Loading...
Thesis
Browse
14 results
Search Results
ThesisItem Open Access Genome wide expression analysis of Multidrug and toxin compound extrusion(MATE) gene in rice for understanding its role in resistance/susceptibility to Rhizoctonia solani AG1-IA(Punjab Agricultural University, Ludhiana, 2021) Rupnaz Kaur; Vikal, YogeshRice (Oryza sativa) is one of the most important staple food crop for approximately 50% of human population across the world and is constantly hampered by sheath blight disease caused by Rhizoctonia solani AG1-IA pathogen. Several cultural, chemical and breeding approaches were applied to combat this disease but till date no major success was achieved. Study of hostpathogens interactions may find a new possible way to understand its molecular mechanism to overcome this disease. Various transporters are well characterized that plays significant role in various stress conditions. MATE gene family is one of them that belongs to secondary transporters group and help in extrusion, transportation of products across membrane via electrochemical gradient way. The current study is aimed to understand role of MATE transporter family towards the resistance/susceptibility in rice caused by R. solani AG1-IA. The commonly grown PR114 (susceptible) variety and ShB-8 (IET-21299) (moderate resistant) line were selected for present study. The R. solani fungus strain was cultured on PDA media and infection was done on targeted rice lines. The RNA was extracted from both infective lines at 0 hr, 24 and 48 hpi and preceded for transcriptome analysis (RNA Seq). The data of RNA Seq dissipated that a set of six differentially expressed MATE genes were identified on the basis of their log2 fold change value. The selected six genes were further validated through RT-PCR. Differential gene expression at different treatments showed that LOC_Os02g45380.1 MATE gene is one of the potential candidate gene involved in ShB resistance/susceptibility. Further in-silico analysis was performed for candidate MATE gene LOC_Os02g45380.1. The evolutionary phylogenetic analysis depicted that candidate MATE gene showed homology to various dicots and monocots. However, in monocots it is highly similar to Oryza nivara which means this gene might be transferred from wild species during natural selection. Various MATE genes are reported in different plant species genome, clustering analysis also suggested that a set of 52 MATE genes were present in rice genome that is supported by previous studies. Clustering of these 52 MATE genes showed that they can be further sub-grouped into four clades on the basis of function as previously reported. The LOC_Os02g45380.1 was present in clade III which may have role in citrate and metal transport. Cis regulating (promoter) analysis of LOC_Os02g45380.1 depicted various cis regulatory elements that are present in UTR which may play significant role in various abiotic and biotic stresses. Protein modelling of LOC_Os02g45380.1 revealed the presence of 11 transmembrane helix which helps to understand its topology. The PPI interaction analysis showed that various interaction molecules and proteins are present that aid in regulation of candidate MATE gene. To identify the variation in genic region of LOC_Os02g45380.1, the gene was amplified and cloned. For functional validation and raising of cis-genic lines, the tissue culture media was standardized for ShB-8 variety.ThesisItem Restricted Identification and Molecular mapping of leaf rust and stripe rust resistance genes introgressed from wild progenitor species in cultivated wheat.(Punjab Agricultural University, Ludhiana, 2021) Jose, Alin Maria; Chhuneja, ParveenWild emmer wheat, Triticum dicoccoides, the tetraploid progenitor of wheat and Einkorn wheat, Triticum monococcum, diploid progenitor of modern hexaploid wheat, are important sources of novel disease resistance genes. T. dicoccoides acc pau 14716, which showed resistance to most common leaf rust and stripe rust races in India, was used to develop introgression line (ILdic) in the background of T. aestivum cultivar WL711(NN). An F4 mapping population was developed by crossing ILdic with T. aestivum cultivar HD2967 and the population was screened against highly virulent Pt leaf rust pathotypes at seedling and adult plant stage and Pst stripe rust pathotypes at the adult plant stages. The population segregated for a single all-stage resistance gene (ASR) against leaf rust and single gene for stripe rust resistance, according to inheritance analyses. Similarly, T. monococcum G1277 showed resistance to the most prevalent Pst stripe rust pathotypes in India. Therefore it was used to create a stable introgression line ILmono into hexaploid wheat background. T. aestivum cultivar HD2967 was used to develop F6 mapping population. ILmono mapping population upon screening against Pst stripe rust pathotypes at adult plant stage segregated for a single gene for stripe rust resistance. For mapping, leaf rust and stripe rust resistance genes in the F4 mapping population derived from HD2967-ILdic, bulk segregant analysis in combination with whole-genome resequencing (BSA-seq) was conducted. Two resistant bulks (RB) and