooded animals, kills virtually every individual it infects and is still a major problem for the developing and developed countries. Confirmatory diagnosis of rabies helps in taking appropriate prophylactic measures and is also essential for epidemiological investigations. The present study was undertaken with an aim to develop synthetic peptide antigens for serodiagnosis of rabies. At the same time antipeptide antibodies were developed for detecting rabies virus antigen. The rabies virus (CVS strain) was maintained in the laboratory by serial mouse to mouse passage. The rabies virus was also grown in BHK-21 and Neuro 2a cells. Additionally, the virus was also isolated from a cow calf suspected to have died of rabies virus infection. The Seller’s staining technique, fluorescent antibody test (FAT), rabies tissue culture infection test (RTCIT) and mouse inoculation tests were performed for confirmation of rabies virus. In case of Seller’s staining, magenta red colored Negri bodies were visible. In case of FAT and RTCIT, apple green colored, grain like fluorescent foci were observed in positive cases. Similarly, the intra-cerebral inoculation of mice with virus, characteristic symptoms were recorded and mice succumbs to death within seven days. The hyperimmune sera (HIS) was raised in mice and rabbits by immunizing with BPL inactivated CVS virus and also using commercially available ‘Raksharab’ vaccine. The virus titration was performed in suckling mice and BHK-21 cells to determine optimum concentration of virus for performing challenge studies. The LD50 of 10-6.5 and 10-3.9 were recorded for intra cerebral mouse inoculation and in cell culture. Thereafter, 20 to 50 LD50 dose of virus was used for various challenge experiments. For developing synthetic peptide antigens, the regions in Glycoprotein, Nucleoprotein and Phosphoprotein of rabies virus were identified based on parameters like hydrophylicity index, surface probability, antigenic index and presence of ordered Beta and turn structure. The identified peptides were synthesized in three different formats. Firstly, B- cell epitopes were synthesized as linear peptides. The chimeric linear peptides were synthesized by combining the B cell epitopes and the T- helper epitopes P & N. appropriate spacer was used between B cell and T helper epitope sequence. Besides this peptide sequences were also synthesized in multiple antigenic peptide (MAP) format in such a way that each MAP molecule contained 4 copies of peptide sequence. In total, 14 different peptides were synthesized by solid phase peptide synthesis method using Fmoc chemistry. The purity of peptides was checked by RP-HPLC and about 90% purity was recorded. For raising antipeptide antibodies, these 14 different peptides were injected in mice at 0,7,14, 28 days and sera were collected at 32 days. Besides, different combinations of these 12 peptides were also injected in mice to assess the role of T helper peptides in generating immune response in mice. The antigenic synthetic peptides detected the rabies virus specific antibodies in the HIS raised in both mice and rabbit. On repeated testing with peptide ELISA, at least 3 times more OD492 nm was recorded in comparison to preimmune sera. Additionally, when these peptides were reacted with convalescent serum from recovered mice, about 2 fold more increase in OD492 values were recorded in comparison to serum from infected mice. When peptides were used in combination to coat the ELISA plate, even better ELISA reactivity was observed, pointing towards suitability of these antigens in sero-diagnosis of rabies. The antipeptide antibodies were raised in mice using synthetic peptide antigen. The peptides were given individually and also in combination with T helper epitopes. Besides, the MAPs were also used for immunization either individually or in combinations with T helper epitope peptide. The antibody titer were estimated and it was found that when immunized along with T helper epitopes, the APA titer increased in case of linear epitopes. Whereas, in case of a combinations of MAPs & T helper epitopes , APA titers upto 102400 were also recorded. This shows the suitability of using T helper epitopes along with either linear or MAPs for generating high titer antibodies in the host. The APAs were also found suitable in detecting native virus by antigen capture ELISA. In double sandwich ELISA virus could be detected with 3 fold more ELISA signals than negative controls. The circular dichorism (CD) spectroscopy was performed to determine the solution conformation of linear, chimeric and MAPs. The CD spectra were recorded in 2 different solvents (water and 90% Tri fluoro ethanol). It was observed that peptides adopted ordered structure in both water and TFE with random coil structure ranging in between 20 to 35 %. When CD spectra of peptides were recorded in helicogenic solvent like TFE, the ordered structure, predominantly helix, was induced in most of the antigenic peptides at the expense of randomness. When peptides were coated on ELISA plate in 90% TFE, higher ELISA signals were recorded indicating that antigenic peptides in more ordered confirmation can serve as