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Theses

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2019 - 201962020 - 202318
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Subject: Biotechnology and molecular Biology × Start date: 2003 × Type: Thesis ×
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Now showing 1 - 9 of 24
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    ThesisItemOpen Access
    Molecular Screening of maize genotypes for lysine, tryptophan and β-carotene using functional markers
    (CCSHAU, Hisar, 2023-07) . Kunal; Yashveer, Shikha
    Maize is the 3rd most significant cereal crop after wheat and rice with high nutritional value. But it is deficient in certain nutrients like essential amino acids lysine, tryptophan and β- carotene. Presence of recessive allele opaque-2 linked with SSR markers umc1066, phi112, phi057 leads to high lysine and tryptophan content. Similarly, presence of crtRB1 gene linked with crtRB1 marker increase β-carotene content. Therefore, this study was aimed to screen opaque-2 and crtRB1 genes in 25 maize inbred lines using these functional markers. These maize inbred lines were grown in greenhouse and DNA was isolated using CTAB method. PCR was performed using umc1066, phi112, phi057 and crtRB1 markers.In addition to this thirty one more markers were used for genetic diversity analysis. Analysis of PCR products was done using UV gel documentation system. Genetic diversity analysis was performed using NTSYS-pc software and high degree of polymorphism was found ranging from 0.37-0.80 and heterozygosity ranging from 0.49 to 0.84. Biochemical estimation of lysine, tryptophan and β- carotene content was done using maize flour. The relation between the amplicon size of PCR product and biochemical estimated content was drawn. It was found that the inbred line LM-17 with the favourable allele 153bp using phi112, 144bp using umc1066, 157bp using phi057 had the highest lysine and tryptophan content. The inbred line HKI-327T with favorable allele (296+543) using crtRB1 marker had the highest β-carotene content (19.86 ppm). The best possible combination is using the LM-17 inbred line because it is having highest lysine content (0.44%) and high tryptophan content (0.20%). In the similar way to increase β-carotene content, HKI-327T is a promising donor line having the highest β carotene content that can be utilized in marker-assisted breeding for introgression of trait for enhanced provitamin A into agronomically superior lines by tracking the favourable alleles. The pyramiding of both these genes with other genes responsible for enhancing the nutritional value of elite maize varieties can provide a novel strategy for biofortification programmes. Thus, this study helps to pave the ways to combat the problem of nutritional deficiency and hidden hunger.
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    ThesisItemOpen Access
    Molecular characterization for targeting foliar blast disease in pearl millet [Pennisetum glaucum (L.) R. Br.]
    (CCSHAU, 2023-04) Kamboj, Aarti; Yashveer, . Shikha
    In the present investigation, 60 pearl millet genotypes were evaluated at experimental field of Bajra Section, CCS HAU, Hisar during kharif season, 2021 for morpho-phenological traits & blast disease and the study of genetic relatedness among them using microsatellite markers. Highly significant mean sum of squares were obtained due to the genotypes for all the traits studied. The traits viz., plant height, ear length, ear diameter, leaf blade width, effective number of tillers per plant, 1000-grain weight, dry fodder yield per plant and grain yield per plant exhibited high heritability (>60%) and high genetic advance as percent of mean (GA%M) (>20%) whereas, the traits ear length, leaf blade width, effective number of tillers per plant, dry fodder yield per plant and grain yield per plant exhibited high Genotypic Coefficient of Variation (GCV) (>20%) and Phenotypic Coefficient of Variation (PCV) (>20%). Correlation coefficient analysis revealed high significant positive correlation coefficients of grain yield with traits viz., plant height, ear diameter and dry fodder yield per plant. As per blast screening, 43 genotypes were found resistant to the disease while 17 genotypes were found susceptible. In order to develop blast resistant hybrids, selected resistant lines were crossed with elite lines of pearl millet and out of the 34 hybrids developed, 32 were found resistant to blast. The increased activities of Phenylalanine Ammonia Lyase (PAL), Peroxidase (POX) and Lipoxygenase (LOX) in the resistant hybrids further confirmed the disease scoring results
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    ThesisItemOpen Access
    Molecular characterization of wheat-Aegilops addition/substitution lines for grain β-glucan content
    (CCSHAU, Hisar, 2023) Prexha; Upendra Kumar
    β-glucan is one of the most important types of soluble dietary fiber proven beneficial for human health, including lowering serum cholesterol and ameliorating type-II diabetes. The 2% or higher β-glucan content in a daily diet is considered adequate for good health. In India, wheat is a staple food crop. To find out the best source of β-glucan, we explored a panel of 37 wild relatives of wheat, 209 accessions of tetraploid and hexaploid wheat, and 17 wheat-Aegilops addition/substitution lines with other associated quality traits. Among all these, high β-glucan content was found in Aegilops species (Aegilops kotschyi and Aegilops peregrina) ranged from 1.67-3.73%, in tetra and hexaploid wheat ranged from 0.30-1.31%, whereas in wheat-Aegilops addition/substitution lines ranged from 0.74-1.76%. The physical mapping of the putative gene(s) for β-glucan synthesis is still unclear. Therefore, one of the putative genes, TaCslF6 has been mapped on the long arm of chromosome 7D of the wheat genome. The physical mapping of TaCslF6 will be helpful for the genetic improvement of wheat for high grain β-glucan. The results of expression analysis of TaCslF6 and TaCslH (performed on different grain developmental stages i.e. 10, 20, 30 and 40 days after anthesis) revealed that gene TaCslF6 was relatively abundant during all the stages of development except 20 DAA. Whereas, a peak of TaCslH transcripts was found at 10 DAA in the selected wheat-Aegilops addition/substitution line (UPHAU-3). A wide genetic diversity found in the panel of 263 wheat genotypes gives ample scope to utilize its variability for crop improvement in population by applying specific and directed breeding approaches to create a “Magic Wheat” for global food and nutritional security.
