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ThesisItem Restricted Molecular mapping of leaf rust and stripe rust resistance genes transferred from Aegilops speltoides to hexaploid wheat(Punjab Agricultural University, Ludhiana, 2020) Angoth, Sravan Kumar; Chhuneja, ParveenLeaf rust and stripe rust are most serious diseases of wheat worldwide. Utilization of resistance cultivars is an effective and economical method to reduce losses from devastating diseases. Aegilops speltoides with SS genome, has been reported to be a valuable source of resistance genes for leaf rust, stripe rust and stem rust. In present study, inheritance studies and molecular mapping of leaf rust and stripe rust resistance genes were undertaken. Stripe and leaf rust resistant accession of Ae. speltoides was crossed with T. durum cv. Aconchi89. F1 was treated with colchicine and an amphiploid was developed which was selfed 2-3 times to develop stable amphiploid. Aconchi89-Ae. speltoides acc. TA1784 amphiploid was crossed and backcrossed with PBW550 and stable BC2F4 introgression lines were developed. Disease resistant introgression line P33-15 was further crossed with PBW550 for mapping population. In F2 population, 161 plants were segregated in ratio of 3:1 for strip rust and in F2:3 populations, plant were segregated in 1:2:1 ratio for resistant and susceptible for leaf rust at adult plant stage. F3 population genotyping was done by dd-RAD-seq, involves cleaving DNA with two restriction enzymes Sph1 and Mluc1 and by using dDocent pipeline, TASSEL V5.0 raw data was processed for processing steps. SNP were identified between parental genotypes P33-15 and PBW550 and 2118 total SNPs were found be polymorphic which were used for linkage map construction. The chromosomes 1D having minimum and chromosome 3B having maximum number of SNPs. Complete linkage map encompassed a total linkage distance of 17875 cM with a SNP density of 0.12 SNPs/cM. With only 569 SNPs the D genome showed least coverage of 4468.48 cM with a density of 0.127 SNPs/cM. A-genome mapped 660 SNPs while B-genome mapped 824 SNPs. Linkage map was further used for mapping genes/QTL for stripe rust and leaf rust resistance using ICI mapping using BIP function. The phenotypic data of the F2 population and genotypic data of the F3 population were used for mapping the stripe rust resistance loci. The QTL mapping was conducted with LOD threshold of 4.0. Ae. speltoides leaf rust resistance gene was mapped on terminal end of short arm of chromosome 7B at a distance of 26cM from nearest SNP marker S7B_733831709. Overall, one gene each for leaf rust and stripe rust resistance have been introgressed from wild progenitor species Ae. speltoides into hexaploid wheat. A SNP based linkage map consisting of 2118 SNP markers has been developed. A major QTL for stripe rust resistance has been mapped on chromosome 3B and a major gene for leaf rust resistance has been mapped on wheat chromosome 7B. Linked markers can be used for transferring these genes to other wheat backgrounds.ThesisItem Open Access INTRODUCTION OF GLYOXALASE I GENE FOR SALT TOLERANCE INTO RICE (Oryza sativa L.) VARIETIES ‘PR118’ AND ‘KITAAKE’ THROUGH PARTICLE BOMBARDMENT(PAU Ludhiana, 2013) Saroj Kumar Sah; Ajinder KaurThe present investigation dealing with introduction of glyoxalase I gene for salt tolerance into rice (Oryza sativa L.) through particle bombardment was carried out using two varieties PR118 (indica) and Kitaake (japonica). Calli were induced from both the varieties on MS medium supplemented with 2,4-D (3.0 mgL-1) + BAP (0.25 mgL-1) + proline (600 mgL-1) + maltose (40 gL-1) + phytagel (3 gL-1). Embryogenic calli were subcultured on shoot regeneration MS medium supplemented with BAP (4.0 mgL-1) + NAA (0.2 mgL-1) + sucrose (30 gL-1) + phytagel (2 gL-1) + agar (8 gL-1). An attempt was made to introduce GlyI gene into both the varieties. Mature seed-derived embryogenic calli were used as explants for all the transformation experiments. Success was achieved in both the varieties. Using GlyI gene, in PR118, out of 2600 calli bombarded, 249 putative transgenic plants were regenerated on medium containing hygromycin (30 mgL-1). Likewise, in Kitaake, out of 615 calli bombarded, 469 putative transgenic plants were regenerated on medium containing hygromycin (30 mgL-1). Among the 249 regenerants of PR118, 13 plants were PCR positive and in case of Kitaake, among 615 plants 41 showed PCR positive results. Further, these plants were grown to maturity in transgenic glass house. Presence of copy number of transgene was done by Real Time PCR. In total, a set of 10 PCR positive plants (5 of PR118 and 5 of Kitaake) 10 samples were analyzed, among them sample number four has single copy gene and rest has multiple copy ranging from 2-14 copies. To conclude tissue culture base line has been established in two varieties PR118 and Kitaake. Using this baseline, a total of 54 PCR positive transgenic plants were developed. The protocol developed here is genotype independent and is suitable for japonica as well as indica varieties.ThesisItem Restricted MOLECULAR PROFILING OF INTROGRESSION LINES DERIVED FROM CROSSES OF O. sativa (L.) X O. longistaminata (A.CHEV. &ROEHR.)