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ThesisItem Restricted Genome editing of Ideal Plant Architecture-1 (IPA1) gene for enhancing tillering in wheat (Triticum aestivum L.)(Punjab Agricultural University, 2023) Islam, Asif; Chhuneja, ParveenWheat is a global staple cereal, and breeding initiatives aim to increase wheat yield to meet consumption and supply needs. Increasing wheat tillers is a key factor in crop yield. Understanding the ideal wheat plant structure helps explain tiller regulation. Ideal Plant Architecture IPA1 gene in rice enhances number of productive tillers. CRISPR/Cas9 based genome editing with Agrobacterium-induced transformation targeting IPA1 wheat orthologs was undertaken in the present study. The guide RNAs (gRNAs) were engineered to target exon 1 of the IPA1 homeologs TraesCS7A02G246500, TraesCS7B02G144900, and TraesCS7D02G245200, simultaneously and subjected to an in-vitro cleavage assay, which identified gRNA 7ABDE1 as a potential candidate. The gRNA cleaved the 1190 bp amplicon containing exon 1 into two 1050 and 139 bp fragments. The gRNA was then injected into the AGL1 strain of Agrobacterium together with the JD633 vector, which delivered the sgRNA and Cas9 components into the plant system. Sanger sequencing with target-specific primers validated the construct assembly's transformation into the wheat genome. A total of 500 wheat immature embryos excised 14 days post-anthesis were used to introduce the Cas9-cassette and after two cycles of Hygromycin selection, 85 putative transformed calli are in the regeneration media. In addition, 54 previously generated GE0 plants for IPA1 gene were characterized for the presence of Cas9 cassette and 284 GE1 plants derived from these GE0 plants were phenotypically investigated and subjected to Sanger sequencing for putative mutations in the target gene. Confirmed genome edited plants will be further studied for the effect of mutations on the plant architecture.ThesisItem Embargo Next-generation sequencing and comparative analysis of onion mitochondrial genome for the identification of gene(s) conferring cytoplasmic male sterility(Punjab Agricultural University, 2022) Solanki, Ravindra; DeepakCytoplasmic male sterility (CMS) is caused by the gene(s) present in the cytoplasm which has been exploited for the production of F1 hybrid seeds in onion (Allium cepa L.). This phenomenon has been previously reported in long day onion varieties, but is still unexplored in short-day Indian onion varieties. In this study, we have used Illumina Hiseq platform for sequencing mtDNA of 97-A (CMS) and 97-B (maintainer) line that resulted more than 10 million high-quality paired-end reads respectively. These reads were processed for de-novo assembly using Spades software that resulted 7 and 105 contigs for 97-A and 97-B respectively. Finally, we obtained a complete circular genome with the help of primer walking technique for 97-A that comprised of 316321bp whereas the genome of 97-B remained linear with 15 scaffolds. Further, the genome annotation revealed that there were all 24 core protein coding genes along with 24 and 28 tRNA genes present in the 97-A and 97-B mitochondrial genome respectively. Further, comparative genome analysis of both the assemblies revealed that the genomes of both 97-A and 97-B are more or less similar except for the chimeric orf725 gene. Pairwise alignment of 97-A orf725 and 97-B COX-1 gene suggested that in 97-A (CMS) line, complete cox1 sequence was present at 5‘end and further extended by 577bp due to change in stop codon that might be the possible cause of sterility.ThesisItem Restricted Understanding the micro RNA control of lignification and its impact on organoleptic traits in guava cultivars(Punjab Agricultural University, 2023) Prabhleen Kaur; Mittal, AmandeepGuava, the climacteric "super fruit" when over-ripens develop a rough taste suggesting lignification as observed in loquat, pear, mangosteen, citrus and others. We analysed white (Allahabad Safeda, Hisar Safeda), pink (Punjab Pink, Arka Kiran and Lalit) and purple (Purple Local) fleshed guava genotypes for their biochemical characteristics of lignification and antioxidant potential at development (Immature vs ready to eat), ripening, over ripening and refrigerated conditions. The lignin content was found higher