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ThesisItem Restricted Genome wide association studies for sorghum shoot fly and red leaf spot resistance(Punjab Agricultural University, 2024) Jasneet Kaur; Dr. R.S. SohuThe present study was conducted for the assessment of shoot fly and red leaf spot resistance and discovery of markers and candidate genes associated with target traits in Sorghum bicolor across years and locations. A diversity set of 121 germplasm lines, acquired from ICRISAT and IIMR, was evaluated under two environments (two locations – Ludhiana (2020-21 and 2021-22) and Faridkot (2021-22)) for shoot fly whereas at Ludhiana for two years kharif 2020-21 and 2021-22 for red leaf spot disease. The germplasm lines were evaluated for nine morphological traits for shoot fly representing- per cent dead heart formation (DH), glossiness(GL), leaf sheath pigmentation (LSP), plants infested with eggs (PWE), number of eggs per plant (PPE), seedling vigour (SV), seedling height (SDH), plant height (PH) and number of tillers (TL). Significant genetic and genotype x environment interaction variance was observed for all test traits. Ten genotypes viz., IC 288122, IS 7657, IS 7663, IS 7745, IS 8155, IS 8282, IS 8256, IS 8348, IS 8230 and SSV 17 were found resistant and gave stable performance across all environments. Resistant genotype IS 8348 was selected for generation of bi-parental cross with susceptible genotype IS 7017. Similarly, disease score and area under disease progressive curve was used for assessment of red leaf spot disease. Significant genetic variance and genotype x environment interaction variance was observed for disease score under both the environments. Four lines viz., IS 7017, IS 8015, IS 8256 and SCL 4 were free from infection, indicating resistance to red leaf spot. Best linear unbiased predictors values (BLUP) of traits were used for genome-wide association study (GWAS) using 42678 SNPs. Linkage disequilibrium decay in population was estimated to be ~50kb. A large number of significant marker-trait associations were identified for all test traits. The genomic regions identified on chromosomes 1, 2, 4, 7 and 10 were previously reported to be associated with shoot fly resistance for trait DH and GL. Annotations helped to predict candidate genes in the genomic regions associated with the significant SNPs. Important candidate genes involved were receptor kinases (LRR, serine/threonine protein kinases, hydrolases and transferases), stress signalling pathways (ethylene, abscisic acid and jasmonic) for shoot fly and (RLP, WRKY, F-box protein, CDP, zinc finger and putative disease resistance protein) for red leaf spot disease. Associative SNPs were converted into KASP markers and S1_15319241 KASP marker associated with DH was validated in the F2 population of cross IS 8348 x IS 7017.ThesisItem Restricted Senescence, nutrient remobilization and yield in wheat germplasm introgressed with Gpc-B1 gene(Punjab Agricultural University, Ludhiana, 2022) Jafar Tanin, Mohammad; Sharma, AchlaThe present breeding efforts are required to evaluate a set of germplasm with the Gpc-B1 gene in their genetic background. However, Gpc-B1 is the only recognized and cloned gene which explains 66% of the phenotypic variation for GPC. Therefore, it is essential to enrich our knowledge regarding its role in protein accumulation in wheat grain. On the other hand, the Gpc-B1 has a negative relationship with grain yield. This has made it difficult to create an elite wheat line that has both high GPC and a high yield. The accelerated senescence due to the presence of this gene shortens the grain filling period. We phenotyped the NILs for different phonological and yield-related traits, including number of days to flowering (DTF), plant height (PH), number of spikelets per spike (SPS), 1000-grain weight (TGW), yield, number of days to maturity (DTM), and grain protein content (GPC). An alpha-lattice design was used to do an analysis of variance, which showed that there were significant differences between the genotypes for all of the traits. The Gpc-B1 gene accelerated the flowering time and maturity time by about one day and two days, respectively, as compared to genotypes having no Gpc-B1 gene in their genetic background. Simultaneously, the same gene made the plant shorter by an average of 4.41 cm. The functional Gpc-B1 gene in the genetic background of the NILs increased the protein accumulation in wheat grain through an accelerated senescence and more efficient nitrogen transportation process, which was initiated earlier. But at the same time, the improvement in GPC could result in a grain yield penalty due to a reduction in grain size. A better understanding of how nutrients or chemicals change the function of wheat plants could help breeding programmes in the future. The NILs were evaluated under KNO3 and salicylic acid treatment and control (no KNO3 and salicylic acid application) conditions with the standard fertilizer application rate recommended by PAU for one year (2020–21). A combined analysis of variance using an alpha lattice