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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 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 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 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.ThesisItem Restricted Mapping and transfer of higher grain length and multiple rust resistance from T.dicoccoides to cultivated wheat(Punjab Agricultural University, Ludhiana, 2022) Manpreet Kaur; Satinder KaurWild emmer wheat, Triticum dicoccoides, the progenitor of modern tetraploid and hexaploid wheats, is an important resource of new variability for disease resistance genes and grain yield traits. T.dicoccoides acc. pau14723 showed resistant to leaf and stripe rust races and was crossed with T.dicoccoides acc. pau4663 (susceptible to leaf and stripe rust) for studying the inheritance and mapping the genes for leaf and stripe rust resistance. Recombinant inbred lines were developed and screened against highly virulent Pt and Pst pathotypes at the seedling and adult plant stages. Inheritance analyses revealed that both the rust infections were controlled by dominant major single gene. For mapping these genes, the markers showing diagnostic polymorphism in the resistant and susceptible bulks were amplified on all RILs. The molecular characterization identified the genes to be present on 1A chromosome of wheat (short arm). MapDisto version 1.7.5. Beta 4 software was used to determining the linkage present between the genes governing resistance and the SSR markers Xbarc148, Xbarc240, Xwmc93, Xwmc818. A total map size of 9.7cM was obtained showing no segregation between Lr and Yr genes and the marker lying closest to the resistance genes was Xbarc148 at 1 cM distance from the resistance genes. The variation in the T. dicoccoides for the yield related traits was studied by crossing the accessions having longer and wider grains with the accessions having shorter grains resulting in three different crosses: T.dico.14723 and T.dico. 4663, T.dico.5219 x T.dico. 4663 and T.dico. TA1027 x T.dico. 5232. The RIL populations developed from these crosses were screened for different traits affecting the yield which include 100 grain wt. (100 Gwt.), spike length (SpL), spike length with awns (SpLWA), no. of spikelets/spike (Splts/Sp), grain length (GL), grain width (GW) and grain area (GA). The interaction between the traits and their contribution to grain yield was studied. The molecular and the phenotypic data was statistically analyzed for the significant contribution of marker alleles. The evaluation of allelic effects of the polymorphic markers for each trait using Kruskal–Wallis test showed that the phenotypic differences in the mean value of GL, GA and GWT were statistically significant for the three different classes of TaGASR7-A and TaGASR7-D. The study suggests the use of variation existing in T.dicoccoides germplasm for wheat breeding programmes.ThesisItem Restricted Genetic transformation of Indica rice for sheath blight resistance using chitinase gene(Punjab Agricultural University, Ludhiana, 2021) Sharma, Manveer; Sandhu, Jagdeep SinghTrichoderma chitinase enzymes hydrolyze β-1, 4-glycosidic bonds of chitin found in the cell wall of Rhizoctonia solani, the causal agent of rice sheath blight. In this study Tvchit42 gene was introduced into rice using Agrobacterium-mediated genetic transformation. The gene construct, carrying Tvchit42 gene under the control of the CaMV35S promoter and the NOS terminator, was mobilised into two Agrobacterium tumefaciens strains, LBA4404 and EHA105, which were used to agro-infect PR121 and Kitaake rice calli, respectively. In PR121 and Kitaake, a total of 99 and 414 plants were regenerated from transformed calli, respectively. The presence of transgene was confirmed by PCR, revealing amplicon of size 1293 bp corresponding to Tvchit42 with transformation efficiency of 0 % and 0.64 % in PR121 and Kitaake, respectively. Real-Time PCR was used to confirm transgene integration and expression in T0 transformants, revealed 2.94-6.14 fold increase in Tvchit42 expression in six Kitaake plants as compared to non-transgenic controls. Moderate resistance was observed in five transgenic plants after inoculation with RS-1 isolate of R. solani, indicating role of Tvchit42 to provide resistance against rice sheath blight disease.ThesisItem Restricted Marker assisted introgression of grain size QTL from Aegilops tauschii Coss. to Triticum aestivum L.(Punjab Agricultural University, Ludhiana, 2020) Preeti Kumari; Chhuneja, ParveenHexaploid wheats shows constrained genetic diversity which can be broadened by exploiting the wild relatives of wheat for introgressing the novel genes or alleles. Aegilops tauschii, the diploid Dgenome progenitor of hexaploid wheat, can be used to increase the genetic diversity both for improving grain yield and nutritional quality of bread wheat. Grain size is one of the important variables influencing the grain yield. To achieve higher wheat yields, breeding efficiency needs to increase which can be done through biotechnological interventions. The present study aimed to validate the markers linked to the QTLs for grain weight, grain length and grain width and to use the markers for marker assisted transfer of these traits from Ae. tauschii to hexaploid wheat using T. durum (PBW114) as bridging species. Four hundred and ninety-five wheat- Ae. tauschii advance backcross introgression lines (GWILT) derived form 13 different Ae. tauschii donor accessions (pau9793, pau9809, pau14328, pau14339, pau14158, pau14169, pau14170, pau14200, pau14232, pau3747, pau3752, pau3760, pau9810) and two recipient wheat cultivars PBW550 and PBW621 were evaluated for various agronomic and quality traits for four consecutive crop seasons (2015-16 to 2018-19). In each generation selections were made and phenotypically superior lines were progressed further. Total