Browsing by Author "Vikal, Yogesh"
Now showing 1 - 10 of 10
Results Per Page
Sort Options
ThesisItem Open Access Conversion of non-aromatic rice to aromatic rice by editing Betaine Aldehyde Dehydrogenase (BADH2) gene(Punjab Agricultural University, Ludhiana, 2022) Navneet Kaur; Vikal, YogeshAroma in rice is a highly appreciated trait and acts as determining factor for market price. Basmati rice varieties possess characteristic aroma, however, are usually low yielding. Attempts for transfer of aroma to high-yielding rice varieties through conventional breeding approaches have not been much successful. CRISPR/Cas9 technology has been established as a revolutionary crop improvement technique. The ribonucleoprotein (RNP) mediated delivery of the CRISPR/Cas9 complex is showing immense potential for transgene-free genome editing in rice. The current research was carried out for editing of OsBADH2 gene through CRISPR/Cas9-RNP complex in PR114, high yielding non-aromatic rice variety. A total of five gRNAs were designed through CRISPR P 2.0 for generation of OsBADH2 mutants. Primers were designed for the assembly of gRNA-DNA template for two gRNA selected and transcribed to gRNA utilizing “GeneArtTM Precision gRNA synthesis Kit”. The cleavage efficiency of RNP complex was detected through in vitro cleavage detection. The gRNA were incubated with Cas9 nuclease enzyme to form RNP complexes and coated onto gold particles for the biolistic delivery into the explants. Embryos excised from mature seeds and calli were used as explants for transformation. A variety of media were tested for callus induction and regeneration in PR114. Approximately, 2000 mature embryos and 381 calli were transformed using gRNA-Cas9 complex delivered through gene gun. A total of 35 plants, created through transformation of gRNA-RNP complex targeting exon 7, were characterized through MSBSPPCR technique. The plants showing positive results were proceeded for Sanger’s sequencing. The sequencing results were analysed through FinchTV software for quality of sequencing. The sequences were aligned using Clustal Omega and NCBI-BLASTn online tools. An addition of nucleotide “A” was found in one plant. The nucleotide sequence was translated through Expasy tool. Through insilico analysis of the amino-acid sequence of mutant frameshift mutation was observed. The mutation resulted in alteration in the last four aminoacids of the peptide chain. The T1 progeny of this mutant plant didn’t show presence of the mutation. Rest of the transformed T0 plants are being screened through restriction digestion assay. The mutants so found will be further analyzed through molecular and biochemical techniquesThesisItem Open Access Fine mapping of qtl-qslb.pau-3.04 for southern leaf blight resistance in maize (Zea mays L.)(Punjab Agricultural University, Ludhiana, 2019) Kirandeep Kaur; Vikal, YogeshSouthern leaf blight (SLB) caused by Cochliobolus heterostrophus (Drechsler), is a serious disease throughout the world where maize is grown under warm and humid conditions, leading to enormous yield losses. Exploiting genes and quantitative trait loci (QTLs) related to SLB is helpful for improving fungal resistance. In earlier study at Punjab Agricultural University, Ludhiana a set of 325 F2:3 families and F4 progenies derived from the cross of CM139 as the resistant (female) parent and CM140 as the susceptible (male) parent were phenotyped for resistance to SLB under field conditions. A total of 172 polymorphic SSR markers were genotyped on F2 population.Three probable QTL viz. qSLB2.1, qSLB3.1, qSLB3.2 were detected for SLB resistance in bins of 2.05-2.08, 3.04 and 3.06-3.09. The marker interval phi099-umc1729 spanning qSLB3.1 was considered as major putative QTL. In present study, the QTL qSLB3.1 spanning region was saturated with more SSR markers on 298 RIL population of the same cross. The detected QTL region (13.7 cM) was fine mapped to 1.2 cM region flanked with marker MSSR1-MSSR20 explaining phenotypic variance of 15.1 per cent at log-likelihood of 6.9. The 1.2 cM region corresponds to 269.3 Kb and comprises only six candidate genes. The candidate gene based markers were designed, to study the differential expression patterns of candidate genes through quantitative real-time