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ThesisItem Embargo CRISPR/Cas9-mediated pectate lyase gene editing for enhanced shelf life of tomato (Solanum lycopersicum L.)(Punjab Agricultural University, 2024) Sumedha; Dr Prashant MohanpuriaTomato is one of the most consumed vegetable crops worldwide. Tomato fruits are having an inherently short shelf life which is directly related to fruit softening, caused by cell wall modifying enzymes. Among which pectate lyase (PL) controls fruit softening and thus PL gene was targeted using CRISPR/Cas9 to enhance shelf life of tomato variety Punjab Ratta. In silico study of PL genes from Solanaceous crops including tomato was attempted and three sgRNAs were designed from exon-2 of PL gene. The CRISPR/Cas9 constructs containing sgRNA-1 and 2 were used for transformation of tomato. A new in planta transformation protocol was developed with 10.49 % overall transformation efficiency. Floral buds of 3-8 days of developmental stages were used for dipping in Agro suspension which resulted in setting of 150 fruits. The extracted T0 seeds were selected on kanamycin. A total of 3240 T1 generation plants were regenerated from transgenic tomato fruits (T0), out of which 1620 were found kanamycin resistant which were used for DNA isolation and PCR confirmation of T-DNA integration. The 425 bp PL gene target region from 170 transgenic tomato T1 plants was PCR amplified. Upon sequencing eight T1 tomato transgenic plants were found with PL gene edits containing T insertion, T insertions as well as T substitutions, GC substitution by CG as well as TT insertion downstream to PAM site. PL gene expression and pectin content were decreased in fruits of mutated tomato plants but morphological and biochemical characteristics were more or less similar.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 Restricted Identification and characterization of genes for regular and irregular bearing in mango (Mangifera indica L.)(Punjab Agricultural University, 2022) Harmanpreet Kaur; Sidhu, Gurupkar SinghThe mango, known as the "King of Fruits," is a member of the Anacardiaceae family and is the most important fruit crop grown in India because of its exquisite flavour. Flowering is a crucial phenophase, since it directly impacts the production. Mango flowering, however, is a complicated phenomenon. Typically, it bears a large crop in one year (on year) and produces little to nothing in the following year (off year). The gene expression analysis for flowering genes in regular (Amrapali and Neelum) and irregular (Dashehari) bearing cultivars was done using RNA-Seq. Illumina technology was used to sequence the cDNA libraries made from the leaves, shoot apex, and inflorescence tissues of Dashehari, Amrapali, and Neelum. For Dashehari, Amrapali, and Neelum, paired-end high-quality clean reads of 117 Mb, 74 Mb, and 24 Mb, respectively, were obtained. Dashehari's de novo assembly generated 67,915 transcripts, 25,776 trinity genes, and N50 value of 1,981. The transcripts were annotated using BLAST2GO and PfamScan, and the biological process, molecular function, and cellular component functional categories of GO were used to group the genes. Major pathways include the sucrose and starch metabolism, tryptophan biosynthesis, trehalose biosynthesis, and phenylpropanoid biosynthesis were found by KEGG and Plant Reactome analyses. From the FLOR-ID flowering database, ortholog transcripts were found using reciprocal blast, and 85 genes relevant to flowering were found. In this study, more genes were found to be up-regulated in leaves of Dashehari bearing tree than in non-bearing tree, in inflorescence than in leaf and apex, and in Amrapali than Dashehari among varieties. In particular, genes associated with photoperiod (CO, GI, FTIP1 and FT), vernalization (FRI4 and VIN3), the circadian clock (LHY1, TIC and PRR7), age (TOPLESS and SPL15), and the hormonal pathway (BR1, EIN3, T6P and GA20OX) were identified. Using qRT-PCR, we validated 18 flowering-related genes for regular and irregular bearing in the three genotypes. All the genes demonstrated greater expression values in leaves of Dashehari bearing tree as compared to non-bearing tree and in Amrapali, which is congruent with the expression values revealed from the transcriptome data. These results will help in the discovery of regulatory regions and factors implicated for regularity in mango fruit bearing, and they will establish the foundation for understanding the cellular and molecular mechanisms involved in regular and irregular bearing fruit varieties.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 Qtl For Heat Tolerance Component Traits In Triticum Durum Desf. X Aegilops Speltoides Tausch. Backcross Introgression Lines And Transfer To Triticum Aestivum L.(Punjab Agricultural University, 2023) Navaneetha Krishnan J; Chhuneja, ParveenWheat, a major cereal crop, is the most consumed staple after rice in India. Frequent episodes of heat waves during the past decade have raised concerns about food security and necessitates the development of heat-tolerant wheat cultivars. A mapping population comprising 315 BC2F10 backcross introgression lines (BILs) was developed by crossing Triticum durum cultivar PDW274 and heat tolerant diploid wild relative Aegilops speltoides accession pau3809 to map QTLs for terminal heat tolerance. The homozygous BILs were evaluated for heat stress tolerance component traits under an optimum environment (OE) and a heat-stressed environment (HE) for the two cropping seasons 2017-18 and 2018-19. Data on spike length, spikelet number per spike, peduncle length, thousand-grain weight, grains per spike, days to heading, days to maturity, grain filling duration, NDVI at heading and plot yield were recorded. Heat tolerance index was calculated for all the traits except plant height and used for QTL mapping. Genotyping-by-sequencing (GBS) of the BILs was carried out, and 3085 polymorphic SNPs were obtained. The QTL mapping for heat tolerance traits detected fifty QTLs on all the chromosomes except 7B. The PVE of the mapped QTLs ranged from 5% to 11.5%, indicating the minor effect contributed by the QTLs on the total phenotypic variability. Several candidate genes reported to play a role in heat stress responses were identified by browsing the 1 Mb physical region flanking the stable QTLs detected under the HE. To introgress the heat tolerance QTLs, three BILs, DS201, DS394 and DS437, which were superior for one or more phenotypic traits, were crossed with the popular bread wheat cultivars PBW723 and PBW725 along with an advanced breeding line CB762 (= PBW550+GpcB1+Yr5). Kompetitive allele-specific PCR (KASP) markers were designed based on the mapped SNPs for targeting the QTLs associated with heat tolerance traits. But these markers failed to differentiate between the donor BILs and the bread wheat parents, making them unfit for marker-assisted selection. Therefore, phenotypic selections were carried out from the BC2F2 generation onwards to select promising lines for important heat tolerance traits. Nineteen BC2F3 lines were selected that performed better than the respective bread wheat parents for one or more of the heat tolerance-associated traits. The selected BC2F3 lines can be advanced further and screened for heat tolerance at multiple locations. A well-developed root system is essential for efficient nutrient and water uptake. The BILs were evaluated for various root architecture traits during the 2019-20 and 2020-21 cropping seasons. The roots were sampled at the maximum tillering stage and data on various root architecture traits namely, total root length, root surface area, root projected area, root volume, average root diameter, number of root tips, number of root forks, number of root crossings and root dry weight were recorded. The QTL mapping for root architecture traits detected twenty-one QTLs for various root architecture traits on chromosomes 1A, 2A, 2B, 3B, 5A and 6B. Scanning of the 1Mb region flanking the mapped SNPs linked to the stable QTLs revealed multiple genes involved in root growth and development. These QTLs can be used in breeding programmes after the development and validation of suitable marker assays.
