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Thesis

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2016 - 20185
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Subject: Agricultural Biotechnology × End date: 2018 × Start date: 2010 × Type: Thesis × Thesis Type: Ph.D ×
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Now showing 1 - 5 of 5
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    ThesisItemOpen Access
    Whole genome de novo assembly of Vigna mungo and Vigna radiata and in silico comparative analysis for marker development
    (Punjab Agricultural University, Ludhiana, 2018) Thakur, Shivani; Satinder Kaur
    Urdbean and mungbean provides nutritionally balanced food for vegetarian population of South and Southeast Asian countries. But, very less genomic studies are reported in these crops. In this study, we have sequenced (Illumina) and assembled whole genome sequences of V. mungo and V. radiata, covering 559.494 Mb and 588.520 Mb of genome respectively. Gene prediction, functional annotation of predicted genes, identification of (protein families, resistance genes, non-coding RNA genes, transmembrane helix, signal peptide), mapman classification of genes and generation of pseudomolecule was done. To increase genomic resources in these crops, in silico polymorphic marker (SSRs and SNPs) was developed from V. mungo and V. radiata genome sequences . To prove the usefulness of these predicted polymorphisms, validation of these newly designed molecular markers was carried on different Vigna genotypes viz. Vigna radiata, Vigna mungo, Vigna umbellata, Vigna mungo var sylvestris and Vigna radiata var sublobata. Among amplified primers, 91 SSRs and 92 KASPs were polymorphic, representing 85% and 93% of the polymorphism. The performance of these markers was evaluated using various statistical parameters defining the usefulness and robustness of primers. This includes effective multiplex ratio (EMR), resolving power (RP), PIC value and Marker index (MI).Thus, development and validation of these newly designed markers has increased genomic resources in V. mungo and V. radiata. This will benefit genetic mapping, assessment of genetic diversity, marker-assisted selection and translational genomics studies in both these crops. Also, these markers will opens the door to genomic research in other Vigna species.
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    ThesisItemRestricted
    Genetic Mapping and Transfer of Brown Plant Hopper Resistance Gene(s) from Oryza nivara Sharma et Shastry to Cultivated Rice
    (Punjab Agricultural University, Ludhiana, 2017) Kishor Kumar; Kumari Neelam
    Brown planthopper (BPH, Nilaparvata lugen Stål) is one of the most destructive insect of rice (Oryza sativa L.) causing significant yield losses annually. Here, we report high resolution mapping of a novel genetic locus, designated as Bph33 on long arm of rice chromosome 4 using an interspecific F2 population derived from a cross between susceptible indica cultivar PR122 and BPH resistant wild species, O. nivara acc. IRGC104646. Inheritance study was performed using F2, F2:3 and F5 populations revealed presence of single dominant gene. A 50K SNP chip (OsSNPnks) was used to construct high density linkage map and QTL mapping. The resistance locus was mapped between two SNP markers, AX-95952039 and AX-95921548 at LOD score of 28.8 contributing 68.3% of total phenotypic variance. The Bph33 locus spanned a physical distance of 91 Kb in Nipponbare reference genome- IRGSP-1.0 pseudomolecule containing eleven candidate genes, out of which one belong to Leucin Rich Repeat (LRR) family protein. In addition to that, two SSR markers, RM16994 and RM17007 co-segregated with the BPH resistance locus. These SSR markers could be used efficiently for markers assisted transfer of this locus into elite rice cultivars.
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    ThesisItemRestricted
    Fine mapping and identification of candidate genes for stripe and leaf rust resistance transferred from Aegilops umbellulata to bread wheat (Triticum aestivum)
    (Punjab Agricultural University, Ludhiana, 2017) Bansal, Mitaly; Chhuneja, Parveen
    Leaf rust and stripe rust are the most damaging diseases of wheat worldwide. Due to domestication and breeding, the narrow genetic base of modern cultivars makes it difficult to combat the changing environmental conditions as well as the emerging new pathogens. To broaden its genetic base, hybridization with wild relatives, landraces and early domesticates is widely used as they harbor rich genetic diversity. In the present investigation, Lr76 and Yr70 transferred from Ae. umbellulata to bread wheat were fine mapped using next generation sequencing technologies. BC-RIL and F2 population derived from the cross of introgression line pau 16057 (syn. T393-4, IC0616573, INGR 15047) with WL711 segregated for a single gene, each for leaf rust and stripe rust resistance. New SSR and STS markers were designed from the IWGSC survey sequence of chromosome 5D to fine map these genes. Lr57-Yr40_CAPS and 5DS-219 were found to be closely linked to the genes, spanning a region of 5.1cM. To further narrow down the region, SNP based markers were designed from flow sorted chromosome 5D sequence of pau 16057 and WL711. SNPs were identified between the two chromosome sequences and validated through KASP technology. Integrating POPSEQ based ordering of wheat contigs information with SNP discovery in pau 16057 and WL711 sequence, the introgressed segment was found to be locating from 0 to 11.94 cM bin. KASP228 and KASP225 are found to be closely linked to Lr76 and Yr70 and can be used in breeding programmes. We have also established that Lr76 and Yr70 are different from previously mapped Lr57/Yr40 genes in the same region on the basis of their location in 11.94 cM and 4.37 cM POPSEQ bin, respectively. In the present investigation, we have used RenSeq which is based upon the knocking down of the major gene and identification of mutations in the NB-LRR encoding genes in all the mutants of the investigated gene. We have identified two NB-LRR encoding contigs in two mutants of Lr76 which needs further validation. In the present investigation, high end sequencing technology like RenSeq and chromosome sorting and sequencing have been used which made it possible to characterize the introgressed alien segment linked to the rust resistance genes.