better ELISA antigens. Overall, in present study peptides were synthesized in different formats and their suitability in detecting antibodies was assessed. Further, the antipeptide antibodies raised against these peptide antigens have shown promising results for the diagnosis of native rabies virus. Basic virological techniques like virus isolation in cell culture and virus titration in vitro and in vivo were used to accomplish research objectives. The immunological techniques like FAT, RTCIT & ELISAs were also performed. Besides this, biophysical techniques like HPLC and CD spectroscopy were also performed to accomplish the desired objectives. The synthetic peptide antigens and epitope specific antipeptide antibodies developed in this study can be promising candidate reagents for the confirmatory diagnosis of rabies.ThesisItem Open Access Deciphering the role of calcium transporters and their regulation in differential spatial and genotype dependent accumulation of calcium in finger millet (Eleusine coracana l. Gartn.)(G.B. Pant University of Agriculture and Technology, Pantnagar - 263145 (Uttarakhand), 2012-08) Mirza, Neelofar; Anil KumarThesisItem Open Access Molecular marker based bulk segregant analysis for blast resistance in aromatic rice(G.B. Pant University of Agriculture and Technology, Pantnagar - 263145 (Uttarakhand), 2010-08) Singh, Balwant; Kumar, J.Basmati is considered as the best quality rice in world market and fetches highest premium. But some of the non-Basmati scented rices, which include some Kalanamak accessions, are much superior to Basmati-types with respect to all other traits except grain length. Many of the traditional small and medium grained scented rices fetch higher price than Basmati in their native areas of cultivation. There is, however, an urgent need to properly characterize and improve these rices for useful traits such as aroma, quality and resistance to biotic (blast) and abiotic (salinity) stresses. Vast diversity in these rices strengthens the necessity to develop designer’s varieties of scented rices with desired traits as per consumers’ requirements, which in turn should offer a great advantage in both national and international market. In the present investigation, thus an effort was made for the improvement of the Kalanamak accessions against rice blast and quality and agro morphological traits. A set of 50 rice accessions, including 24 Kalanamak accessions, was characterized on the basis of agro-morphological characters, quality traits and SSR molecular markers. The mean values of the traits were found to be significant, indicating that there were significant differences in different morphological and grain quality characteristics of the scented rice genotypes selected for the study. The mean value of morphological trait, plant height varied from 100.83 to 177.4, number of panicles varied from 6 to 12, panicle length varied from 24 to 34, 50% flowering varied from 93 to 147.67 and days to maturity ranged from 123 to 183.67. The mean value of quality trait, L/B ratio ranged between 2.5-3.0, alkali spread value between 4-5, elongation ratio between 1.8-2.2 and strong aroma were found among most of the genotypes studied. With collective score of all the morphological and quality traits, genotype PB-1 was found to be most suitable genotype for improvement of Kalanamak accessions. Five Kalanamak accessions viz., 3216, 3327, 3119, 3131 and 3122 were also found suitable and selected for breeding programme. Cluster analysis clearly differentiated all the accessions into different subgroups. Molecular marker analysis clustered blast resistant genotypes in the same group with more than 80% similarity and rest of the genotypes showed about 50% similarity. The parents selected on the basis of their resistance against blast under controlled conditions, wherein isolates from different geographical regions were used for challenge inoculation, also showed similar resistant pattern at the blast hot spot at Hawalbagh (Almora). The pattern of blast resistance found in the parents 3131SN, 3327 and 3131SN was found to be similar to the IRRI differential lines, IRBLks-F5 (gene Pik-s), IRBLz-Fu (gene pi-z), and IRBLz5-CA (piz-5). Molecular marker analysis confirmed the presence of above mentioned genes in the parents. Chi-square values for disease resistance were good fit to the Mendalian ratio of 3:1 among all the F2 populations. Significant correlation was found between most of the markers and resistance phenotype, however PVE ranged from 9.52% for marker lc-19 in population B to 37% for lc-22 for population C. Bulk segregant analysis proved close association of the resistance gene(s) with their respective makers. All the segregants showed discrete pattern of segregation for blast resistance. Screened morphological traits showed continuous segregation among all the progenies. All the F2 populations viz: A, B, C, D, E and F showed resistance to blast to a good extent. Along with blast resistance, population E showed reduction in plant height and population C and E showed early maturation. The segregants found promising for the desired traits would be advanced to the next generation.