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ThesisItem Restricted Linkage map construction in guava F1 population of Allahabad Safeda x Arka Kiran using molecular markers(Punjab Agricultural University, Ludhiana, 2018) Jindal, Manish; Mittal, AmandeepGuava is a perennial fruit tree grown in tropical and sub-tropical regions of the world. As of now, there are about 160 cultivars available in India. Crop improvement work attempted in India has resulted in release of several superior selections or hybrids. However, the maximum area under guava cultivation is occupied by Allahabad Safeda. Being a cross-pollinated tree (25.7 to 41.3 % cross pollination) guava has a heterozygous genome. Molecular mapping can help us to find the relative positions of the markers as well as the markers co-segregating with the trait of interest that could finally be transferred to the cultivated species. In the present study we have attempted Linkage map construction in guava. A cross between white fleshed Allahabad Safeda and colored fleshed Arka Kiran was attempted in Fruit Science, PAU. We genotyped Allahabad Safeda and Arka Kiran using 167 genomic SSR, 22 EST based and 5 apple color specific markers. Forty eight markers showed polymorphism out of 194 total markers. Polymorphic markers applied on a population of 73 F1 individuals showed segregation. Pattern of marker segregation in the population was scored and analysed with software, MAPDISTO version 1.7.7.0.1.1 (XL 2007) and a genetic linkage map was constructed using stringency parameters of LOD score and recombination frequency set to 3.0 and 0.35, respectively. Out of 48 polymorphic markers, thirty markers were mapped on different linkage groups of guava genome and 6 linkage groups were obtained. The genetic linkage map covered a total of 538.68 cM of the guava genome. Fruits were not set on 2 year old F1 trees so color segregation was studied on leaves as a proxy for fruit color trait. Color in young and mature leaves was measured using miniature leaf spectrometer. The data recorded in terms of anthocyanin reflective index 1 (ARI1) was analysed with the help of mapping software. Two markers mPgCIR93 and mPgCIR21 were mapped to linkage group 2 on positions 54.3 cM and 10.3 cM for leaf color traits at young and mature stage.ThesisItem Open Access Molecular mapping of high grain number QTLs in aromatic rice line RNR-2354(Punjab Agricultural University, Ludhiana, 2018) Meelu, Manpreet; Kumari NeelamBasmati, the unique quality aromatic rice, is nature‟s gift to the Indian sub-continent. Basmati rice is harmonious combination of aroma, kernel expansion, fluffiness and palatability. Compared to indica varieties, Basmati rice is low yielding primarily because of lower number of grains (70 to 80) per panicle. Therefore, present study is planned to increase the grain number of Punjab Basmati 3 (PB-3). It is a semi-dwarf variety having resistance to the bacterial blight disease of rice (xa 13 and Xa 21 genes), possesses extra- long slender and translucent grains with strong aroma and good cooking and eating quality characteristics. RNR-2354, one of the rice aromatic rice line was found having very high grain number (150- 200) per plant while evaluating and characterizing 400 aromatic rice germplasm lines from all over India. With an objective of improving grain number of PB-3, a cross between PB-3 and RNR-2354 was done to generate F1, F2 and F3 progenies. Phenotypic data were recorded for grain number, grain length and grain width in F2 population and F3 progenies. A total of 5 QTLs designated as qGN-1, qGN-2.1, qGN-2.2, qGN-3 and qGN-5 were mapped on chromosome 1, 2, 3 and 5 using QTL Cartographer mapping software. This information could be further used for narrowing down and dissecting the regions harboring putative QTLs.ThesisItem Open Access Molecular mapping of Fusarium wilt resistance in muskmelon (Cucumis melo L.)(Punjab Agricultural University, Ludhiana, 2018) Jaideep Kaur; Sarao, Navraj KaurMuskmelon is an important vegetable crop of the Cucurbitaceae family grown in temperate and tropical regions of the world. Fusarium wilt is one of the major constraints for melon production around the world. Yield losses were reported as high as 100%. It primarily infects the roots resulting in blockage of uptake of water and nutrients, thus killing the plant. Various resistance genes (Fom-1, Fom-2, Fom-3 and Fom-4) specific for specific races have been reported so far. In order to readdress the production bottlenecks in melon within reasonable time frame, deployment of genomic tools such as marker assisted breeding is urgently required. Therefore, this study is frame worked to map the fusarium wilt resistance gene using SSR markers. Two genetically diverse parents “Punjab Sunehri” and “KP4HM15” were crossed and F1 was selfed to generate a F2 mapping population of 154 plants. Phenotypic evaluation on F2:3 families indicated that resistance to fusarium wilt is controlled by single dominant gene, which might be of race 0, 1 or 0, 2. For molecular analysis, a total of 527 simple sequence repeat (SSR) primer pairs were screened for parental polymorphism following bulk segregant analysis (BSA). Linkage analysis indicated that four SSR markers CMCTN35, DM0096, CSWCTT02 and ECM181 were linked to the Fom gene. Marker DM0096 was closest to the gene at 17.6cM distance and the marker CSWCTT02 was mapped at 22.7cM distance from the gene. The four markers were mapped on melon chromosome 5. There is a need to design more flanking SSR markers between DM0096 and CSWCT02 region in order to minimize the distance from the Fom gene and its transfer into the elite cultivar (Punjab Sunehri) for providing durable resistance against fusarium wilt disease. The markers provide further perspectives for fine mapping and map based cloning of the Fom gene.ThesisItem Open Access INTROGRESSION OF