(PAU Ludhiana, 2012) Nguyen Le Van; Kuldeep SinghRice (Oryza sativa L.) is staple food for more than a third of the world’s population, and we need to produce 30% more rice to meet the demand in next 25 years. Utilization of wild species for enhancing productivity is one of the several approaches proposed for meeting food requirements. In this study we evaluated a number of BC2F6 introgression lines (ILs) generated from the crosses between O. sativa cv PR114 X O. longistaminata acc IRGC 104301 and using DNA markers identified regions introgressed from the wild species. A set of 45 ILs and three checks were evaluated in a complete randomized block design with three replications for two years. Data were recorded on 11 agronomic traits. Several ILs showed significant increase or decrease over the recurrent parent in yield components like plant height (PH), days to flowering (DF), tiller number (TN), panicle length (PL), spikelet per panicle (SPP), spikelet fertility (SF), grain length (GL), grain width (GW), thousand grains weight (TGW) and plot yield (PY) compared to the recurrent parent PR114. Ten ILs showed significant increase in SPP during both the years over the recurrent parent and the increase ranged from 9.3 – 41.0 per cent. Likewise, five ILs showed significant increase in grain length ranging from 4.95 – 10.1 per cent, two ILs showed significant increase (16.0%) in TGW, and nine ILs showed significant increase (6.4-24.1%) in grain width. Parent polymorphism between PR114 and donor species O. longistaminta was done using a set of 366 SSR markers spanning all the 12 linkage groups and 92 polymorphic SSRs were used for characterizing the ILs. The alien segments introgressed in each of the ILs varied from 1.09% to 13.04%. Alleles which showed positive effect from O. longistaminata were observed in chromosomal regions associated with DF, PL, SPP, GL and GW traits. Alleles associated with negative effects were also observed for PH, TN, SF, PY and grain size traits. The ILs showing significant increase for one or more traits have been identified and these could be used for mapping QTL introgressed for these traits, using bi-parental crosses.ThesisItem Open Access MARKER ASSISTED INTROGRESSION OF THE opaque2 (o2) GENE INTO ELITE MAIZE (Zea mays L.) INBRED LINES(PAU Ludhiana, 2012) Ravneet Kaur; Yogesh, VikalMaize (Zea mays L.) is deficient in the essential amino acids, lysine and tryptophan. The low nutritive value of maize endosperm protein is genetically corrected in Quality Protein Maize (QPM), which contains the opaque 2 (o2) gene along with numerous modifiers for kernel hardness. Two normal inbred lines viz. LM12 and LM13 were targeted for conversion into high quality protein versions using gene based SSR markers located within o2 using two generation marker-based backcross breeding program. DMR7 and CML165 were used as the QPM donor parents. In BC2F1 foreground selection for o2 gene was done using SSR marker phi057 in cross 1 (LM12/DMR7//2*LM12) and with SSR marker umc1066 in cross 2 (LM13/CML165//2*LM13). Out of the total 188 BC2F1 plants of cross 1, 87 heterozygous plants were obtained and similarly in cross 2, 114 heterozygous individuals were obtained out of 206 plants. The BC2F1 plants having o2 allele in heterozygous form were further screened using o2 gene flanking SSR marker and the plants either single or double recombinants, were identified. Whole genome background selection on the single or double recombinants using 104 SSR markers identified three plants with 83.7 to 91.0% recurrent parent genome content in each cross and were selfed to generate BC2F2 population. The three BC2F2 families were subjected to foreground selection and phenotypic selection for kernel modification. Fifty six plants in cross 1 and thirty nine plants in cross 2 having o2 allele in homozygous condition were selfed to generate BC2F3 progenies. The kernels from BC2F2 ears were segregated for hardness of endosperm and showed different levels of modification. In BC2F2 kernels the tryptophan concentration ranged from 0.56% to 0.97% for cross 1 whereas for cross 2 it ranged from 0.58% to 0.91%. 