in immature fruits which varied from 2.54 % FW in Punjab Pink to 1.70% FW in Purple local concomitant with high G-POX activity and H2O2 concentration. At harvest maturity lignin content gets reduced along with a reduction in the G-POX activity, making this the most preferable stage from consumer aspect. As the ripening proceeds, lignin content again begins to rise along with high G-POX and PPO activity which altogether might be responsible for development of unpleasant flavor and rough taste. We found refrigeration of fruits tends to retard lignification process than storage under ambient conditions. High anthocyanin content of PL could be a possible reason leading to high CATLASE activity and increased H2O2 content. miRNAs negatively control the expression of target genes. We identified 1183 conserved miRNAs from in-house developed guava genome of cv. Allahabad Safeda. qRT-PCR expression analysis in guava cultivars reveal that laccases LAC4, LAC5, LAC17, CSE and POX 63 play significant role in the lignification process and were inversely related with their corresponding miRNA397b-5p, miRNA397b-3p, miR419 and miR11418. Higher expression of L-APX and few MYBs with downregulation of corresponding miRNAs miR828, miR858, miR159 in PL at various stages of post-harvest might be contributing to anthocyanin development in PL.ThesisItem Restricted Identification Of Candidate Gene For Genetic Male Sterility Gene, ms-1 IN MUSKMELON (Cucumis melo L.)(Punjab Agricultural University, 2023) Goyal, Aashima; Navraj KaurHybrid breeding is preferred in muskmelon as hybrids are uniform, higher yielding and early maturing. Genetic male sterility is one of the most extensively exploited pollination control mechanisms for hybrid breeding in muskmelon. However, a codominant molecular marker is required to rogue out heterozygous male fertile plants in the female line before pollination. Previously, during the molecular mapping of the male sterility ms-1 gene, six putative candidate genes were identified in 400kb region on chromosome six. The present study was conducted for the identification and validation of male sterility ms-1 candidate gene through gene expression analysis and SNP genotyping. Two contrasting parents for the ms-1 trait namely, MS-1 inbred line (male-sterile) and KP4HM-15 inbred line (male-fertile) were used. The flower buds of four different sizes referring to different stages of microspore development were used for gene expression analysis using real time PCR. Of all the six putative candidate genes the expression of PDI gene was significantly higher in male fertile parent KP4HM-15 than male sterile parent MS-1. Insilico analysis also revealed the missense mutation within its protein coding region confirming the PDI gene to be most likely the candidate gene for ms-1. Further, the two parents were used to generate the F2 mapping population. Ten samples each with male-sterile genotype (msms) and male-fertile genotype (MsMs) were pooled separately, to construct male sterile bulk and male fertile bulk respectively. One polymorphic KASP marker was able to distinguish both male sterile and fertile parents and the extreme bulks. The SNP site for this polymorphic KASP marker was present in the exonic region of PDI gene. Thus candidate gene (PDI) and the KASP marker linked to male sterility gene ms-1 were identified and validated which can be used for early selection of male sterile plants.ThesisItem Restricted Inheritance and Mapping of Ascochyta Blight Resistance Derived from Chickpea (Cicer Arietinum L.) Cultivar ‘PBG 7’(Punjab Agricultural University, 2023) Manisha Rani; Ajinder KaurIn the present study, a total of 208 RILs derived from cross HC 5 ᵡ PBG 7 were evaluated for inheritance studies and mapping of Ascochyta blight resistance during rabi season 2021-22 in Experimental Area of Pulses Section, Ascochyta blight nursery and Molecular Biology Laboratories, Punjab Agricultural University, Ludhiana. The plant material was planted in α-lattice plot design with two replications for assessment of important agronomic traits. The RIL population segregated in the ratio 3 (Resistant): 13 (Susceptible) for Ascochyta blight disease and indicated that the Ascochyta blight resistance was governed by two genes and there was epistatic interaction between two genes, with dominant