two susceptible bulks (SB) were prepared separately for performing BSA-seq for mapping leaf rust and stripe rust resistance genes. A 10Mb candidate region on chromosome 7A with three highly significant SNPs linked to leaf rust resistance and 61 annotated genes was identified. It was observed that out of 61 annotated genes, 12 genes were involved in the defense mechanism against diseases and considered as candidate putative genes for leaf rust resistance gene in ILdic. Similarly, a 20Mb region with two highly significant SNPs was identified on chromosome 3A to be associated with stripe rust resistance and was identified to carry 49 annotated genes. Out of 49 annotated genes, four genes were found to be involved in defense mechanisms against diseases in which one gene had an NBS-LRR domain and therefore considered as candidate putative genes. The current study discovered useful leaf rust and stripe rust resistance genes that are effective against several rust races and can be deployed in the wheat breeding program.ThesisItem Open Access Molecular mapping of Karnal Bunt resistance genes transferred from Aegilops tauschii and Aegilops triuncialis in the hexaploid wheat background(Punjab Agricultural University, Ludhiana, 2021) Thomas.T, Libin; Chhuneja, ParveenKarnal bunt is a disease of wheat that can jeopardize global grain industry due to its quarantine status. Deployment of resistant varieties is the most effective way of constraining this disease. The present study was conducted to develop mapping populations and to identify quantitative trait loci (QTL) that confer resistance against Karnal bunt in two RIL populations. Population 1 (226 lines) was derived from cross between resistant interspecific introgression line IL422 (BC3F8 (PBW114/Ae. tauschii acc14195//4*WH542) and HD2967 while population 2 (115 lines) was derived from cross between resistant interspecific introgression line IL455 (BC1F11 (WL117/Ae. triuncialis/CSS/ 2*PBW343) and HD2967. Both the populations were advanced upto F5 generation through single seed descendent method. High intensity phenotyping for Karnal bunt was done in F4 (1 replication) and F5 (2 replications). About 120 lines from population 1 and 100 lines from population 2 were genotyped with high density SNPs markers by genotyping by sequencing using ddRAD sequencing. The SNPs obtained were filtered for polymorphism between two parents, maximum allele frequency of 5% and maximum missing of 20%. This criterion brought down the total number of SNPs to 13053 (IL422/HD2967 population) and 11341 (IL455/HD2967 population). The binning function of Ici mapping software was used to filter the redundant markers. A linkage map was developed from these binned SNPs using the Ici mapping software. Four QTLs in population 1 (2B, 3B and 5A) and two QTLs in population 2 (5A and 5B) were mapped with high LOD score which explained significant phenotypic variation. The major QTLs mapped on 2B, 3B, 5A and 5B were novel which using linked SNP markers can be in deployed in the development of resistant wheat varieties.ThesisItem Open Access Generation of interspecific hybrids in chickpea ( Cicer Arietinum L.) and their confirmation through morphological and molecular analyses(Punjab Agricultural University, Ludhiana, 2021) Ritika; Ajinder KaurThe present study was conducted on ―Generation of interspecific hybrids in chickpea (Cicer arietinum L.) and their confirmation through morphological and molecular analyses‖. A total of 12 different crosses (using 546 flower buds) were attempted (C. arietinum C. reticulatum: L552 × EC396375, PBG7 × EC396375, PBG7 × EC426536, PBG7 × ILWC292, GPF2 × EC396375, PDG3 × ILWC292; C. reticulatum C. pinnatifidum: ILWC141 × C. pinnatifidum 49; C. reticulatum C. reticulatum: PB64 × ILWC292; C. arietinum C. bijugum: L552 × ILWC0, PDG3 × ILWC0; C. arietinum C. judaicum: L552 × C. judaicum 185B, PDG3 × C. judaicum 185B). A mixture of plant growth regulators (GA3 120 ppm + NAA 30 ppm + Kin 15 ppm) were applied to the base of pedicels of crossed flowers to prevent flower drop and enhance pod setting. A total of 76 ovules were rescued and cultured, however no plants were obtained. A total of 42 seeds were obtained from crosses left under natural conditions that were sown to raise 27 putative F1 plants (C. arietinum C. reticulatum: 25 plants, C. reticulatum C. pinnatifidum: one plant, C. reticulatum C. reticulatum: one plant). The remaining cross combinations belonging to C. arietinum C. bijugum and C. arietinum C. judaicum did not result in any F1 plant. Twenty two plants from C. arietinum C. reticulatum and one each from C. reticulatum C. pinnatifidum and C. reticulatum C. reticulatum crosses were proved to be hybrids through morphological traits viz. plant height, growth habit, number of secondary branches, number of pods and seeds per plant, and seed yield. Eighteen plants obtained from crosses C. arietinum C. reticulatum and one plant from C. reticulatum C. pinnatifidum were proven to be hybrids on the basis of molecular data of five polymorphic SSR markers, namely TA2, TA43, TA89, TA116 and TS45. The molecular and morphological data of F1 plants corroborated with each other to