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    ThesisItemOpen Access
    Identification of microRNAs targets in response to salt stress in wheat (Triticum aestivum L. em. Thell)
    (CCSHAU, Hisar, 2023-03) Sharma, Kritika; Yashveer, Shikha
    MicroRNAs are small endogenous non-coding RNAs of about 20-24 nucleotides that play an important role in transcriptional and post transcriptional gene expression via directing mRNA cleavage, translational repression, chromatin remodeling, and DNA methylation. RECent advances in high-throughput sequencing and bioinformatics tools have enhanced the study related to miRNA discovery and their putative roles in different molecular, biochemical and physiological processes. Development of miRNA and their target related markers have been identified in wheat and other crops which can be used for genetic diversity, population studies and genotype identification in crop improvement programmes of wheat. In this study, a total of 1,14,216 targets were predicted for eight hundred ninety four miRNAs using psRNAtarget tool. About one thousand five hundred twenty-two SSR markers were developed from twenty three targets and validated by insilico PCR. Expression studies were also conducted to study the relative expression of six genes S-adenosylmethionine synthase (ADENO29), Cysteine proteinase inhibitor (CYS4), lipoxygenase (LIPO37), Vacuolar cation/proton exchanger (VAC9), Triticum aestivum Salt Response Gene (TaSRG) and stress repressive zinc finger protein 1 (TaSRZ) in six wheat genotypes; WH1105, HD2967 and WH711 (Salt sensitive) and KRL 210, WH157 and Kharchia65 (salt tolerant). Expression of vacuolar cation/proton exchanger gene was found to be high in all genotypes under salt stress (ECe 8dS/m). This gene was targeted by aof-miR12159, ath-miR5025, ath-miR5029 and osa-miR160e-3p miRNA. So these four miRNA might be salt responsive miRNA in wheat. Further studies may possibly reveal the mechanism of these miRNAs under salt stress.
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    ThesisItemOpen Access
    Characterization and transcriptome analysis of transgenic pigeon pea (Cajanus cajan (L.) Millsp.) plants with LecRLKgene for salt tolerance
    (CCSHAU, Hisar, 2023-01) Mehla, Sheetal; Upendra Kumar
    Receptor kinases are one of the most potent cell signaling molecules that work against different kinds of stresses, however, very few studies are there on the characterization of the Lectin Receptor-like Kinase gene in plants for regulating salt stress. Biochemical analysis of increased concentrations of antioxidant enzymes like catalase, peroxidase and osmolytes like proline and total soluble sugar deciphered the role of OsLecRLK in maintaining the osmotic balance, ion homeostasis, and keeping a check on ROS production. Also, we observed a significant decrease in membrane injury index and an increase in the physiological parameters like Relative water content, chlorophyll content and photosynthetic rate confirming the role of OsLecRLK in providing salt tolerance in transgenic pigeon pea plants. Analysis of phenological and yield attributes confirmed that the mechanism governed by this gene is ultimately maintaining the vigor of the pigeon pea plants. A potential increase of 23-26% in the harvest index of transgenic lines conferred the completion of the pigeon pea life cycle in a normal pattern with very minimal effects of salt stress. OsLecRLK expressing pigeon pea lines were found to perform much better than wild types starting from vegetative till the completion of the reproductive phase. Through comparative transcriptome analysis, the intrinsic mechanism responsible for providing salt tolerance in transgenic pigeon pea plants with the OsLecRLK gene was unveiled. The relative expression analysis of histone deacetylase1, acyl CoA, ascorbate peroxidase, peroxidase, glutathione reductase and catalase genes were found higher and the expression of indole acetic acid synthetase, calmodulin and ketoacyl genes were found much lower in transgenic in comparison to wildtype pigeon pea plants under salt stress.