design was conducted, which indicated significant variation among the studied traits, including DTF, PH, SPS, GPS, DTM, HI, GPC, Yield, and TGW. The potassium nitrate application significantly affected the PH, GPS, DTM, TGW, yield, HI, chlorophyll content in flag leaf at anthesis state and 30 DAA, and other traits. Salicylic acid application significantly affected the number of grains per spike (GPS), 1000-grain weight (TGW), yield, and number of days to maturity (DTM) as compared to the control experiment. This delay had no effect on GPC in NILs, but it did increase yield and traits related to yield, like TGW and GPS. A pooled analysis of variance using an alpha lattice design was conducted, which showed significant variation for all the traits under study. High GPC variation in lines with the same genetic background (PBW550) led to a lot of questions. To find the reasons for this variation, we isolated the high quality RNA of two high GPC NILs and two low GPC NILs. Differential gene expression analysis was carried out on each NIL as a control. A total of 1418 differentially expressed genes (DEGs) were discovered for the selected NIL across all three stages, out of which 1117 genes were upregulated while 301 genes were downregulated. This suggests their probable role in seed development.ThesisItem Embargo Genetic enhancement of white maize (Zea mays L.) through conventional and molecular approaches for yield and amylose content(Punjab Agricultural University, 2023) Arora, Arushi; Sujay RakshitMaize is a widely grown crop with enormous potential in food, feed and industries. White maize is preferred staple food crop in India. Combining ability and SNP data was used to develop heterotic groups in white maize. Two heterotic groups with 7 and 19 inbreds were developed. These can be used to develop high-yielding white maize hybrids. The top high-yielding hybrids were subjected to analysis of food quality parameters like physical, proximate, textural, pasting and sensory properties and compared with local landraces. The hybrids, WHM 1, WHM 2 and WHM 8 performed better in various food quality parameters. Further, high amylose maize offers several advantages in food and industrial sectors. Parents of two maize hybrids, viz., HM 5 and HM 12, were converted to a high amylose version (43.20 to 58.10% in comparison to HKI 1344 with 26.39 %, 40.40 to 56.60% in comparison to 23.29 in HKI-1348-6-2 and 40.40 to 56.30 % in comparison to HKI 1378 with 22.25%, amylose contents) through marker-assisted backcross breeding. These maize lines would be the first highest amylose lines developed in India. To elucidate the role of several other genes/modifiers involved in controlling the amylose content, transcriptomic analysis of lines with differential amylose content was performed. Total of 43 differentially expressed genes (DEGs), 19 core and 24 were involved indirectly. Pul gene, several β-glucosidase, sucrose, hydrolyses and Nudix hydrolyze genes were involved in regulating the amylose contents besides ae1 gene. Functional annotations and expression networks also identified several pathways linked to amylose content.ThesisItem Embargo Pyramiding QTLs for heat stress tolerance in bread wheat variety PBW 343(Punjab Agricultural University, 2023) Relan, Ashima; Johar SinghWheat is a major cereal crop that is considered to be a source of basic calories and protein for more than 80% of the world population. Heat is the major abiotic stress limiting wheat production around the globe and its importance is expected to increase because of the expected climate change. The present study was conducted to pyramid various QTLs with complementary action for heat stress tolerance and combine them with stripe rust resistance. To accomplish these objectives, first KASP primers for SNP markers linked to grain yield and 1000-grain weight under heat stress conditions in wheat were developed. To pyramid the complementary QTLs, a double cross F2 population of 144 progenies was developed between a widely adapted variety, PBW 343 and two donor parents viz., Giza 168 and Sunstar. Based on phenotypic evaluation of the progenies under normal and heat stress conditions along with molecular marker analysis, five progenies numbered as 25, 55, 69, 79 and 94, were selected that had yield and other component traits better than the recurrent parent both under normal as well as under heat stress conditions. With the objective to combine heat tolerance with stripe rust resistance, 97 BC1F2 progenies from a cross between a rust donor parent (PBW 723) and two heat tolerant doubled haploid (DH) lines were evaluated at seedling stage for rust resistance. Ten of these 97 progenies were selected which had higher stripe rust resistance than the donor parent and grain yield than PBW 343.ThesisItem Embargo Mapping QTLs and identification of candidate genes for forage quality and biomass-related traits in pearl millet [Pennisetum glaucum (L.) R. Br.](Punjab Agricultural University, 2023) Harmanpreet Singh; Bhardwaj, RuchikaThe present investigation was undertaken with the objectives to evaluate