PCR (qRT-PCR). Two of the candidate genes viz. GRMZM2GO88371 and GRMZM5G862219 showed 3.48 and 6.40 fold higher expression in CM140 at 48h and 72h respectively. GRMZM2GO88371 and GRMZM5G862219 have their biological function in lipid metabolism and β oxidation respectively that is involved in the defense pathway and may be one of the potential candidate genes conferring resistance against C. heterostrophus. The SLB QTL linked flanking markers were employed for mobilization of qSLB3.1 QTL into the background of CM140 through marker assisted backcross breeding (MABB). The F1s of cross (CM139 X CM140) were backcrossed to recurrent parent to generate BC1F1 population. A total of 64 plants out of 420 were selected on the basis of foreground selection. Recombinant selection for the carrier chromosome was done to identify single and double recombinants. The plants having maximum recurrent parent recovery for carrier chromosome were selected and backcrossed with CM140 to generate BC2F1 generation. The data generated from this study can serve as valuable genomic resource for maize breeding programmes. It will enable the researcher to multi-thronged and focused approaches for sustainable development of new genotypes by pyramiding it with other desirable genes using MABB.ThesisItem Restricted Fine mapping of quantitative trait loci associated with waterlogging stress tolerance in Maize (Zea mays L.)(Punjab Agricultural University, Ludhiana, 2018) Loveleen Kaur; Vikal, YogeshWaterlogging stress, second most serious constraint after drought to maize production, causes about 25-30% production losses every year. Mapping of QTLs conferring waterlogging tolerance is crucial to develop ‘climate resilient’ maize varieties. The present investigation focussed on identification and fine mapping of putative QTLs for yield components reported in F2 population on chromosome 1, 7, 8 and 9 under waterlogging conditions using SSRs. 260 RILs derived from parental cross of I110 (WLS) and I172 (WLT) were sown in α-lattice design in three replications to evaluate various phenotypic and quantitative traits. Waterlogging stress was applied to two replications at V7 stage for 15 days at ponding depth of 10.0 ± 0.5 cm treating third as control. Statistical analysis of phenotypic data indicated significant differences between stressed and control replications for almost all traits. A total of 5 QTLs designated as qSpad2S1, qSpad2S2, qNELC, qCobwt on chromosome 1 and qPHS on chromosome 8 were obtained in this study. The QTL window has been significantly reduced to an average distance of 5-8 cM from the earlier distance of 30 cM achieving the objective of fine mapping to an extent. More number of markers will be required to further saturate and dissect the regions harbouring putative QTLs. The eventual aim is to precisely locate and isolate novel genes influencing stress response to develop ‘climate ready’ cultivars of maize.ThesisItem Restricted Genetic analysis and molecular mapping of components of resistance to shoot fly in maize(Punjab Agricultural University, Ludhiana, 2016) Arshpreet Kaur; Vikal, YogeshShoot fly (Atherigona naqvii) is a major insect of maize causing yield losses up to 45 per cent. The insect resistance in maize is complex and quantitative in nature. Therefore, the present study was undertaken using 107 F2:3 families from the cross between CM143 as the resistant (female) parent and CM144 as the susceptible (male) parent and data was recorded at different time intervals of inoculation. The disease reaction data showed normal distribution clearly indicating that the inheritance of shoot fly resistance in CM143 is under the control of quantitative loci. F2:3 families alongwith parents were evaluated for various component traits (like deadheart, oviposition, leaf injury, leaf dimensions, seedling vigor, leaf glossiness etc.) that could be involved in shoot fly resistance. Parental polymorphism survey was done with 643 SSR markers and 195 were found to be polymorphic. A linkage map of 1181.11 cM length was constructed with 105 polymorphic SSR markers on 107 F2 individuals. Both genotypic data and mean values of phenotypic data of each component trait was analysed using QTL cartographer and QTLs on chromosome 1, 2, 4 and 9 were