of two hundred and forty-nine highly promising lines were identified which showed better performance in all the four years. These lines offer a good opportunity to study the genetic loci which may be responsible for variation in grain size and other related traits. The pooled correlation coefficient among traits for wheat-Ae. tauschii AB introgression lines reveals a positive correlation of thousand grain weight (TGW) with grain filling duration (GFD), length (LN), width (WD) and yield per plot (YD) but grain number per spike GNpS showed negative correlation with TGW. Tiller number per meter showed positive correlation with overall yield. TGW depends more on grain width than grain length. Seventeen introgression lines derived from Ae. tauschii accessions pau14169, pau3760, pau9809 showed high level of resistance for stripe rust in the range of 5MR-10MR. In previous study, three Ae. tauschii accessions pau14232, pau14231 and pau9796 which were found to have high grain size were used in present study for transfer of grain size QTLs to hexaploid wheat cv. PBW725 and advance breeding line BWL4478 using T. durum (PBW114) as bridging species. Two molecular markers GS7D and CWI21 which have been reported to be associated with grain size were used in the present study for marker assisted selection at different stages (BC1F1, BC2F1, and BC2F2) and selection of grain size introgressions. One hundred and seventy-one introgression lines in BC2F2 generation were developed and evaluated genotypically as well as phenotypically for grain weight. All these lines showed significant grain weight improvement over the respective recurrent parent PBW725. The selected introgression lines can further be used in wheat improvement programme and mapping yield component traits.ThesisItem Restricted Genetic and biochemical analysis of bitterness in citrus(Punjab Agricultural University, Ludhiana, 2020) Priyanka; Sidhu, Gurupkar SighCitrus is the economically important fruit crop belongs to the family Rutaceae. Citrus fruit have abundance of health promoting substances which has various antimicrobial, antiseptic and anticancerous properties. The consumer’s acceptability of citrus juice gets lowered due to the problem of delayed bitterness. Previously identified LGT gene responsible for delayed bitterness was confirmed in the citrus germplasm which was present in all the studied citrus germplasm. The expression of this gene is highest during late fruit development stage and seeds exhibit the highest expression as compared to peel. Among three tissues studied, the highest gene expression was observed in the peel of grapefruit during late fruit development stage. Among studied genotypes, seeds of pummelo had the highest gene expression during late fruit development stage. The expression of this gene increases from early to late fruit developmental stage irrespective of the three tissues studied. The major delayed bitterness causing components are limonoids which occurs in glucoside and aglycone forms were estimated. Among all the commercial recommended scions, Pummelo had highest limonin aglycone content and glucoside content in leaves, peel and seeds. The flavonoid content was also highest in pummelo in all three tissues studied. The effect of citrus rootstocks on morphological, vegetative, physico-chemical, physiological traits and leaf nutrient status in three mandarins (Kinnow, Daisy and W. Murcott) was studied. The total phenolics, antioxidants, vitamin C content increased from early to late fruit development stage. The fruit and vegetative characters also followed the similar trend from early to late fruit development stage. The physiological characters decreased from early to late fruit development stage in all three mandarins irrespective of the rootstock. Leaf nutrients status of three mandarins show variability on different rootstocks studied.ThesisItem Open Access Molecular mapping of yellow mosaic virus resistance in bitter gourd (Momordica charantia L.)(Punjab Agricultural University, Ludhiana, 2021) Gurpreet Kaur; Navraj KaurBitter gourd (Momordica charantia) is a popular cultivated vegetable in India and other countries. The susceptible parent ‘Punjab-14’ and the resistant parent ‘PAUBG-6’ were crossed to obtain F4 mapping population comprising 101 individuals with the objective of mapping yellow mosaic disease resistance in bitter gourd. In the present study, genotypingby-sequencing (GBS) approach was used to develop the genetic linkage map. The map contained 3,144 single nucleotide polymorphism (SNP) markers, consisted of 15 linkage groups and spanned for 2415.2 cM with an average marker distance of 0.7 cM. By adopting the artificial and field inoculation techniques, F4:5 individuals were phenotyped for disease resistance in Nethouse (2019), Rainy (2019) and Spring season (2020). The QTL analysis using the genetic map and phenotyping data identified three QTLs qYMD.pau_3.1, qYMD.pau_4.1 and qYMD.pau_5.1 on chromosome 3, 4 and 5 respectively with phenotypic variation explained (PVE) from 13.5 to 22.1%. In addition, nine horticultural important traits including days to appearance of first female flower, days to appearance of first male flower, days to fruit maturity, node to appearance of first female flower, seed hardness, seed number, fruit length, fruit diameter and fruit weight were evaluated using quantitative data. As a result, twelve QTLs responsible for these traits were also identified. In another experiment, the relative gene expression of begomovirus genes in susceptible and resistant hosts was studied to understand the mechanism of virus resistance. The expression analysis indicated the high expression of AC4 gene in resistant plant provided the information on the efforts of virus to combat the resistance of plant. The information generated in this study is very useful in future for fine-mapping and for marker-assisted selection for these traits.
