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ThesisItem Embargo Mapping of Genomic Regions Associated With Anaerobic Germination In Rice Under Direct-Seeded Cultivation Conditions(Punjab Agricultural University, 2024) Verma, Vikas Kumar; Dr. Nitika SandhuRice (Oryza sativa L.) is mainly grown by transplanting rice seedlings into standing water. However, seed transplanting method faces hurdles due to the increasing shortage of water and labour. There is a need of transition from transplanting method of cultivation to direct-seeded cultivation, a method that not only conserves water but also offers additional advantages. To identify the genomic regions associated with anaerobic germination, we used 384 breeding lines derived from seven biparental F3:4:5:6 mapping populations, where IR14D155, IR15D120, IR13F474, IR10F365, IR 127152-2-10, IR 127155-1-22, IR 93312-30-101-20-13-66-6 used as donors and PR126 as recipient parent for anaerobic germination. Significant phenotypic variations have been observed for most of the traits measured in the present study. The study revealed 12 significant marker-trait associations for percent seed germination, distributed across six chromosomes (3, 4, 5, 6, 7, and 9). Notably, a cluster of notable single nucleotide polymorphisms on chromosome 3 consistently appeared over multiple years. Specifically, a 6.9 Mb genomic region spanning from 21089181 bp to 28017712 bp on chromosome 3 was associated with percent germination at 15 and 21 days after sowing. Similarly, a 6.4 Mb genomic segment from 18028538 bp to 24492161 bp on chromosome 6 displayed associations with percent germination at 15 and 21 days after sowing, with specific SNPs (S6_18028538 and S6_24492161) associated with these respective time frames. Additionally, one marker trait association was detected for days to 50% flowering on chromosome 1 and six for grain yield on chromosome 1, 2, 5, 8 and 10.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 Characterization of Bt RNAi cotton plants produced through Agrobacterium-mediated genetic transformation for whitefly resistance(Punjab Agricultural University, 2024) Choudhary, Ankit Kumar; Dr. Prashant MohanpuriaCotton is India's important cash crop, and it plays a significant part in the country's industrial and agricultural economy. Whitefly (Bemisia tabaci) has been one of the most threatening polyphagous pests of cotton and no whitefly resistance germplasm is available in India. Also, there are no Bacillus thuringiensis (Bt) toxins known to be effective against whitefly. Generating RNAi cotton plants expressing dsRNA of vital gene of whitefly can be a novel approach to produce whitefly resistance in available Bt cotton variety. In present work, screening was done to check the presence of integrated RNAi cassette in genomic DNA of BtRNAi cotton plants in T1 generation. For this DNA from true leaves of BtRNAi cotton plants were isolated and PCR with RNAi cassette specific primers resulted in total seven positive plants. Non-chimeric nature of these seven BtRNAi cotton plants also confirmed through DNA-PCR in different branches which resulted in same 208 bp amplicon. Further expression of RNAi cassette (sense fragment of Vitellogenin gene) in seven PCR confirmed BtRNAi cotton plants (T1 generation) was seen. For this total RNA was isolated and cDNA was prepared, and expression of RNAi cassette (208 bp) was observed using qPCR. Upon insect bioassay fecundity rate on leaves of wild cotton plants was much higher (doubled) as compared to confirmed BtRNAi cotton plants and showed the effect of VG gene silencing in whiteflies which were fed on these plants. This proved the development of some tolerance/resistance against whitefly in these produced BtRNAi cotton plants through RNAi.ThesisItem Embargo Saturating genomic regions spanning QNR6.1 and QNR12.2 associated with root-knot nematode resistance in rice(Punjab Agricultural University, 2023) Shabneek Kaur; Yogesh VikalM. graminicola, a potent organism causing the root-knot nematode (RKN) disease, can be found in most of the areas in India. Due to lack of effective chemical control, most effective strategy to combat the RKN is finding new resistant sources and its deployment in elite rice cultivars. Tagging and fine mapping is a pre-requisite for transfer of RKN resistant genes through marker-assisted selection (MAS). Among the wild varieties, O. glaberrima has been proved to be a resistant donor for nematode resistance. A cross was generated between O. glaberrima acc. IRGC 102206 and O. sativa cv. PR121 to transfer the genes for resistance into the commercial cultivar. The potential QTLs, through BSA QTL-seq approach were identified and two of the QTLs qNR6.1 and qNR12.2 were considered major QTLs. Therefore, present investigation aimed to fine map these QTLs identified for nematode resistance. Phenotypic evaluation of BC2F2 and BC2F3 progenies for the morphological traits such as gall number, root length and plant height is done. The genetic studies for gall number revealed that nematode resistance is governed by single dominant gene in O. glaberrima. The BC2F2 and BC2F3 progenies segregated in the desired ratio of 3:1 and 1:2:1, respectively for single gene inheritance. A total of 37 SSR and 33 KASP markers targeting qNR6.1 region and 16 SSR markers targeting qNR12.2 region were surveyed for parental polymorphism. The 17 polymorphic markers were genotyped on BC2F2 population. The total map length obtained was 1.034 Mb which was saturated from the original region of 3 Mb. Two QTL peaks flanked by markers NRK_13 & NRK_20 and NRK_21 & RM7158 were detected within the qNR6.1 region explaining 29.87% of total phenotypic variance. The new detected region needs to be saturated with more markers so that MAS can be followed for root-knot nematode resistance rice breeding programs. A total of 42 potential candidate genes were detected within the QTL region and probably six of them have putative role in nematode resistance. Further, role of candidate genes could be explored to understand the rice root knot nematode resistance mechanism.