detected for various component traits explaining phenotypic variance from 2.15 to 19.43 per cent with LOD score ranging from 2.6 to 5.83. The QTL detected on chromosome 9 for oviposition and deadheart in the present study is syntenic to regions of chromosome 10 of sorghum which was also accounted for deadheart and oviposition. Fine mapping of identified QTL regions may provide useful information of genes underlying quantitative shoot fly resistance that can be further used in for genetic and breeding studies.ThesisItem Restricted Genetic homology and differentiation in some species of vigna(Department of Genetics College of Basic sciences & Humanities PAU, Ludhiana, 1992) Vikal, Yogesh; Satija, C. KThesisItem 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 High resolution genetic mapping of xa43(t) gene for bacterial blight resistance in rice (oryza sativa l.)(Punjab Agricultural University, Ludhiana, 2020) Bhatia, Sukhpreet; Vikal, YogeshBacterial blight (BB) caused by Xanthomonas oryzae pv. oryzae (Xoo), is one of the epidemic diseases in rice causes significant yield losses worldwide, especially in Southeast Asia. Due to the lack of effective chemical control, most effective strategy to combat the BB disease is the finding of new resistance sources and its deployment in the elite rice cultivars. Since the durability of resistance is always at stake due to the rapid change in the pathogenicity of the Xoo pathogen, there is a need to identify and characterize novel BB resistance genes. Tagging and fine mapping is pre-requisite for transfer and pyramiding of BB resistance genes through marker-assisted selection (MAS). Therefore, the present investigation was undertaken to develop high resolution genetic map of novel bacterial blight resistance gene, xa43(t) identified from Oryza rufipogon acc. CR100098A. The backcross introgressed lines (BILs, BC1F7) were developed from cross between PR114 and Oryza rufipogon acc. CR100098A. The genetic studies using BC1F2 population and BC1F3 progenies showed single recessive locus conditioning resistance to the Xoo pathotype #7 (PbXo-7). The BILs were segregated in the ratio of 197 resistant : 195 susceptible which is consistent with the expected allelic frequency of 1:1 ratio for single gene inheritance. The xa43(t) gene was tagged with two SSR markers viz. RM27154 and RM2136 on long arm of chromosome 11 using bulked segregant analysis. To fine map the gene, 85 SSR and 86 KASP markers of chromosome 11 were surveyed for parental polymorphism. A total of 22 SSR and five KASP markers were found to be polymorphic between parents. The polymorphic markers were analysed on 392 BILs and genetic analysis placed the xa43(t) BB resistance gene between two flanking SSR markers, PAU11_39 and PAU11_44 within an approximately 447 kb region. Bioinformatic analysis revealed a total of 73 genes within that region and seventeen of them were putative candidate genes involved in biotic stress resistance. The novelty of xa43(t) gene was confirmed by testing the linked markers on other cultivars as well as by surveying the linked markers of other BB resistance genes present on chromosome 11 on parents and bulks.ThesisItem Open Access Identification and characterization of high temperature stress responsive genes in maize (Zea mays L.)(Punjab Agricultural University, Ludhiana, 2020) Ashok Babadev Jagtap; Vikal, YogeshHeat stress due to climate change is an emerging issue for maize breeders as it largely affects the yield. The present study focuses to elucidate molecular mechanisms and underlying genes, or QTLs associated with heat stress tolerance in maize. The transcriptional studies of maize leaves, pollens and ovules using heat stress susceptible and tolerant inbred lines, namely LM11 and CML25, respectively revealed a total of 2,164 (1127 up-regulated and 1037 down-regulated) differentially expressed genes (DEGs) between LM11 (HS) and CML25 (HT) samples, with 1151, 451 and 562 DEGs were identified in comparisons of corresponding leaf, pollen and ovule samples, respectively. Functional annotations of DEGs showed that many of them were related to transcription factors (TFs) viz. AP2, MYB, WRKY, PsbP, bZIP and NAM, heat shock proteins (HSP20, HSP70 and HSP101/ClpB), as well as genes related to