50% opaque BC2F3 lines were evaluated for agronomic traits for selection of single plant progenies to generate BC2F4 progenies.ThesisItem Open Access CYTOGENETIC CHARACTERIZATION AND MOLECULAR TAGGING OF LEAF RUST RESISTANCE GENE TRANSFERRED FROM Aegilops peregrina INTO HEXAPLOID WHEAT(PAU Ludhiana, 2012) Deepika Narang; Satinder KaurLeaf rust is the most damaging disease of wheat worldwide. Wild germplasm of wheat is known to be a rich source of different useful genes. Ae. peregrina acc. pau 3519, a non-progenitor tetraploid species with UUSS genome, was found to be an excellent source of resistance for various diseases. In the present investigation, WL711-Ae. peregrina introgression lines (ILs) were characterized through Genomic in situ hybridization and no hybridization signal was detected in any of the IL. F2 population derived from the cross of introgression line 973 with WL711, was tested at seedling stage and segregated in a ratio of 191 resistant:63 susceptible plants with 2=0.0052(3:1). At the adult plant stage, the population segregated into 185 resistant: 65 susceptible with 2=0.0053(3:1). The progeny testing in F3 confirmed the transfer of a single gene for leaf rust resistance. Molecular characterization of the IL973 with the SSR markers indicated Ae. peregrina specific introgression on homoeologous groups 1, 2, 5 and 7. Bulked segregant analysis was done using 79 polymorphic markers and one of the SSR marker Xcfd50, detected IL973 specific allele in resistant bulk. This marker has already been reported to be linked with known gene Lr58 from Ae. triuncialis on long arm of chromosome 2B. STS marker Xncw-Lr58 amplified on whole F2 population and it was mapped at a distance of 1.7 cM from leaf rust resistance gene using computer software MapDisto. Nullitetrasomic analysis confirmed the introgression of Ae. peregrina leaf rust resistance gene (LrAP) on the wheat chromosome 2D. LrAP gene studied during the present investigation is either a new gene or an allele of the already known gene Lr58 will be established through allelic tests in subsequent studies.ThesisItem Open Access INTRODUCTION AND EXPRESSION OF ANTIFUNGAL CHITINASE AND β-1, 3-GLUCANASE GENES IN ARBOREUM COTTON TO INDUCE RESISTANCE AGAINST Fusarium oxysporum(PAU Ludhiana, 2013) Aradhna Sonik; Jagdeep Singh, SandhuIn the present study, immature embryos of diploid cotton (Gossypium arboreum L. cv. RG8) were transformed with three recombinant plasmids viz., H1Z, H2Z and pBI121 containing -1, 3-glucanase, chitinase and GUS gene cassettes respectively, under the control of CaMV 35S promoter and NOS terminator, with an objective to induce resistance against Fusarium oxysporum. The immature embryos were excised from 18-day old cotton bolls obtained during early, mid and late flowering seasons of the diploid cotton plants, cultured on germination medium (GM) [basal MS medium containing proline (560mg/l), BAP (2mg/l), GA3 (40mg/l), activated charcoal (0.20%), omnatax (500ppm) and casein hydrolysate (500mg/l)] followed by incubation under dark at 28±2oC. After five days of culture establishment, the germinating embryos were transferred under 16 hr(s) photoperiod during which they elongated into shoots and developed true leaves. Root multiplication was induced by placing elongated shoots on root induction medium (1/2 strength MS medium fortified with 0.20% activated charcoal). The plantlet formation efficiency of the embryos cultured during the mid flowering season was found to be significantly higher (37.67%) compared to the embryos excised during late (32.80%) flowering season and at par with the efficiency of embryos excised during the early season (36.28%). The three recombinant plasmids–H1Z, H2Z and pBI121, after being confirmed for the presence of gene cassettes by PCR and restriction digestion were introduced in the immature embryos using particle bombardment. The integration of gene cassettes was analysed in transient histochemical GUS assay and PCR, 48 hr(s) post bombardment. The results revealed 91.6% bombarded embryos expressing GUS gene, whereas 66.6% embryos had the co-integration of H1Z and H2Z plasmids containing -1, 3-glucanase and chitinase coding region respectively. The bombarded embryos were cultured on media GM supplemented with 210mg/l ampicillin for five selection cycles of 20 days each. After five selection cycles, 12 plantlets survived and the plantlet formation efficiency of 0.65% was obtained, which was significantly higher than the efficiency of non-bombarded embryos (0%). The PCR analysis, 100 days post bombardment, showed co-integration of -1, 3-glucanase and chitinase genes in eight plantlets with a transformation efficiency of 0.43%, while the remaining 4 plantlets showed integration of either of the genes. These putative transgenic plantlets are currently growing in the glass-house.ThesisItem Restricted GENETIC DIVERSITY AMONG Gossypium arboreum L. GERMPLASM LINES AS DETERMINED BY SSR MARKERS AND MORPHOLOGICAL CHARACTERISTICS(Punjab Agricultural University, Ludhiana –, 2013) Lovepreet Singh; Pathak, DharminderThe present study was undertaken to study the genetic diversity among Gossypium arboreum germplasm lines using molecular and morphological markers. Sixty four germplasm lines from diverse eco-geographical regions of India were taken for evaluation. Forty SSR primer pairs belonging 11 of the 13 A-genome chromosomes were