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 Fine mapping and cloning of Brown planthopper (BPH) resistance introgressed from Oryza nivara to O. sativa(Punjab Agricultural University, 2023) Pavneet Kaur; Kumari NeelamBrown planthopper (Nilaparvata lugens Stål; BPH), is one of the major constraints of rice productivity in Southeast Asia. Here, we report fine mapping of a novel BPH resistance gene Bph34 from the wild species of rice Oryza nivara accession IRGC 104646 that localized to a 91 kb region of Nipponbare reference genome IRGSP-1.0 containing 11 candidate genes. Phenotypic screening of 250 F9 RIL population and 1000 F2:3 progenies against BPH biotype 4 revealed 1:1 and 1:2:1 segregating ratio, respectively and hence confirmed the single gene control of Bph34. Based on genotypic data of F9 population, we observed 26, 25, 2, 8, 11, and 11 recombinants at LOC_Os04g35180, LOC_Os04g35190, LOC_Os04g35210, LOC_Os04g35240, LOC_Os04g35260, and LOC_Os04g35280, respectively. This indicates that LOC_Os04g35210 stands out as a promising candidate among these 11 genes. Further, presence of only 3 breakpoints among F2:3 progenies robustly support the candidacy of LOC_Os04g35210 as Bph34. The qRT-PCR-based transcript abundance assay indicates a notable increase in the expression of LOC_Os04g35210 in resistant plants at 48 hours of post-BPH infestation. The sequencing of the coding domain sequence of the locus LOC_Os04g35210 indicated a single exon of 2706 bp in O. nivara and 2712 bp in PR122. A gene construct carrying CDS of Bph34 resistance gene was subjected to validation using colony PCR, plasmid PCR followed by double digestion of 6 positive plasmids. The recombinant plasmids with desired fragment size will be preceded for mobilisation into Agrobacterium strain and generation of T0 plants. Fundamentally, this research identifies and characterizes a novel Bph34 resistance gene, paving the way for its application in plant breeding initiatives.ThesisItem Restricted Genomic Assisted Mapping Of Nematode (Meloidogyne Graminicola) Resistance From Oryza Longistaminata(Punjab Agricultural University, 2023) Jessica Kaur; Yogesh VikalIndia's rice production averages 195.43 million tonnes over 46.379 million hectares but is threatened by the destructive nematode Meloidogyne graminicola. This parasite can cause yield losses of up to 70% in lowland, upland, and deep-water rice, posing a significant risk to rice cultivation. Managing M. graminicola in rice fields typically involves flooding, nematicides, and crop rotation, but these methods have limitations. Exploring resistance in rice genotypes offers a promising alternative to reduce crop losses. This present study aims to identify the gene/QTLs responsible for resistance against the root-knot nematode (Meloidogyne graminicola) sourced from Oryza longistaminata acc. IRGC89153, by utilizing the F2 generation resulting from a cross between Oryza sativa cv. PR121 and Oryza longistaminata acc. IRGC89153. The F2 and F3 generations were screened in triplicates during the Kharif 2022 and Kharif 2023 respectively, within nematode-infested sick plot, where the initial nematode population density was 3 J2/gram of soil. After screening, the soil revealed a final nematode density of 10 J2/gram, signifying a threefold rise in the M. graminicola population from the initial density. These nematodes induce the development of root galls, impeding root growth, disrupting physiological processes, and obstructing the transport of water and nutrients through the root's vascular system. This leads to observable above-ground symptoms, including stunting, reduced vigour, chlorosis ultimately resulting in significant yield losses. Morphological trait analysis, encompassing parameters like plant height and root length, disclosed significant differences between infested and controlled (non-infested) conditions. Based on screening, resistant and susceptible bulks were made from the F2 population and along with parents were sent for whole genome re-sequencing. The analysis identified QTLs for nematode resistance on chromosomes 4 (qNR4.1) as a major QTL and a minor QTL on chromosome 10 (qNR10.1) with differing SNP counts and densities. The development of SNP-based molecular markers from these QTL regions will aid in identifying markers closely associated with rice root-knot nematode resistance. These markers will facilitate the accelerated improvement of genotypes for nematode resistance through marker-assisted breeding.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.