photosynthesis (PsaD and PsaN), antioxidation (APX and CAT) and polyamines (Spd and Spm). KEGG pathways analyses explicated that metabolic overview pathway and secondary metabolites biosynthesis pathway, with involvement of 264 and 146 genes, respectively were highly enriched in response to heat stress. A bioinformatics pipeline was used to call and type SNPs from RNA-seq reads and applied it to transcriptomic data of LM11 and CML25. A total of 554,423, 410,698 and 596,868 polymorphic SNPs were identified respectively among leaf, pollen and ovule of the LM11 and CML25. A total of 100 genome-wide SNP based KASP assay markers were developed and validated as well as subsequently genotyped on 90 F2 individuals derived from the cross of LM11 × CML25. The success of SNP conversion rate was 71%. In addition, F2 population and their parental inbreds were genotyped using 94 polymorphic SSR markers. The 175 F2:3 families during late March (Spring 2017) were evaluated for heat stress under field and glass house conditions. Heat stress significantly affected all the morpho-physiological and yield contributing traits. Grain yield was positively associated with ear weight, number of kernels per ear, pollen viability, pollen shedding durations and chlorophyll content. Furthermore, secondary traits like membrane thermostability, days to anthesis and silking, anthesis-silking interval, canopy temperature, leaf firing and tassel blast showed significant negative impact on grain yield in both field and glass house conditions under heat stress. Both genotyping and mean phenotypic data of each component trait was analyzed for single marker analysis (SMA) and composite interval mapping (CIM) using WinQTL Cartographer. A linkage map of 1857.1 cM in total length was constructed by applying both SSR and SNP markers. A total of 11 QTLs were detected for 7 traits on chromosomes 1, 3, 4, 6, 7 and 9 with phenotypic variance ranged from 8.67 to 29.62 per cent. Four of these QTLs, qKPE6.1, qPV6.1, qCC9.2 and qLF4.1, accounted for above 15 per cent of phenotypic variation, and might be considered as major QTLs for heat tolerance. The data generated in present investigation laid the foundation for future work to uncover genes and mechanisms critical for the development of heat-resilient maize using genetic and biotechnological approaches.ThesisItem Open Access Mapping and transfer of GENES/QTLs for nematode (Meloidogyne graminicola) resistance from Oryza glaberrima into Oryza sativa L.(Punjab Agricultural University, Ludhiana, 2020) Gurwinder Kaur; Vikal, YogeshRice root knot nematode (Meloidogyne graminicola) is one of the emerging constraints for rice production, causes about 50% yield losses in field conditions and 98% yield losses in pot experiments. The identification and exploitation of resistant rice genotypes is one of the economic and sustainable option to minimize the yield losses caused by M. graminicola infestations. The present investigation was undertaken to identify the QTLs associated with rice root knot nematode resistance as well as the transfer of resistance to elite rice cultivars. A total of 42 accessions of O. glaberrima along with O. sativa cultivar PR121 were screened in triplicates during kharif 2016 and kharif 2017 in nematode infested sick plot at initial nematode population density of one juvenile per gram of soil. Reproduction factor of rice root knot nematode among O. glaberrima accessions was found to be <1 while in O. sativa cultivar PR121, reproduction factor was >1. Out of 42 accessions, three accessions (IRGC102196, IRGC102538 and IRGC102557) were found to be highly resistant, thirty-three accessions were resistant, and six accessions were moderately resistant. Data on different morphological traits (plant height, root length, fresh shoot weight, fresh root weight, dry shoot weight and dry root weight) revealed that no statistically significant differences were found among O. glaberrima accessions whereas PR121 exhibited significant reduction in all growth parameters in nematode infested sick plot as compared to controlled conditions. The BC1F1 population derived from the cross of O. glaberrima acc. IRGC102206 × PR121 was used for mapping of rice root knot nematode resistance. The BC1F1 plants were screened against