employed to assess diversity at the molecular level. LD 694 and RAC 024 were observed to be quite diverse from each other. G. hirsutum accession TM1 and G. herbaceum accession RAHS 14 formed separate clusters. PIC value of the markers ranged from 0.11 to 0.79. The dissimilarity coefficient for G. arboreum ranged from 0.08 to 0.29. Mahalanobis D2 distances statistics was used to cluster the genotypes into six groups. Cluster I was the largest with thirty three genotypes followed by cluster II which consisted of 25 genotypes. Cluster III and IV both included two genotypes, whereas, cluster V and VI were having one genotype each. The maximum inter-cluster distance was 97.85 (cluster IV & V). The maximum intra-cluster distance was 30.68 (cluster I). Both the estimates of genetic distances did not find any relationship between genetic diversity and eco-geographical distribution. Several morphological characters such as plant body colour, flower colour, leaf shape, presence/ absence of leaf nectar did not have any impact on the clustering pattern of the genotypesThesisItem Restricted Analysis of detoxification and phenolic metabolism mediated through nitric oxide and hydrogen peroxide under ABA and water stress in wheat(Punjab Agricultural University, Ludhiana, 2016) Rattanpreet Kaur; Vikramjit KaurTwo wheat cultivars PBW644 (drought tolerant) and PBW343 (drought sensitive) were compared under PEG/ABA for ROS/nitric oxide (NO)-regulation of antioxidant metabolism. Endogenous ROS/NO under ABA/PEG increased germination and growth, such effect was higher in PBW644. In PBW343, reduced growth under PEG was improved by exogenous ROS/NO. ABA/PEGupregulation of GPX and GST involved ROS/NO signalling which was similar in both cultivars under ABA but higher in PBW644 under PEG. PEG increase of acid protease was higher in PBW343 while of alkaline protease was higher in PBW644. ABA/PEG-upregulation of proteases involved ROS/NO-signalling. Redox state of glutathione under PEG/ABA involved ROS/NO signalling which was induced in PBW343 while in PBW644, it was induced when exogenous ROS/NO applied under ABA (high stress level). Increase of ROS/NO under ABA/PEG was signal, it was higher in PBW644. Such signals were increased by ROS/NO-signalling. Moreover, ROS/NO-signalling also decreased ROS under ABA/PEG. Oxidative toxicity was lesser in PBW644 under PEG, was reduced by ROS/NO signals in both cultivars. However, exogenous ROS/NO under ABA could also increase oxidative toxicity. Under PEG, PBW644 showed higher upregulation of cell wall peroxidase (CW-POX) while lesser soluble peroxidase (S-POX). However, under ABA, it showed higher levels of both CW-POX and S-POX. ROS/NO signals under PEG/ABA contributed to increase both types of POX in both cultivars. PEG increase of phenolics and ABA/PEG increase of lignin was higher in PBW644. Under PEG/ABA, ROS/NOsignalling increased phenolics and decreased lignin in PBW343 while in PBW644, it increased both in shoot while decreased both in root. PEG increased flavonoids. Under PEG/ABA, ROS/NO signals may increase flavonoids.ThesisItem Open Access Effect of nitrogen fertilization on rice grain quality and the biochemical composition of straw(Punjab Agricultural University, Ludhiana, 2016) Amandeep Kaur; Sharma, NeerjaA field experiment was conducted to evaluate the effect of nitrogen fertilization on rice grain quality and the biochemical composition of straw. The main plots included nitrogen levels of 0, 90,120 and 150 kg ha-1 for non-basmati and 0, 40 and 60 kg ha-1 for basmati varieties, while the subplots included non-basmati varieties viz., PR 111, PR 122 and Pusa 44 and basmati varieties viz., Basmati 370 and Pusa Basmati 1509. In rice grain, crude protein content, head rice recovery and gel consistency were positively related with increase in nitrogen levels in both non-basmati and basmati varieties. Thousand grain weight of only the basmati varieties expressed similar response. However a negative relationship for amylose content and elongation ratio with increase in nitrogen fertilization in all the cultivars was observed. There is significant interaction between nitrogen levels and all physiochemical characters of the grain except alkali spreading value in all the cultivars. In rice straw, increase in the level of nitrogen fertilization increased the crude protein from 2.28 to 7.41% in nonbasmati and 5.16 to 6.28% (dry matter basis) in basmati varieties whereas the neutral detergent fibre, acid detergent fibre and cellulose content decreased in both non-basmati and basmati varieties. The straw hemicellulose, acid detergent lignin, silica, ash, crude fat, crude fibre and organic matter exhibited significant interaction with different nitrogen levels in both non-basmati and basmati varieties. Therefore, use of optimum nitrogen levels will improve the nutritional quality of grain for human consumption and straw as feed resource.
