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Chaudhary Charan Singh Haryana Agricultural University, Hisar
Chaudhary Charan Singh Haryana Agricultural University popularly known as HAU, is one of Asia's biggest agricultural universities, located at Hisar in the Indian state of Haryana. It is named after India's seventh Prime Minister, Chaudhary Charan Singh. It is a leader in agricultural research in India and contributed significantly to Green Revolution and White Revolution in India in the 1960s and 70s. It has a very large campus and has several research centres throughout the state. It won the Indian Council of Agricultural Research's Award for the Best Institute in 1997. HAU was initially a campus of Punjab Agricultural University, Ludhiana. After the formation of Haryana in 1966, it became an autonomous institution on February 2, 1970 through a Presidential Ordinance, later ratified as Haryana and Punjab Agricultural Universities Act, 1970, passed by the Lok Sabha on March 29, 1970. A. L. Fletcher, the first Vice-Chancellor of the university, was instrumental in its initial growth.
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ThesisItem Open Access Molecular characterization for targeting foliar blast disease in pearl millet [Pennisetum glaucum (L.) R. Br.](CCSHAU, 2023-04) Kamboj, Aarti; Yashveer, . ShikhaIn 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 resultsThesisItem Open Access Molecular characterization of wheat-Aegilops addition/substitution lines for grain β-glucan content(CCSHAU, Hisar, 2023) Prexha; Upendra Kumarβ-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.ThesisItem Open Access Identification of microRNAs targets in response to salt stress in wheat (Triticum aestivum L. em. Thell)(CCSHAU, Hisar, 2023-03) Sharma, Kritika; Yashveer, ShikhaMicroRNAs 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.ThesisItem Open 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 KumarReceptor 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.ThesisItem Open 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, PushpaSoil 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.ThesisItem Open Access Dissecting the role of OsRuvB transgene for salt tolerance in transgenic chickpea (Cicer arietinum L.)(CCSHAU, Hisar, 2023-03-15) Chaudhary, Rinku; Kharb, PushpaChickpea (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.ThesisItem Open Access Characterization of bacteriophages against ESKAPE pathogens and assessment of their synergy with antibiotics(Chaudhary Charan Singh Haryana Agricultural University hisar, 2022-09) Vashisth, Medhavi; Yashveer, ShikhaThe current study was carried out to isolate and characterize bacteriophages with therapeutic potential against ESKAPE pathogens and their synergy assessment with the routinely used antibiotics to control the infection caused by drug resistant strains. A total of 36 different bacteriophages were isolated and characterized for their morphology, physiological stability, molecular & proteomic profile, lytic spectrum and lytic efficiency using spot and EOP assays. Phages φSA116, φKP205, φAB182, and φPA180 were the most lytic out of all the phages isolated during this study. Further, eight bacteriophages were tested with an array of antibiotics to control the growth of drug resistant strains of ESKAPE pathogens using disk embedded double agar overlay method, time kill curve assays in liquid infection models, mutant frequency assays, and in eradication of biofilms. After preliminary screening by double agar overlay method, phages φSA115 (p<0.0001), φSA116 (p<0.0001), φKP202 (p<0.001), and φAB182 (p<0.001 & p<0.0001), showed synergy with sub-inhibitory concentrations of β-lactams (CEP, CAZ, CTX) and polymyxins (PB, CL) to inhibit the bacterial growth as observed by reduced bacterial turbidity compared to the individual treatment with phages and antibiotics. The synergistic effect of phages φKP205 (p<0.0001), φPA176 (p<0.0001), and φPA180 (p<0.0001) with β-lactams (AT, CAZ and PI) was even more pronounced in terms of greater reduction in bacterial turbidity. However, with some of the antibiotics (AK, GEN, AZM, TE), antagonistic activity, in form of smaller sized plaques of bacteriophages was observed. The combination of φPA180 with AT and PI demonstrated the strongest suppression of resistance evolution. A cocktail of φAB182, φKP205, and φPA180 phages, with colistin was demonstrated to successfully eradicate multi-species biofilms of ESKAPE pathogens. Thus, combination therapy of phages with antibiotics presents a potential alternative to control difficult-to-treat infections caused by resistant strains of ESKAPE pathogens.ThesisItem Open Access Genome-wide association mapping in barley for terminal heat tolerance and malting quality(CCSHAU, Hisar, 2021-07) Verma, Swati; Yashveer, ShikhaBarley (Hordeum vulgare L.), is one of the major cereal crops and has been cultivated since ancient times in different parts of the world. In particular, heat stress at the post-heading stage causes considerable yield reduction due to the stress. Understanding the genetic variation, changes in physiological processes, and level of genetic diversity existing among genotypes are needed to produce new cultivars not only having a high tolerance to heat stress, but also displaying high yield. So to address this challenge, a panel of 316 diverse barley genotypes (AM2017) were procured. It was evaluated under two conditions timely-sown (TS) and late-sown (LS) conditions in two seasons of 2017-18 and 2018-19 at CCSHAU, Hisar. Ten agro-morphological, four physiological, and five grain malting quality traits were studied. Genetic diversity and population structure were explored in the AM2017 panel after genotyping it with the 50 K iSelect Illumina Barley SNP array. A set of 36,793 SNP markers, covering a genetic distance of 991.82 cM with an average marker density of 37.09 SNPs/cM, was obtained after quality filtration. The gene diversity (GD) and Polymorphic Information Content (PIC) at the genome level were 0.362 and 0.289, respectively. The phenotypic results showed a clear reduction in trait performance under the LS condition. Marker-trait associations (MTAs) were estimated using the compressed mixed linear model. Based on the estimated MTAs and linkage disequilibrium (LD) decay observed in the genome, putative QTL was identified as associated with the traits studied. The most robust QTL observed under both sowing conditions were further studied and validated using the previously reported QTL. Several traits were found to have QTL that was not found to be reported. The combination of analyses using SNPs and changes in physiological traits provided useful information on genomic regions taking part in heat stress tolerance.ThesisItem Open Access Molecular approaches for detection and forecasting of wheat yellow rust(CCSHAU, Hisar, 2020-03) Rizwana Rehsawla; Yadav, Neelam R.Yellow or stripe rust, caused by Puccinia striiformis f. sp. tritici (Pst), is a devastating airborne disease that affects bread wheat in the major wheat growing regions of India. The understanding of the origin, evolution, pathogenicity, avirulence/virulence behaviour of Pst is very important for the development of more effective management strategies to combat the disease spread. To understand inter and intraspecific phylogenetic relationship among Indian Pst pathotypes, multigene sequence analysis was done. Molecular marker study along with sequencing technology was used to collect information, which was more effective than virulence characterization. The molecular diversity analysis among 13 different Pst pathotypes showed two major cluster formations at similarity coefficient of 0.78. Sometimes, all three wheat rust or two rusts in combination occur simultaneously in the field. Under such conditions identification and differentiation of the yellow rust is needed for precise identification and high throughput DNA based detection protocols. To address this problem PCR based markers were developed which can specifically detect and differentiate Pst from two other rust species of Puccinia and other wheat pathogens. DNA-based methods such as conventional PCR have revolutionized plant disease detection; they are not very reliable at asymptomatic stage. Therefore, a simple and reproducible LAMP assay was developed which could detect the pathogen at an early stage i.e. 3rd day of post infection without any visible sign of pathogen attack on the leaf sample using LAMP primers available in public domain. Four novel sets of LAMP primers from ketopantotate reductase gene were also designed for Pst detection which worked successfully. Biosensing by electrochemical and SPR for the detection of yellow rust was also undertaken. Electrochemical based sensing was done using different sequences of Pst specific genes as probes. Linear response over wide DNA concentration range from 10 pg/μl to 115ng/μl was obtained with a high sensitivity, accuracy and reproducibility. The lowest detection limit was observed for microRNA like RNA 1 gene i.e. 10 pg/μl. Electrochemical DNA based biosensing was developed to distinguish between the yellow rust susceptible and resistant wheat genotypes using TaATG8j gene sequence. Linear response over wide DNA concentration range from 1 ng/μl to 50 ng/μl was obtained with detection limit of 4 pg/μl. The SPR biosensor demonstrated high specificity and long shelf life thus promising its application in Pst diagnosis. The developed biosensor exhibited a high sensitivity (0.18°/ (ng/μl)), good linearity, low detection limit (1 ng/μl) and high specificity over a wide concentration range of DNA (1–150 ng/μl).