allele of a gene (suppressor gene) at one locus suppressing the action of both dominant and recessive alleles of second gene at a completely different locus. For mapping of AB resistance, sequencing based Bulk Segregant Analysis approach was used and four potential QTLs (3 on chromosome no. 4 and 1 on chromosome no. 6) were identified. For validation of identified QTLs, 47 SNP based KASP markers were designed and validated on parents and bulks. Out of 47 KASP markers, 15 showed polymorphism (9 markers for chromosome no. 4 and 6 markers for chromosome no. 6). Using the information of position of these markers on linkage groups, 2 genomic regions (1.75 Mb on chromosome no. 4 and 0.212 Mb region on chromosome no. 6) associated with AB disease response were tagged. Upon statistical analysis of phenotypic data, it was observed that a total of 96 RILs and 98 RILs were significantly better than both the parents for number of pods per plant and total seed yield respectively.ThesisItem Restricted Optimization of gene expression assays and growth medium composition for developing an in vitro flower induction system in sugarcane(Punjab Agricultural University, 2023) Gongati Pavani; Malhotra, Pawan KumarFlowering is necessary in sugarcane not just for their species propagation in nature also important for the crop improvement. The erratic and unpredictable flowering in the cultivated canes is hindering the breeding programmes. While in the commercial fields, flowering in canes depletes the sucrose reserves accumulated in the stalk there by posing the detrimental impact on sucrose juice quality and quantity. So, either to regulate the flowering bypassing the negative impact on the sucrose yield or to standardize the speed breeding protocols, there is a need of studying the flowering in sugarcane. The present work explains relative gene expression analysis done using q RT-PCR in Saccharum spontaneum, Saccharum robustum, Saccharum officinarum species and also in cultivated varieties after the photoperiod treatment. All the three Saccharum species reported a significant upregulation for the FT3 and FT4 genes, suggesting that FT3 and FT4 are the dominant PEBP genes involved in flowering mechanism of sugarcane. The photoperiod treatment given to the cultivated cane varieties resulted in elevated levels of FT1, making FT1 as the PEBP member to act dominantly in the canes during 12hr 30min of day-length. Marcotting procedure was followed by various research groups in sugarcane but, mortality of the marcotted canes is one of the drawbacks. In the marcotting procedure followed in this present study, IBA 2500ppm resulted in optimum rooting percentage. To minimize the mortality rates after detaching the marcotted canes different compositions of nutrient media were tested. Out of all, marcots that were established in the 2 litres of MS medium supplemented with 100g of sucrose and 200ppm of GA3 were able to survive for 54 days and also reported stem elongation especially in the nodes present in the top half portion of the marcots. This present study yielded satisfactory results giving insights into the molecular aspects of the flowering in sugarcane and also optimized the survival rates of the marcots after their detachment.ThesisItem Restricted Mapping of Sheath blight resistance QTL(s) from a land race ShB-8 in rice(Punjab Agricultural University, 2023) Rasool, Sheezana; Neelam KumariSheath blight is the second most devastating disease of rice worldwide, caused by necrotrophic fungus Rhizoctonia solani Kuhn. The crop losses due to sheath blight can go up to 60%, affecting both grain yield and quality of rice. The finest control strategy is to develop genetically resistant variety and mapping of QTLs is a valuable starting point in this direction. In present study, we report mapping of ShB resistance QTLs from a land race ShB-8 in rice. The population under study was evaluated under field conditions during Kharif 2021 and 2022. A positive correlation between disease score, lesion height and relative lesion height was observed. Disease score was negatively correlated with plant height and heading date during both seasons. A total of 28 QTLs were mapped for nine disease variables using 4622 single nucleotide polymorphic markers. Among the mapped QTLs 22 were major QTLs distributed over