crtRB1 AND LcyE GENES FOR HIGH β-carotene INTO QUALITY PROTEIN MAIZE (QPM)(Punjab Agricultural University, Ludhiana, 2018) Amandeep Kaur; Malhotra, Pawan KumarMaize (Zea mays L.) being the queen of cereals deficient in Vitamin A which causes the malnutrition and major health problems. Quality protein maize has enhanced level of the amino acids, lysine, and tryptophan over normal maize varieties. However, QPM varieties are low in provitamin A, a precursor of vitamin A which can lead to vitamin A deficiency in human. In the present investigation, the grain quality of QPM inbred is further enriched for β-carotene by introgressing of crtRB1 and LcyE gene through marker assisted backcross breeding. Rare natural genetic variation of crtRB1 and LcyE gene enhances β-carotene in the kernel by blocking its conversion to further components. Traditional yellow maize though contain high kernel carotenoids, but the concentration of provitamin A is quite low (<1.5µg/g) as compared to the recommended level (15µg/g). Development of biofortified maize enriched in provitamin A, lysine and tryptophan thus holds significant potential in the alleviation of micronutrients. Marker assisted stacking of crtRB1, LcyE and o2 were undertaken in the genetic background of QLM13, inbred of PMH1 hybrid. Foreground selection was carried out using gene-specific primers on BC2F1 population of QLM 13 and background selection was carried out using SSR markers to check the recovery of recurrent parent genome. The plants of favorable alleles (crtRB1 and LcyE) and 88.5-90.1% recurrent parent genome recovery were selected and selfed to generate BC2F2 population. Foreground selection was carried out on BC2F2 population using crtRB1 and LcyE gene-specific markers and plant carrying favorable homozygous allele were selfed to generate BC2F3 progenies. Quality analysis for determination of β-carotene and tryptophan analysis was carried out on BC2F3 progenies. The introgressed BC2F3 progenies possessed a high concentration of provitamin A (1.29–11.75 µg/g) as compared to recurrent parent QLM13 (4.69 µg/g). The selected lines of high beta-carotene and tryptophan content were crossed to reconstitute PMH1 hybrid. Introgressed inbred having contrast for pigmentation in glume base and silk with respective to recurrent parents possess great utility for registration and unambiguous identification in the field.ThesisItem Restricted Fine mapping and expression analysis of stripe rust resistance genes derived from Aegilops geniculata(Punjab Agricultural University, Ludhiana, 2018) Shivendra KumarWheat production in temperate area is significantly affected by rust diseases, among which stripe (or yellow) rust caused by Puccinia striiformis Westend f. sp. tritici and leaf (or brown) rust caused by Puccinia triticina Eriks. is major threat to production. Non-progenitor Aegilops species with substantial amount of variability for stripe rust resistance genes has been exploited to a limited extent. A tetraploid non-progenitor species (UUMM genome), namely Aegilops geniculata accession pau3549 is found to be resistant to stripe rust. A stripe rust resistant introgression line-ILT598(BC2F8 Ae. geniculata acc. pau3549/CSS//3*WL711) was already available. In the present investigation an F5 population was derived from the cross of ILT598 with wheat cultivar WL711(NN). Inheritance studies in F5 population revealed that stripe rust resistance is controlled by a single dominant gene, temporarily designated as YrAg. Mapping of YrAg was done by using SNP based KASper marker linked with two group of linked gene on chromosome 5DS viz Lr57-Yr40 and Lr76-Yr70 and one marker was designed from candidate gene of comp_121307_c0_seq4 derived from ILT598. Molecular mapping using F5 population mapped YrAg at a distance of 3.3cM from KASP comp_121307_c0_seq4 towards distal end of chromosome 5D.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 limonoid glucosyltransferase gene cloned from kinnow mandarin and its overexpression in E. coli(Punjab Agricultural University, Ludhiana, 2018) Patel, Ekta Ashok; Mohanpuria, PrashantKinnow mandarin (Citrus reticulata Blanco) is a commercially important fruit crop of north western India. Its fruits and juices are rich source of health promoting phytochemicals especially limonoids. Despite to the cause of delayed bitterness, citrus limonoids can also be valued for human health because of their recently demonstrated anticancerous properties. Citrus limonoid glucosyltransferase (LGT) encoding natural debittering enzyme which is mainly responsible for conversion of all limonoid aglycones (mostly bitter) to their corresponding glucosides (non-bitter). Keeping the importance of citrus limonoids for human health and for delayed bitterness cause, in silico characterization of CrLGT gene (KP306791) from Kinnow mandarin was done. This CrLGT gene sequence was shorter than LGT reported from other mandarin because of presence of several indels. Homology detection based on BLASTN and BLASTP showed 94-99% identity with related Citrus species. The evolutionary tree construction based on PSPG motifs present in glucosyltransferases at Cterminal revealed some glucosyltransferases deficient in motifs while in CrLGT sequence it was shifted near to the N-terminal. The expression of CrLGT was seen in DE3 at 37°C with 0.7mM IPTG and RG2 bacterial strains at 25°C and 37°C with 0.5mM and 0.7mM IPTG concentrations. In order to detect the importance of citrus limonoids such as limonin, limonin glucopyranoside and obacunone, obacunone glucosides for making anticancerous drugs in future, in silico screening of cancer targets was attempted using Autodock v1.5.6. This study proved satisfactory cancer targets and predicted the list of complexes for anticancer potential for future use after validation at in vivo level.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.
