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ThesisItem Open Access Molecular Screening of maize genotypes for lysine, tryptophan and β-carotene using functional markers(CCSHAU, Hisar, 2023-07) . Kunal; Yashveer, ShikhaMaize 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.ThesisItem Open 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 KumarLodging 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.ThesisItem Open 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, NitaMustard 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.ThesisItem Open Access In-silico characterization and expression analysis of genes responsible for Zn uptake in wheat-Aegilops lines(CCS HAU, Hisar, 2022-07) Raja Kumar; Upendra KumarZinc deficiency is the most ubiquitous micronutrient deficiency in the world affecting nearly 2.6 billion peoples resulting in disease such as growth retardation, hypogonadism, immune dysfunction. It is a particular problem in developing countries, where most people rely on cereals as their staple food. One-third of the world’s cereal growing area is deficient in Zn. Wheat (Triticum aestivum L., 2n = 6x = 42) belongs to family Gramineae is a major cereal crop grown all over the world constitutes approximately 30% of the total cereals consumed by world’s population making it a major source of minerals. Therefore, it is imperative that wheat cultivars with improved concentrations of Zn is necessary to be developed to alleviate malnutrition among peoples. Lack of genetic variation in wheat is a major drawback in mineral biofortification and bioinformatics tools can be used as a potential resource in investigation of genes related to Zn homeostasis. In the present investigation 7 Zn regulatory gene families namely NAS, NAAT, DMAS, ZIP, ZIFL, YSL and TOM had been identified and analyzed for their importance in biosynthesis of phytosiderophores, their uptake and translocation under Zn deficiency. Chinese spring, an elite wheat landrace and UPHAU-3, a wheat-Aegilops addition/substitution line were grown hydroponically under Zn sufficient and deficient conditions. The relative expression of NAS, NAAT, DMAS, ZIP, ZIFL, YSL and TOM measured in UP HAU-3 and CS revealed that DMAS, ZIP and TOM genes are the most potential genes majorly responsible for Zn uptake and transport by release of phytosiderophores under Zn deficiency. The content of phytosiderophores released in the root exudates induced under Zn deficiency was found 17.41% more higher for UPHAU-3 as compared to CS. Therefore, this comparative study represents that under Zn deficiency UPHAU-3 was more potential genotype for more Zn uptake and transport in comparison with Chinese spring, which can be used as a promising wheat genotype for further studies to make Zn biofortified wheat crop for mitigating Zn malnutrition in human population.ThesisItem Open Access Morpho-physiological, biochemical and protein profiling under drought stress in selected chickpea (Cicer arietinum L.) varieties(CCSHAU Hisar, 2022-07) Yama Keerthi Sree; Lakra, NitaChickpea (Cicer arieitnum L.) is an important food legume crop of the family Fabaceae with high protein levels that is widely grown in rainfed areas prone to drought stress. It is a self-pollinated cool season crop with true diploid (2n=16) nature. They are relatively cheaper and higher source of protein. About 90% chickpea crop is grown in rainfed conditions without depending on irrigation. In the present study, two varieties of chickpea namely ICC 4958 and HC-6 were grown under 3 field capacities (100% FC, 50% FC and 25% FC). Sampling was taken 3 times i.e., 15, 30 and 45 days after sowing (DAS). Parameters like morphological, physiological, biochemical, metabolite and protein profiling of the two varieties was done. Under morphological parameters, shoot length, number of branches number of leaves, number of flowers declined as the drought level increased. Under physiological parameters, relative water content (RWC), cell membrane stability (CMS), chlorophyll content decreased in both the varieties as field capacity decreased. Under biochemical parameters, proline, sugar, glycine betaine, superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX) and glutathione reductase (GR) increased as drought stress increased. Under metabolite analysis, MDA (Malondialdehyde) content, total anthocyanin, flavonoid content, phenolic content increased as drought progressed. We also performed protein profiling of the two varieties using SDS-PAGE (Sodium dodecyl-sulfate polyacrylamide gel electrophoresis) to differentiate the expression analysis of the two varieties.ThesisItem Open Access Evaluation of advanced wheat lines for stripe rust resistance and grain quality(CCSHAU, Hisar, 2019-08) Bhawna; Bhawna; Yadav, Neelam R.; Yadav, Neelam R.Biofortification of staple foods like wheat without any yield penalty is of utmost importance. Advanced lines introgressed with Gpc-B1 allele were used in the study. The Gpc-B1 allele co-segregated with Yr36 yellow rust resistance gene in all the advanced lines. All WH711-derived advanced wheat lines confirmed for the Yr36/Gpc-B1 allele. Seven advanced lines (WL-8-1, WL-8-2, WL-8-3, WL-8-5, WL-8-6, WL-8-7, WL-8-8) were confirmed for Yr18 while six advanced lines (WL-8-1, WL-8-2, WL-8- 6, WL-8-7, WL-8-8, WL-8-9) were positive for Yr17 gene. All five PBW343-derived advanced lines confirmed for the Yr36/Gpc-B1 allele presence. Three advanced lines (PL-8-2, PL-8-3, PL-8-4) displayed the presence of the Yr18 gene. Two advanced lines (Pl-8-3, PL-8-5) confirmed presence of the Yr17 gene. All the advanced wheat lines derived from WH711 as well as from PBW343 displayed very high levels of resistance against yellow rust and better agronomic characteristics than the parents. Positive correlation was observed in yield and yield related traits. The hectoliter weights of PBW343-derived lines indicated their superiority in terms of good flour recovery than PBW343 and comparable to the checks. WH711- and PBW343- derived advanced wheat lines show better gluten quality and high quantity in sedimentation test. Gpc-B1 allele introgression provided high GPC in WH711 derived advanced wheat lines. Iron and zinc contents of both WH711-derived and PBW343-derived advanced wheat lines were at par when compared to either parent and checks, indicating successful bio fortification of both micronutrients. Two of the WH711-derived lines are being further tested in IPPSN/station trials.ThesisItem Open Access Development of an efficient protocol for micropropagation of strawberry (Fragaria ananassa Duch.)