chromosome 2, 4, 6, 8, 9, 10, 11 and 12, and 6 were minor QTLs distributed over chromosome 3, 4, 9, 11 and 12. The QTLs mapped for disease score colocalized with QTL for relative lesion height. Only one QTL was mapped for LH which localized with QTL for three other traits with same peak marker. This region was found to contain more than 8 NB-LRR genes. The genotypes which consistently showed disease resistance can be further utilised for future research. The QTLs identified can also be further used in the molecular analysis of mapping populations and enhancement of ShB resistance in rice.ThesisItem Open Access Introgression profiling of F4 population derived from the cross of Zea mays × Teosinte spp. mexicana using SSR markers(Punjab Agricultural University, Ludhiana, 2022) Ashmita; Sharma, PritiTeosinte is the wild ancestor of maize also known as God of grains. Teosinte populations could serve as a genetic bridge for transferring genes from one maize population to the next. The present investigation was carried out using 109 recombinant inbred lines (RILs) derived from the cross of Popcorn and Teosinte. RILs along with parental lines were grown in three replications and data was recorded for morphological traits like days to maturity, number of cobs, number of tillers, seed rows per cob and cob length. Analysis of variance showed significant variation among all traits. Highest positive correlation was observed between number of rows per cob and cob length. Genotyping was done using SSR markers from maize database spanning all the maize chromosomes. Out of 250 markers used, 35 were polymorphic. Introgression profiling of all the lines along with parents was done to analyze the introgressed regions of Popcorn and Teosinte in each RIL. Introgression profile of different lines were inferred from consensus of genotypic and morphological data which revealed that marker bnlg1297 was found to be common among the lines having more number of tillers and cobs. For lesser number of maturity days marker bnlg1131 was present in most of the lines. Therefore, these genomic regions might be associated with trait. Further, more number of markers can be used to identify the regions associated with different traits.ThesisItem Restricted Mapping of leaf rust and powdery mildew resistance from Aegilops triuncialis derived disomic substitution line in wheat(Punjab Agricultural University, Ludhiana, 2022) Dhillon, Gurmanpuneet Singh; Satinder KaurWheat (Triticum aestivum) is the leading cereal crop supplying 20% share of the proteins and energy in the human diet. However, it faces significant yield constraints due to losses caused by various diseases, especially rusts and powdery mildew. To manage these losses, incorporation of genetic resistance is the best strategy. In present study a disomic substitution line [DS5Ut(5A)] having adult plat durable resistance to leaf rust (LR) and complete resistance to powdery mildew (PM) was used for mapping of LR and PM resistance. A set 95 chromosome segment substitution lines (CSSLs) generated from a cross of disomic substitution line [DS5Ut(5A)] with Ph1b suppressor Pavon Ph1b followed by crossing with LR and PM susceptible cultivar WL711. The CSSLs were screened against LR and PM for two years in which 25 lines showed resistance against LR (≤ 20MR) and 2 lines showed resistance against PM (≤ 3). Four CSSLs were identified showing resistance against both leaf rusts and powdery mildew which will serve as a useful resource to transfer the respective resistance to susceptible cultivars to develop all stage resistant elite cultivars. Genotyping of CSSLs through genotyping by sequencing (GBS) identified 693 single nucleotide polymorphisms (SNPs) loci specific to chromosome 5A-5U. Introgression profiling detected presence of 26.84% to 55.70% genome of 5U chromosome of Aegilops triuncialis in CSSLs. Genomic region providing resistance against LR races Puccinia triticina (Pt) pathotypes 77-5 (THTTS) and 77-9 (MHTTS) were found to be linked with SNP named S5A_582102813. Similarly genomic region providing resistance against mixture of Blumeria graminis f sp tritici (Bgt) pathotypes found to be associated with S5A_173251584 and S5A_180049756. The linked SNPs will be helpful in marker assisted transfer of these two important gene into elite wheat background.