M. graminicola in triplicates and genotyped using 84 polymorphic SSR markers. Both phenotypic data and genotypic data was analyzed and a total of 13 QTLs associated with gall number, gall index and different morphological traits were mapped on chromosomes 1, 3, 4, 5, 6 and 8. Out of 13 QTLs, three QTLs associated with gall number were identified; two QTLs designated as qGN4.1and qGN4.2 were mapped on chromosome 4 and one QTL, qGN6.1, was mapped on chromosome 6. QTL associated with gall index (qGI6.1) was found to be co-localized with qGN6.1. Similarly, qGN6.1 QTL was co-localized with plant height and root length. Due to less coverage by SSR markers, we simultaneously performed BSA-QTLseq (Bulked segregant analysis coupled with QTL-seq approach) analysis and identified QTLs for nematode resistance on chromosomes 1, 2, 3, 4, 5, 6, 11 and 12. The genomic interval of all QTLs was narrowed down to 1–2 Mb using QTL-seq analysis. The development of SNP based molecular markers from identified QTL regions will further help to saturate the linkage map and to identify closely linked markers to rice root knot nematode resistance. The identified markers will further fasten the improvement of genotypes for rice root knot nematode resistance through marker assisted breeding approach. Based on introgressed genomic regions from O. glaberrima carrying QTLs for nematode resistance the BC1F1 resistant plants were selected and backcrossed to generate BC2F1 and subsequently BC3F1 progenies for transfer of nematode resistance in the background of PR121. The data generated from this study can serve as valuable genomic resources for rice breeding programmes.ThesisItem Open Access Marker Assisted Pyramiding of Southern Leaf Blight Resistance QTLs (qSLB3.1 and qSLB8.1) in Maize Inbred CM140(Punjab Agricultural University, Ludhiana, 2022) Prabhmeet Kaur; Vikal, YogeshSouthern corn leaf blight (SLB) caused by Cochliobolus heterostrophus is the most destructive disease of maize. It is predominant in warm and humid climate and can result in yield lossesof about 40 percent. The present study was undertaken to pyramid SLB resistance QTLs (qSLB3.1 and qSLB8.1) in the background of CM140 using backcross populations developed from crosses LM5 (resistant parent) × CM140 (susceptible parent) and CM139 (resistant) × CM140 (susceptible). CM139 is the donor of qSLB3.1 while LM5 is the donor of qSLB8.1. The BC2F1 populations along with three parental lines were evaluated for SLB disease reaction by inoculating plants at 7-9 leaf stage and the disease data were recorded twice at an interval of 15 days. The disease data showed considerable variation for SLB resistance within the backcross populations. The BC2F1 progenies were subjected to foreground selection with flanking SSR markers for the identification of target QTL. The individuals which were heterozygous for both the markers were selected and possessed a disease score of less than or equal to 3 were selected. Three plants from each cross (CM139 × CM140 and LM5 × CM140) were inter-crossed for the generation of F1s. The F1 plants were assessed using the four SSR markers (two for each QTL) to check for the hybridity. The plants having both QTLs were self-pollinated for raising the F2 population and for the selection of pyramided QTLs both genotypic and phenotypic selection was conducted. The genotypic selection of the F2 population was carried out using SSRs and the individuals were categorized on the basis of presence of both the marker alleles (homozygous for each QTL), presence of a single QTL (qSLB3.1 or qSLB8.1), and no QTL present. A total of twelve plants were obtained which were having both the QTLs with homozygous donor alleles. The F2 population was also evaluated phenotypically for SLB resistance as well as flowering characteristics. The plants that were homozygous to both the QTLs (qSLB3.1 and qSLB8.1) showed enhanced level of SLB resistance. The present results indicated that additive effects of the QTLs for SLB resistance played an important role among these plants. Our findings also revealed non-significant association between flowering timings and SLB disease ratings. The pyramided lines so generated can accelerate the process of marker assisted breeding in maize for SLB resistance.