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ThesisItem Restricted Allele mining for phosphorus starvation tolerance gene (pstoli) in wild species of rice(Punjab Agricultural University, Ludhiana, 2016) Thakur, Shiwali; Neelam KumariIn present study, 182 accessions of Oryza rufipogon having different countries of origin along with O. sativa cv. PR114, PR121, PR122, PB3 and Vandana (positive control) were screened with flanking, co-dominant and dominant (InDel specific) markers to assess the variability throughout 90 kb insertion region of Kasalath. Previously reported SSRs markers OsPupK-4, OsPupK-05, OsPupK-20, OsPupK-29, K-41, K-42, K-43, K-46, K-48, K-52, and K-59 were applied. Based on analysis, it is inferred that most of the O. rufipogon accessions under study had 90 kb insertion present, whereas O. sativa cv. PR114, PR121, PR122 didn't have amplification with K-46, a diagnostic marker for PSTOL 1. Further, the primers were designed for full length amplification of PSTOL 1 gene (1 kb) and sequencing of 69 representative accessions of O. rufipogon with Vandana were performed. Sequence alignment led to the detection of 74 single nucleotide polymorphism among O. rufipogon accessions when compared to Vandana/ Kasalath. Out of this, 21 turned out to be false positives on manual curation and ultimately fifty three true SNPs were observed in the transcribed region of PSTOL1 gene. Transitions were more common than transversions. There were 39 transitions and 14 transversions observed. The most common SNP was A/G SNP. Based on the nucleotide diversity, a total of 17 haplotypes were formed. Haplotype II forms a major group with 41 accessions of O. rufipogon including Vandana and Kasalath whereas other 16 Haplotype groups had O. rufipogon accessions ranging from 1 to 3. Further, protein sequences were also studied in order to detect if any functional variation is pressent. A total number of 28 conversions for amino acid at different positions with comparison to reference sequence were found. The most common SNP was Lysine/Arginine. Validation of O. rufipogon accessions carrying PSTOL1 gene was done in P-deficient soil along with checks and it was found that O.rufipogon accessions had higher tolerance against P starvation when compared to check Vandna under stress conditions. F1 plants were generated by cross-pollination between cultivars and O. rufipogon accessions in order to transfer the novel alleles of PSTOL1 gene to elite cultivars. Thus, this study found out that allelic variation for locus PSTOL1 is present in Wild germplasm.ThesisItem Open Access Characterization of DREB1A putative transgenic sugarcane plants using molecular techniques and tolerance assay(Punjab Agricultural University, Ludhiana, 2017) Kaura, Varun; Malhotra, Pawan KumarTransgenic sugarcane plants of variety CoJ88 augmented with DREB1A transgene were screened physiologically to test the susceptibility and tolerance for abiotic stresses (drought, cold and salt). The PCR positive plants (Twenty-two sugarcane lines) grown in pots were subjected to drought stress at grand growth phase and evaluated physiologically (viz. Chlorophyll content, Relative water content (RWC) and Chlorophyll fluorescence) in TC1 stage (Tissue culturally generated clonal stage 1). The values for chlorophyll content exhibits a variation of (1.78-7.72) in transgenic lines as compared to control plants (1.07) whereas chlorophyll fluorescence shows a minimal effect of drought stress in transgenic lines (0.694-0.755) with respect to control plants (0.628). On the basis of aforementioned parameters six (96, 93, 90, 82, 59 and 108) best-performing lines were selected for sowing in next stage. The selected lines were subjected to drought and salt stress (50 mM, 75 mM, 100 mM, 200 mM and 300 mM). After the appearance of stress symptoms (chlorosis, wilting, leaf rolling and necrosis) the physiological and biochemical parameters (Proline and LPO content) were recorded. In TC2 stage (Tissue culturally generated clonal stage 2) transgenic plants demonstrate a nominal effect of salt (1.16-1.61) and drought (1.10-1.49) stress on chlorophyll content values as compared to control (1.03 for salt stress and 0.9 for drought stress) plants. The MDA content varies from (3.57-6.02) under salt and (4.72-7.08) drought stress in transgenic lines with respect to control plants (4.97 for salt stress and 5.93 for drought stress). Three transgenic lines (93, 82 and 96) reveal a higher increase in proline content (4417.5, 2816.8 and 3965.3) under salt stress. On the basis of above-mentioned