support hybridity of 19 F1 plants. Generation of maximum number of plants from C. arietinum C. reticulatum cross revealed that genetic variability found in C. reticulatum can be utilized for the improvement of cultivated chickpea. All plants were obtained without the use of embryo rescue.ThesisItem Restricted Mapping of some important agronomic traits in pigeonpea(Punjab Agricultural University, Ludhiana, 2021) Bhandari, Geetika; Ajinder KaurThe present investigation was performed to study the genetics and mapping of the agronomic traits, namely determinate type growth habit, early maturity and short stature in an intraspecific population of pigeonpea derived from cross Pusa 992 (female parent) × AL 2205 (male parent). During kharif 2020, 222 F2 plants were evaluated for growth habit, days to maturity and plant stature along with other agronomic traits. A wide range of variation was observed in F2 population for all traits; 36 plants having early maturing, short statured and determinate type growth habit were harvested separately to raise F2 derived F3 progenies during kharif 2021. These were also evaluated for plant stature, days to maturity and growth habit. The inheritance studies on F2 and F3 populations confirmed monogenic recessive control of determinate type growth habit, early maturity and short plant stature. From a set of 232 SSR primer pairs, 54 markers gave parental polymorphism between two parents. Six bulks for three traits under investigation were made and bulk segregant analysis was carried out on F2 population using 54 polymorphic markers. BSA studies revealed three SSR markers viz. CcM2753, CcM2754 and CcM0268 distinguishing tall/short stature bulk, late/early bulk and IDT/DT bulk, respectively. Single marker analysis was then used to establish marker-trait association for the traits. R2 value of 43.5% demonstrated significant association of marker CcM2753 with short plant stature, while R2 value of 71.8% indicated highly significant association of marker CcM2754 with early maturity. The marker-trait association explained the presence of variability in the phenotype (plant stature and days to maturity) due to variation in genotype. Further, BSA and F2 genotypic data clearly showed the association of marker CcM0268 with determinate type growth habit. Our study highlighted the successful inheritance of agronomic traits viz. determinate type growth habit, early maturity and short plant stature from the male parent AL 2205. The identified molecular markers can be used for marker assisted selection in pigeonpea.ThesisItem Open Access Development of Water use Efficient Transgenic Sugarcane through Expression of Wheat TaVAP Transgene(Punjab Agricultural University, Ludhiana, 2021) Gill, Jashandeep Singh; Sandhu, Jagdeep SinghThe present study entitled “Development of Water use Efficient Transgenic Sugarcane through Expression of Wheat TaVAP Transgene” was carried out on Sugarcane variety Co238. pBI vector carrying TaVAP gene was mobilized into Agrobacterium tumefacians strain EHA 105. Transformation was carried out using invitro Agrobacterium mediated transformation. Sugarcane spindle leaf roll segments were raised on solid MS medium containing NAA (5.0 mg/l) + Kin (0.5 mg/l). The Agrobacterium broth at 1.0-1.2 was used for agroinfection of explants in presence of 9.8mg/l acetosyringone. The agroinfected explants were transferred on solid MS + 5 mg/l NAA + 0.5 mg/l Kin + 500 mg/l Cefotaxime for 21 days. Then explants were rooted on solid MS + 5 mg/l NAA + 0.5 mg/l Kin + 500 mg/l Cefotaxime for next 21 days. A total of 38 putative transgenic plants were developed. PCR verification of these 38 putative transgenic plants were analysed using TAVAP gene specific primers. The PCR did not detect the presence of TaVAP in putative plants.ThesisItem Open Access Fine mapping of male-sterility ms-1 gene using whole genome resequencing of muskmelon (Cucumis melo L.)(Punjab Agricultural University, Ludhiana, 2021) Simranjot Kaur; Sarao, Navraj KaurMuskmelon (Cucumis melo L.) is a warm season crop, cultivated in tropical and temperate regions of the world. Five types of male-sterility genes have been reported in this crop, out of which ms-1 and ms-5 has been utilized in hybrid seed production programmes. The identification of molecular markers linked to these male-sterile genes may find practical applications in hybrid seed production and marker-assisted backcross (MABC) programmes. The previously found SSR markers linked to ms-1 gene are quite at a distance to be considered as tightly-linked. Therefore, the current study was aimed at fine mapping the genomic loci harbouring the ms-1 gene using whole genome resequencing approach. A cross between male-sterile MS-1 and male-fertile KP4HM-15 was used to raise an F2 mapping population for the construction of two contrasting bulks namely, male-sterile bulk (MSB) and male-fertile bulk (MFB). These bulks along with both the parents were sequenced and analyzed using SNP-index method for identifying the ms-1 associated region. A 400-kb candidate region on chromosome six was identified using this method which lied within the previously mapped 3.2 Mb region using SSR markers. This