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    ThesisItemOpen Access
    In silico characterization and transcriptome analysis of transgenic pigeon pea [Cajanus cajan (L.) Millsp.] with OsRuvB gene for salt tolerance
    (CCSHAU, Hisar, 2023-01) Redhu, Neeru Singh; Kharb, Pushpa
    Soil salinity reduces the productivity of many agricultural crops, including legumes, which are highly sensitive. In this study, OsRuvBL1a, a DNA helicase, was computationally characterized and analyzed through transcriptomics to elucidate its potential pathway for imparting salt tolerance in transgenic pigeon pea. The sequence with locus ID Os01g0837500 was retrieved. DNA binding (from amino acid residues 1-15, 63-67, and 218-224), RNA binding (from amino acid residues 2-13 and 219-223), and Protein binding (from amino acid residues 9-14, 295-297 and 427-429) sites were predicted. NCBI-CDD predicted it as a member of AAA+ ATPase family and TIP49 (TBP interacting) superfamily. SOMPA used self-optimized prediction method for predicting 49.89% of alpha helix, 14.95% of beta strand, 29.67% of coils and 5.49% of beta turn. RuvB-like helicase (PDB-Id: 2C9O) with 99% query coverage and 73% sequence identity was selected as a template. MODELLER, SWISS-MODEL, Phrye, I- TASSER, Robetta, and IntFOLD predicted model with -15187.475, -19170.473, -12729.712, -18859.584, -21191.418, and -15212.321 kJ/mol energy, respectively. Molecular dynamic simulation was carried out using GROMACS-2019 software for representative structure of all models. RMSD between pre-simulated and simulated models of MODELLER, SWISS-MODEL, Phrye, I- TASSER, Robetta, and IntFOLD were observed to be 1.207, 1.148, 1.201, 1.274, 1.063, and 1.101 (Å), respectively. Even though little change in RMSD was observed with 2C9O template against simulated and pre-simulated models, still the simulated models gave a good understanding of its tertiary protein structure. A total of 19,59,13,490 (~190 million) raw reads were generated by Illumina HiSeq sequencer from transgenic and non-transgenic samples. The 100% of reads were mapped to the pigeon pea genome. The reads were mapped to a total of 21676 transcripts out of which 9407 genes were differentially expressed. The gene ontology of annotation uniquely identified 78 biological processes, 51 cellular component and 128 molecular functions in differentially expressed genes. A total to 378 genes with significant differential expression were identified with >= 3.0 for –log10(p adjusted value). Downstream analysis revealed 32 key genes with potential role in acquiring salt tolerance. 21 genes were found upregulated and 11 were found downregulated belonging to pathways like ABC transporters, flavonoid biosynthesis, fatty acid elongation, tropane, piperidine and pyridine alkaloid biosynthesis and circadian rhythm – plant among others. Although, the present study identifies key genes and pathways involved in the OsRuvB mediated salt stress tolerance in transgenic pigeon pea, more research is required to identify the exact molecular mechanism and the underlying signaling pathways of OsRuvB.
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    ThesisItemOpen Access
    Dissecting the role of OsRuvB transgene for salt tolerance in transgenic chickpea (Cicer arietinum L.)