the genetic variability in the RIL mapping population along with the mapping of QTLs and identification of candidate genes for fodder quality and biomass related traits in pearl millet. The RIL mapping population comprised of 273 lines derived from the cross between Jakhrana S8-28-2-P4 (high biomass and low forage quality) x RIB 335/74-P1 (low biomass and high forage quality) was evaluated at two locations in Punjab viz., Ludhiana and Abohar in an alpha lattice design with two replications. Significant variability was observed for all the forage quality and biomass related traits at both the locations. Transgressive segregants were recovered for all the traits and stable lines for in vitro organic matter digestibility (RIL166, RIL54, RIL188, RIL57, RIL126) and total biomass (RIL256, RIL173, RIL133, RIL235, RIL179) were identified by modified AMMI stability analysis. The linkage map was constructed using 755 single-nucleotide polymorphisms (SNPs) markers spanning a length of 3080.44cM with the presence of SNP at 4.08 cM regular distance interval. Total 40, 42 and 25 QTLs were identified for Ludhiana, Abohar and across the locations, respectively for various fodder related traits. A genomic interval with flanking markers S6_234379851- S6_64109715 was found to be associated with IVOMD, CP and ME at Ludhiana, Abohar and across the locations.Major QTLs were mapped for these traitsshowing phenotypic variance ranged from 10-34%. For total biomass, major QTLs were detected on LG 1, 2, 3 and 4 and with a phenotypic variance ranged from 14-40%. The expression studies revealed the nine key candidate genes viz., BHLH 148, Resistance to phytophthora, Laccase 15, cytochrome P450, PLIM2c, GRF11, NEDD AXR1, NAC 92 and TF 089 based on differential expression between the parents in leaf tissues at active vegetative stage and reproductive stage. These genes were identified from cross taxa analysis across different crop species. The key candidate genes identified in the present study were found to regulate the cell wall biosynthesis and had a direct influence on the quality of fodder. Therefore, QTLs and candidate genes identified from this investigation would be further useful for the development of superior pearl millet genotyped for forage quality and biomass.ThesisItem Restricted Identification and characterization of QTLs governing key nitrogen fixation and nutrient traits in chickpea (C. arietinum L.)(Punjab Agricultural University, Ludhiana, 2022) Sethi, Sorabh; Sarvjeet SinghThe present study was conducted with an objective to identify and characterize the genetic factors underlying two broadly categorized traits viz., nutrient availability and nitrogen fixation related traits in chickpea and their relevance with economic yield in chickpea. The analysis of variance (ANOVA) revealed significant variation for all the traits under study among all genotypes across the years and environments (location) and also in pooled data. Measurement of skewness and kurtosis indicated presence of duplicate and complementary gene interaction for different traits. Correlation analysis indicated positive and significant correlation of seed yield with pods per plant. Similar association was observed between days to 50 % flowering and days to maturity. Apart from agronomic traits, no significant correlation was observed between nutrient and nitrogen fixation related traits. However, significant and positive correlation was observed among Fe and Zn content traits and also among different nitrogen fixation traits, but the pattern was not consistent for all the years and locations. For genotypic analysis, genotypic data was generated using genotyping by sequencing (GBS-ddRAD) approach. Total of 25,233 filtered SNPs were obtained based on 2X genomic coverage but post filtering, 520 SNPs were eventually used for generation of linkage mapping and bi-parental QTL mapping using QTL ICi mapping software. Total 14 QTLs have been identified, out of which four QTLs have been identified for nutrient traits i.e., three for grain Fe content while one QTL for Zn content. Six QTLs were identified for nodule number trait while for leghemoglobin content, total of four QTLs identified. In our study, no statistically significant QTL were identified for chlorophyll content and nodule dry weight. Promising lines for grain iron, zinc content and nitrogen fixation traits were identified which can be used as donor parents in chickpea breeding programme and developed markers can be used in marker assisted selection (MAS) for identifying desirable genotypes having allele of interest.ThesisItem Restricted Molecular characterization of basmati introgression lines for spikelet per panicle QTL (qSPP2.1) AND transfer of neck blast resistance in basmati rice(Punjab Agricultural University, Ludhiana, 2021) Navdeep Singh; Mangat, Gurjit SinghThe present day Basmati rice cultivars have lower yield potential particularly due to lower spikelet per panicle. In this study, Basmati introgression lines (BasILs) have been used which were developed by backcrossing of Punjab Basmati 3 as recurrent parent with