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    ThesisItemOpen Access
    Fine Mapping and identification of candidate gene(s) for leaf rust and stripe rust resistance introgressed in Triticum aestivum L. from Aegilops peregrina
    (Punjab Agricultural University, Ludhiana, 2017) Narang, Deepika; Chhuneja, Parveen
    Ae. peregrina acc. pau 3519, a non-progenitor species (UUSS) maintains high level of resistance to leaf and stripe rust. Two sets of homozygous WL711-Ae. peregrina introgression lines (ILs) viz. IL pau16061 harboring LrAp and IL pau16058 harboring LrP–YrP were crossed with wheat cv. WL711 to generate mapping populations. Inheritance studies, fine mapping of rust resistance genes with SNP/STS markers and identification of candidate genes through Resistance Gene Enrichment Sequencing (RenSeq) were undertaken in the present investigation. F2.3 population of WL711/ IL pau16058 segregated into 260 resistant: 517 segregating: 226 susceptible (χ21:2:1=3.26) against leaf rust and 257 resistant: 520 segregating: 226 susceptible (χ21:2:1=3.281) against stripe rust races indicating transfer of two dominant co-segregating rust resistance genes. In the initial analysis, LrP–YrP genes were mapped on 5DS showing linkage with XLr57/Yr40–CAPS16, Xbarc130 and XTa5DS_44573. High resolution genetic map spanning 4.28cM including eight SNP markers, one SSR and two STS markers was generated. Marker loci BS00163889 flanked distally (co-segregating) and gsnp_5ms_4641 proximally (seven recombinants) to LrP–YrP. SNP markers designed from nucleotide binding and leucine rich repeats (NLRs) depicting independent variations in mutants were placed away from LrP–YrP. Inheritance studies on F6 RILs of WL711/IL pau16061 indicated transfer of a single major dominant gene conditioning seedling resistance. In initial analysis, GISH on IL pau16061 established that LrAp has been introgressed from the Sp genome of Ae. peregrina to the long arm of a pair of homoeologous chromosomes of wheat. Bulked Segregant Analysis (BSA) placed the alien segment onto chromosome 2BL as indicated by linkage of LrAp with markers Xncw-Lr58-1 and Xcfd50. For fine mapping of LrAp, five co-dominant SNP markers and one dominant marker based on presence/absence variations were designed directly from NLRs. These markers positioned the LrAp gene distally on the alien segment and indicated homology with the 6BL chromosome of wheat. The marker Ren_1191 showed co-segregation with LrAp. Linkage of both 2BL and 6BL specific markers with LrAp indicated translocation of a 6Sp alien segment onto the long arm of wheat chromosome 2B. The identification of co–segregating markers for LrAp and LrP–YrP in the present study will facilitate marker–assisted pyramiding with other genes.
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    ThesisItemRestricted
    Transfer And Molecular Characterization Of Aegilops Triuncialis Leaf Rust And Powdery Mildew Resistance Genes In Triticum Aestivum L. Background
    (Punjab Agricultural University, Ludhiana, 2016) Sagar, Preeti; Chhuneja, Parveen
    Chromosome engineering is efficient method for transfer of alien chromosome to wheat. Methodology adopted allowed the identification and utilization of unexploited tertiary gene pool to transfer agronomic important traits. In the present study homoelogous recombination is induced using Pavon cv. ph1b carrying non functional Ph1 locus. ph1b homozygous BC1 plants were identified using two PCR based markers PSR2120 and PSR574. The presence of ph1b specific amplicon signified BC1 plant carrying functional Ph1 locus, absence of PCR amplification indicated deletion of thePh1 locus (presence of ph1b). only those BC1 plants were selected which were carrying ph1b and 5U chromosome and backcrossed with Wl711 for ph1b to recover functional PH1 and Leaf rust (Lr), Stripe rust (Yr) and Powdery mildew (Pm) are important diseases of common wheat. DS5U(5A) substitution line carrying 5U chromosome and is having resistance against biotic stress. Adult plant screening allowed the identification of moderate to highly resistance plants among F1 and F3 progenies derived from the cross Ae. triuncialis DS5U(5A)/2*Pavon ph1b//WL711 (NN). F1 and F3 population was screened using SSR markers Xgwm129, Xwmc705, Xwmc446, Xwmc445 and 5U specific indel markers were designed and applied to identify the introgression lines. Set of 14 CSSL lines were identified, which represents complete 5U chromosome in a 5A background of WL711. Average, lines were recovered with 85.88% recurrent genome and 11.66% donor genome. FISH is a powerful tool to track alien chromosome and is used to assign different chromosome to the respective homoelogous group.