    (CCSHAU, Hisar, 2023-03-15) Chaudhary, Rinku; Kharb, Pushpa
    Chickpea (Cicer arietinum L.), a self-pollinated diploid (2n = 2x = 16), is one of the most important grain legume with a genome size of approximately 740 Mbp. Chickpea is an annual grain crops with global production of 18.7 million tons, including 11.38 million tons in India. Chickpea is highly sensitive to salinity and its growth is highly affected. Soil salinity decreases the productivity of legume crops including chickpea, which is known for its sensitivity towards salinity. About 20 days old T3 transgenic chickpea seedlings were subjected to 100mM NaCl stress. The seedlings were harvested after 72hrs of NaCl stress and stored in RNA later for trancriptome analysis. A total of approx. 154 million raw reads were generated in all four samples with the total raw data of 49.14GB using Illumina Sequencer. 97493out of 214131 transcripts were observed as uniquely annotated, out of which 24767 genes were differentially expressed and highest number of DEGs were identified from membrane part i.e.15332 DEGs. Molecular function annotation of DEGs listed catalytic activity, transcriptional regulation activity, protein kinase activity, binding activity (heme, ADP), protein serine/threonine kinase activity with the highest members. Some significantly upregulated genes like aldehydehyde dehydrogenase, NADH dehydrogenase, histone, glutamine synthase, Calmodulin, Chlorophyll a,b binding protein, , MAPK, proline-rich protein, MYB48, MYB88, ABC transporter, GABA transporter, zinc finger protein and sucrose synthase were observed. These genes are already reported to play an important role in salt tolerance in various crops, but still more investigation is needed to understand the exact mechanism of salinity tolerance imparted by OsRuvB in transgenic chickpea.
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    ThesisItemOpen Access
    Identification and characterization of Meta-QTLs, ortho-MQTLs, and candidate genes for the traits associated with lodging tolerance in common wheat (Triticum aestivum L.)
    (CCSHAU, Hisar, 2023-05) Nikita; Upendra Kumar
    Lodging is an abiotic stress greatly affected by environmental conditions. It is a state of permanent displacement of stem from its vertical position which can reduce yield up to 80% in crops. A meta analysis of QTLs related to lodging and its associated traits was conducted in the present study to find the accurate and robust genomic positions concerning lodging tolerance in wheat. 86 MQTLs were identified using 1269 initial QTLs collected from 107 earlier studies. A high-density consensus map was generated containing 1,51,945 markers spanning a total length of 11,277.087 cM with an average marker density of 13.47 markers per cM length of map. The marker density for individual sub genome was 29.58 for A, 17.46 for B and 5.55 for D. In the resulting MQTLs there was a 4.13 fold reduction in confidence interval than the initial QTLs. 12 Breeders’ MQTLs were selected based on high PVE value, large LOD score, the greater number of initial QTLs involved and low confidence interval. 10 ortho-MQTLs were found using the Breeders’ MQTLs based on synteny and collinearity among wheat and maize. 678 gene models were found in the Breeders’ MQTL regions out of which 53 genes had an expression value of greater than 5 TPM. The most likely candidate genes belonged to the following families: F-box protein, SANT/Myb domain transcription factor, Pectinesterase inhibitor domain, glycoside hydrolase family, protein kinase domain, zinc finger domain, cytochrome P450, UDP-glucuronosyl/UDP-glucosyltransferase, glycosyltransferase 61 etc. 34 genes were found in wheat which orthologous of already reported genes encoding for lodging tolerance in rice, maize and Arabidopsis. This meta-analysis defines the most stable and consistent genomic locations associated with reliable molecular markers and candidate genes for lodging tolerance in wheat.
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    ThesisItemOpen Access
    Physiological and biochemical changes in selected Brassica juncea varieties under drought stress (Brassica juncea L. Czern. & Coss.)
    (CCS HAU, Hisar, 2022-08) Sharma, Reena; Lakra, Nita
    Mustard is the major oilseed crop that can assist meet India's demand for edible oil, and it ranks third after soybean and oil palm as a source of edible oil. As a result of the drought, its development and seed yield output have been seriously hindered. Therefore, the present study aimed to screen for drought tolerance at different stages among six (RH1566, RH725, RH749, RH725, RH761 and RH30) varieties of Indian mustard. These six varieties were grown as hydroponic in green house and for further analysis four varieties (RH30, RH 8812, RH1566 and RH725) were selected. Screening was done using PEG-6000 treatment to maintain different drought level. Various morpho-physiological (chlorophyll content, root and shoot length, RWC, osmotic potential, Electrolyte leakage), Biochemical, Metabolites were analysed at seedling and pot level. By investigating this study, we found the increasing trend of protective osmolyte (sugar, proline, glycine betaine) and increased activity of oxygen scavenging enzymes (Catalase, ascorbic peroxidase, superoxide dismutase) in severe stress (50 Field capacity %) as compared to its control. Plants under drought stress indicated a significant increase in antioxidative enzyme but this increase was more pronounced in drought tolerant variety. The content of ascorbic acid, carotenoids, total glutathione content were also recorded higher in tolerant variety than in sensitive variety under drought stress. We also studied PAL enzyme activity and reported increased activity under drought stress. This study helps Plant breeders and plant physiologists to design particular ways to alleviate the negative impacts of drought and maximise Indian mustard crop output by better understanding the processes of drought tolerance in Indian mustard.