parameters, three transgenic lines (93, 82 and 96) subjected to drought, salt, and cold stress were selected for molecular studies at RNA level through reverse transcription-PCR by using DREB1A gene specific primers. The quantitative gene expression study was done by using fifteen different stress responsive genes p5CS1, APX1, CAT, GST, HSP, LEA, NAC1, NAC5, NAC6, SOD, NHX1, NFY, NCED, ERA1, and SOS1. In contrast to proline estimation, p5CS1 gene reveals an up-regulation of 65.2 fold and 61.1 fold in transgenic plants subjected to salt and drought stress respectively. Some other genes that showed an up-regulation in transgenic plants subjected to salt and drought stress were CAT, NAC1, NAC5, NHX1 and SOS1.ThesisItem Restricted Integration of antifungal β-1,3-glucanase gene in japonica rice variety Kitaake(Punjab Agricultural University, Ludhiana, 2017) Sandhu, Dilpreet Kaur; Sandhu, Jagdeep SinghAgrobacterium mediated genetic transformation was carried out in japonica rice cultivar ‘Kitaake’ for integration of β-1,3-glucanase gene. The callus obtained from mature seeds on culture media supplemented with 3 mg/L 2,4-D, 0.25 mg/L BAP, 600 mg/L proline, 300 mg/L casein hydrolysate, 40 g/L maltose and 3 g/L clarigel was co-cultivated with Agrobacterium tumefaciens strain LBA4404 carrying H1 plasmid with pBI121 backbone harbouring β-1,3-glucanase gene. The Agrobacterium broth at O.D-0.25-0.3 was used for infection and the co-cultivation was performed for two days in the presence of 100 µM acetosyringone. The 38% regeneration was obtained after infection on media containing 4 mg/L BAP, 0.2 mg/L NAA, 30 g/L sucrose, 8 g/L agar, 2 g/L clarigel supplemented with 250 ppm cefotaxime. Out of 1215 calli subjected to infection, 368 calli regenerated and produced 620 plantlets after rooting. When transferred to glasshouse, the surviving 500 plants were analysed by PCR which revealed 16 PCR positive plants. Their T1 generation was grown and the 10 lines were found PCR positive. The reverse-transcriptase PCR analysis showed the expression of gene in 5 plants. RT-PCR positive plants were further analysed by qRT-PCR using actin as the reference gene. The results revealed the 1.5 fold increase in expression of β-1,3-glucanase gene in two lines.ThesisItem Restricted Phosphorus use efficiency in wheat and identification of the associated genes(Punjab Agricultural University, Ludhiana, 2017) Hari Gowthem G; Chhuneja, ParveenAgriculturally, phosphorus (P) is the second most essential element for the growth and development of the crop; due to its key role in various biochemical reactions in the plant system such as phosphorylation, DNA replication, membrane formation, etc. The population boom demanding a leap in the quantum of production of food grains increases the need for the external application of the nutrients in the form of fertilizers. Phosphatic fertilizers being the costliest among all the chemical fertilizers makes it difficult for a marginal farmer to meet his farm demands. This situation could be managed by breeding crops which would use phosphorus efficiently. A panel of 42 wheat genotypes consisting of 10 bread wheat genotypes, 16 synthetic wheat and 16 Triticum dicoccoides accessions was assembled for assessing phosphorus use efficiency and underlying genes. Thirty of these genotypes were analysed in a pot experiment at two Phosphorus treatments. The panel was evaluated for various agro-morphological traits such as phosphorus concentration in straw and seeds, shoot dry matter, number of grains and yield and Phosphorus deficiency tolerance index (PDTI) of every trait was determined for the panel. The synthetic wheat PBW114-Ae.tauschii acc. pau14200 was found to be an efficient genotype at phosphorus starvation as its relative growth at phosphorus starvation was 68.5%, 66.2%, 92.1% and 151.1% more in case of shoot dry matter, number of tillers, yield and number of grains per plant, respectively. T. dicoccoides were observed to be better at Phosphorus uptake as compared to other genotypes. In rice OsPstol1(Phosphorus starvation tolerance), a gene for phosphorus use efficiency, has been cloned and characterized which is responsible for a three-fold increase in phosphorus-uptake and grain-yield in rice. A similarity search was conducted in the wheat genome sequence (IWGSC) for the OsPstol1 orthologous sequences which led to identification of six homologous sequences, two at chromosome 5AS, one each on 3B, 3AL, 3DL and 6DS. Genes were predicted and primers were designed for in vitro amplification and matching transcripts for these sequences