(CCSHAU, Hisar, 2020-07) Swati Rani; Upendra KumarStrawberry (Fragaria × ananassa Duch.) is the most important fruit worldwide. Micropropagation is mainly for the clonal multiplication.Strawberry cvs. Grenada and Petaluma were micropropagated for rapid shoot and root multiplication. Meristematic part and leaf primordia is source of explants. Foe the sterilization process, the explants were dipped in two to three drops of Tween 20 per 100 ml for 10 minutes with distilled water, 0.5% Bavisitin & Streptocycline for 30 mintues with antimicrobial supplement and 0.1% HgCL2 for 2 minutes gave the maximum aseptic cultures. After the surface sterilization of meristemetic part of plants 3-5 mm long was used as a explant. Multiplication stage results indicate that highest auxiliary buds were observed when MS medium supplemented with 1 mg/l and 1.5 mg/l BAP in Petaluma and Grenada. At rooting stage, it was clear visually that MS medium supplemented with 0.5 mg/l of BAP with 1.5 mg/l of IBA in both the cultivars gave the best results of enhanced number roots with higher length and number shoots with higher length per explants. The best result for root multiplication indicating the use of IBA with ( 1.0, 1.5 mg/l) concentration as compared to other treatments.The highest response for the shoot multiplication was obtained with MS containing 1.5 mg/l and 1.0 mg/l BAP respectively. The present research is very useful in commercializing the new cultivars of strawberry into north Indian conditions which gives higher yield of fruits.ThesisItem Open Access ―Phenotypic and molecular characterization of HD 2967 x Kharchia 65 F5 and backcross generations for salt tolerance in wheat (Triticum aestivum L. em. Thell”(CCSHAU, Hisar, 2019-08) Saini, Pooja; Yashveer, ShikhaSoil salinity is emerging environmental stress that reduces rate of germination, delays emergence and affects almost all stages of growth and development of wheat plants which ultimately leads to reduction in grain yield of crop.The present investigation focused on phenotypic and molecular characterization of selected wheat (Triticum aestivum L. em. Thell)genotypes of F5 and backcross (BC1F4, BC2F3, BC3F1) generations derived from HD 2967 X Kharicha 65 for salinity tolerance on the basis of presence of both Nax loci and their agronomic performance at salt stress level 8 dS/m. A total of 92 plants confirmed the presence of Nax1 locus at a band size of 210 bp consisting of 16 F5, 41 BC1F3, 9 BC2F2 and 27 BC3F1, a total of 53 plants confirmed the presence of Nax2 locus at a band size of 225 bp comprising of 6 F5, 35 BC1F4, 8 BC2F3 and 4 BC3F1.Overall, plants comprising of 6 F5,26 BC1F4, and 4 BC3F1 of HD 2967 x Kharchia 65were found to have both the Nax loci. On the basis of phenotypic and genotypic variations, 56 high yielding plants were selected for genotyping using polymorphic SSR markers. A total of 180 SSR primers were used to study the polymorphism between parents HD 2967 and Kharchia 65, out of which 27 SSR primers showed polymorphism. Polymorphism information content (PIC) value in the studied genotyped ranged from 0.191 (xgwm480) to 0.425 (wmc3) with an average value of 0.339. Cluster tree analysis using NTSYS-pc software, F5 and backcross (BC1F4, BC2F3, BC3F1) generations showed that all the selected plants were inclined toward recurrent parent HD 2967 representing higher similarity with HD 2967. From above analysis, high grain yielding and salt tolerant plants were selected such as plant number 259Q3c, 271Q3c (F5); 202P9b, 303P2b and 202P3b (BC1F4); 241R2c and 243R1a (BC2F3); 25(3)1b and 24(1)1c (BC3F1) and these plants could be further backcrossed with the parent HD 2967 to develop salt tolerant wheat lines.ThesisItem Open Access In silico identification and physical mapping of gene(s) responsible for β-glucan in Bread Wheat (Triticum aestivum. L)(CCSHAU, Hisar, 2020-07) Dhamija, Aryan; Upendra KumarDietary fibers from plant cell wall are an essential component of healthy foods. Higher intake of dietary fiber reduces the risk of diet related chronic disease like type 2 diabetes, obesity and also improves gastrointestinal health. In human diets, cereal fiber is the largest contributor to total dietary fiber consumption. The soluble dietary fiber (1-3) (1-4) mixed linked β-D-glucan from cereal grains is a valuable component of a healthy diet. In the present investigation, the gene responsible for β-glucan in hexploid wheat was identified using both in silico and molecular methods.The in silico analysis confirmed that CslF6 gene in wheat was located on the chromosome 7A. Sequence similarity search was conducted between HvCslF6 and wheat survey sequences displayed a similarity of 96% with chromosome 7A. The structure of this gene had 3 exons and codes for a protein of 945 amino acids. Homology modeling of putative CslF6 protein was described as Probable cellulose synthase A catalytic subunit 8.Different cytogenetic stocks of Chinese spring revealed that CslF6 gene was located on centromeric region of 7AL (FL=0.29). Identified CslF6 in wheat can be utilized to make β-glucan efficient wheat and can be used in biofortification program.