candidate region harboured nine highly-significant SNPs and 28 annotated genes. The nine highly-significant SNPs can be used to develop KASP markers for the further improvement of genetic map. It was observed that out of 28 annotated genes, six genes were involved in imparting male-sterility in other crops and therefore these six genes were regarded as candidate putative genes. It was also noticed that the genes fasciclin-like arabinogalactan protein 1 (FLA) and ABC transporter G family member 26, each harboured a highly-significant SNP. Also, one of gene named protein-disulfide isomerase (PDI) contained a missense variation from „A‟ to „T‟ at 36,921,314 bp position. This missense mutation in the PDI gene might be responsible for the male-sterility in ms-1 plants.ThesisItem Restricted Exploring Matrilineal (MLT) Gene Based in Vivo Haploid Induction of Wheat(Punjab Agricultural University, Ludhiana, 2021) Khanna, Simran; Sandhu, NitikaDouble haploid technology has been one of the extensive used technologies to produce haploids commercially. The objectives of the present research include 1) validation of MTL gene mutants using allele-specific primer assay and evaluation of the efficacy of identified mutants for haploidy induction 2) pyramiding of mutations in homeoalleles of MTL gene located on A, B and D wheat genomes. There were multiple mutations in the lines and 45 mutant lines (24 Cadenza and 21 Kronos) were procured having mutations in the TaPLA1 gene. Out of 45 mutants, 16 were validated by KASP assay. The mutant lines were tested for haploidy induction based on pollen viability test and seed set factor. Crosses were attempted among the Cadenza (hexaploid) mutant lines and Unnat PBW343, Kronos mutant lines (tetraploid) and PBW114, Kronos mutant lines and SGPD36. However, single/ individual mutations were not enough to induce haploidy or any chromosomal elimination, so pyramiding of the homeoalleles was attempted in order to complement the mutations of MTL gene present on the three homeologous genomes. Crosses were attempted for Cadenza and Kronos among the mutants to complement single copy mutations (A/B, B/D and A/D), followed by double copy mutants (A/B//B/D) in case of Cadenza mutants and their seed set was observed. The 49 out of 263 F2 in case of crosses involving Cadenza mutants lines and 102 out of 731 F2 in case of Kronos mutants showed less seed set. The F2 from double copy mutants of cadenza are in field for further evaluation.ThesisItem Open Access Whole transcriptome assembly and annotation of wild wheat Aegilops speltoides for identification of novel heat stress tolerance genes(Punjab Agricultural University, Ludhiana, 2021) Seni, Sushmita; Satinder KaurWheat, a major staple food crop of the world, is relatively sensitive to the changing climatic conditions, especially the high temperature. The comprehensive mechanism of heat stress response at the molecular level and exploitation of candidate heat tolerant genes in the wild species of wheat Aegilops speltoides have not been exclusively studied. Using transcriptome data, we made comparative RNA-Seq analysis for the gene expression profiles of heat stress tolerant genes in diploid wheat Aegilops speltoides acc. pau3809, tetraploid wheat Triticum durum cv. PDW274 and hexaploid wheat Triticum aestivum cv. PBW725. cDNA libraries generated from flag leaf tissues of AS3809, PDW274 and PBW725 were sequenced using Illumina technology and approx. 22M, 10M and 14M high-quality clean reads were obtained respectively. De novo assembly was generated for all three genotypes to avoid any bias and we obtained 94,946; 68,873 and 76,404 genes and N50 value of 1409, 904 and 998 bp for AS3809, PDW274 and PBW725 transcriptome respectively. Annotations were performed using BLAST2GO, PfamScan and MapMan and the identified genes in the three wheat transcriptomes were divided into 53 GO functional categories under the biological process, molecular function and cellular component. OrthoMCL was used to find ortholog transcripts across the three wheat genotypes. In this investigation, we analyzed the expression abundance of heat shock transcription factors (HSFs) which are known for their roles in plant tolerance to abiotic stresses. 12 HSF orthologs classified to class A, B and C were studied for comparative expression abundance in the three wheat transcriptomes. The expression of all the HSFs under study was observed to be highest in AS3809, followed by PDW274 and least expression was observed in PBW725. We validated 8 HSFs under control and heat stressed condition in the three wheat genotypes using qRT-PCR. Six out of eight HSF genes showed higher expression values in AS3809 sample (both in control and HS) as compared to the other two genotypes which corroborates with the expression values obtained from the transcriptome data. Our results demonstrate that the HSFs have higher gene expression in the wild wheat Aegilops speltoides as compared the cultivated wheat and they play positive roles in plant tolerance to heat stress. The HSFs from Aegilops speltoides may be used as a candidate gene for genetic modification of cool-season plant species for improving heat tolerance.