were mined from wheat transcriptome database from a Phosphorus starvation study. Amplifications in the germplasm panel showed the presence of homologous sequences in most of the genotypes. Investigation was proceeded with the first hit contig of the BLAST result ‘5AS_IWGSC_1484262’ for allele mining which resulted in the identification of seven SNPs forming five haplotypes which further translated to four different polypeptide sequences. The synthetic wheat PBW114-Ae.tauschii acc. pau14200 could be a source for the genes responsible for enhanced phosphorus use efficiency in wheat and its relation with the wheat orthologs of OsPstol1 could be deduced by further investigating through expression analysis and the promoter sequence analysis of the ortholog.ThesisItem Restricted 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 NeelamBrown 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.ThesisItem Restricted 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, ParveenLeaf 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.ThesisItem Open 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, ParveenAe. 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.ThesisItem Open Access Characterisation of transformed sugarcane carrying anti-fungal β-1,3-glucanase gene(Punjab Agricultural University, Ludhiana, 2016) Nayyar, Shivani; Sandhu, Jagdeep SinghThe present study entitled “Characterisation of transformed sugarcane carrying anti-fungal β-1,3-glucanase gene” was carried out to characterize in planta transformed sugarcane plants. The objective of this study was to induce red rot resistance caused by C. falcatum. The in planta transformed T0 putative transgenic plants (10) were propagated through plant to row progenies leading to formation of 295 axillary bud setts, out of which 127 setts sprouted representing the clonal generation 1 (CG1). The PCR analysis on T0 putative transgenic plants of CG1 (127 clones) revealed the amplification of 1764 bp β-1,3-glucanase transgene in six putative transgenic plants. The RT-PCR pointed towards the differential expression of transgene in all the six plants and the relative β-1,3-glucanase transgene expression by qRT-PCR confirmed that three plants had 3.197, 4.280 and 4.438 fold increase in transgene expression as compared to non-transformed plant. Bioassay for red rot on six qRT-PCR positive plants with Cf08 pathotype demonstrated that three plants showed resistance against red rot and with Cf09 pathotype demonstrated that two plants showed moderate resistance against red rot. The CG2 plant analysis through qRT-PCR revealed similar relative transgene expression in the clones suggesting absence of chimerism.ThesisItem Open Access Androgenesis response studies in Guava (Psidium guajava L.)(Punjab Agricultural University, Ludhiana, 2017) Bhupinder Singh; Mittal, AmandeepDevelopment of haploids and doubled haploids through in vitro androgenesis greatly accelerates the process of achieving homozygosity in crop plants including fruit trees. In tree species including Citrus, Papaya, Neem, Mulberry, Custard apple and Apple, androgenesis have been attempted successfully to overcome self in-compatibility barriers so as to raise homozygous trees. In current study we attempted haploid production in three genotypes of guava viz. Allahabad safeda, Purple Guava and Punjab Pink. Anthers with microspores at mid to late uninucleate stage on N6 basal media containing 1% PVP showed maximum callus induction of 16.3±0.27% in Allahabad Safeda followed by 10±0.41% in Punjab Pink and 4.6±0.27% in Purple Guava. The stereomicroscopic evaluation of callus indicated the callus emergence from microspores. The callus induction frequency was enhanced from 1.7±0.27% to 27.7±1.18% by cold (4 ºC) and mannitol (8%) pre-treatment for 3-7 days to unopened stage IV flower buds. 77% calli survived on best regeneration media, nourished with liquid form in the mother test tube without mechanical disturbance for 1 month. One callus exhibited secondary structure on regeneration media. Isolated microspores showed increase in size on MS and N6 media containing 2,4-D (2.5 mg/L) and Kinetin (2.5 mg/L) and few anomalies on MS media fortified with BAP (0.2 mg/L) and Kinetin (0.2mg/L). 2/50 ovaries underwent callusing after 4 months of in vitro culturing on 1% PVP containing basal MS media and callus finally underwent necrosis after one week of sub culturing on MS media fortified with BAP (0.2 mg/L) and Kinetin (0.2 mg/L). Haploid nature of callus can further be evaluated by counting the chromosome number in the cells.