O. longistaminata derived introgression line LIL-427 having significantly higher spikelet per panicle QTL (qSPP2.1) as donor parent. A total of 115 BasILs in BC1F5 and BC2F4 generations were evaluated in replicated trial for yield and quality traits during the kharif season 2019 at the experimental area, School of Agricultural Biotechnology, PAU, Ludhiana. Data was taken from five randomly selected plants for all the traits except plot yield and averaged. Range of variation was observed among BasILs for yield and quality traits viz; plant height, number of tillers per plant, spikelet per panicle, spikelet percentage, 1000 grain weight, grain yield, milled rice recovery (MRR), head rice recovery (HRR), grain length (GL), grain breadth (GB), length breadth ratio (L/B) and kernel length after cooking (KLAC). Analysis of variance revealed significant variation for all the traits except KLAC. A number of BasILs were identified having desirable variation for combination of different yield and quality traits. Mean spikelet per panicle in BasILs ranged from 26.7-195.4 as compared to recurrent parent Punjab Basmati 3 (81.0). Based on this variation, 46 BasILs containing 23 BasILs with significantly higher and 23 with significantly lower spikelet per panicle were selected to know the proportion of parental genome contribution with the help of 117 polymorphic SSR markers. Chromosome-wise graphical genotypes for each individual were prepared using GGT v 2.0 software which represented a clear-cut distinction between recurrent and donor parent genome per chromosome. The proportion of the recurrent parent genome ranged from 21.0-54.7 % with an average of 40.1 % for all BasILs. The proportion of the donor genome for the BC2F4 and BC1F5 progenies ranging from 37.4-75.2 % with an average of 52.5 %. The Punjab Basmati-3 accounted for an average introgression of 40.1 % in BC1F5 and 67.7 % at BC2F4 progenies. The estimated average of LIL-427 genome was 52.5 % in BC1F5 and 21.8 % in BC2F4. The 46 BasILs were further analyzed with Punjab Basmati 3 gene specific markers viz. aroma (Badh 1 & Badh 2), bacterial blight resistance (xa13 & Xa21) and amylose (RM 190). Of these, six lines were found to be positive for all the genes. Since neck blast caused by Magnaporthe oryzae remains to be the major production constraint, therefore, another experiment was undertaken with the objective to incorporate two major QTL namely qBL3 and qBL7 in the genetic background of Pusa Basmati 1121. Based on evaluation of 69 BC1F2 plants of cross Pusa Basmati 1121 X INGR15001 with SSR markers RM 141738 & RM 3484 for the presence of qBL3 and qBL7, 24 plants were found to be positive for qBL3, 5 plants for qBL7 and one plant positive for both of the QTLs. Selected BasILs containing desirable variation for yield and quality component traits and backcross progenies showing presence of neck blast resistant QTLs can be further used in breeding programme to improve yield potential of basmati rice.ThesisItem Open Access Genome-wide association mapping for Fe, Zn and yield traits in pigeonpea(Punjab Agricultural University, Ludhiana, 2021) Harpreet Kaur; Inderjit SinghIn the present study, an effort was made to understand the complex nature of yield and quality traits and genome-wide association studies (GWAS) was undertaken to identify QTLs associated with traits. The analysis of variance (ANOVA) revealed significant variation among all genotypes in both the years and pooled data. Measurement of skewness and kurtosis indicated presence of duplicate and complementary gene interaction for different traits. The seed yield exhibited highly positive and significant correlation with number of primary branches per plant, number of secondary branches per plant and number of pods per plant for both the years and pooled data across two years. Path analysis identified pods per plant, number of primary and secondary branches as the major contributing traits to grain yield in both the years and pooled data. Yield per plot and pods per plant exhibited high heritability along with high genetic advance showing additive gene effect and could be considered during selection. In genotypic analysis, a total of 7366 filtered SNPs were used for genome-wide association studies through ddRAD-Seq. A total of 30 highly significant associated SNPs in 2018-19, 23 significant associated SNPs in 2019-20 and 25 significant associated SNPs in pooled data were identified through combination of two or more than two models for different traits. Most of the SNP loci were found to be environment and trait specific but few stable and consistent SNPs were identified. Some of the SNPs associated with days to maturity, yield per plot and grain iron content were observed to be consistent in 2018-19 and pooled conditions. The candidate regions were defined by the average LD decay distance or the LD block. Biological function of the identified candidate genes revealed their role in plant growth and development, organogenesis, pollen development and source-sink relationship. These genes were found to be present on chromosome 02, 03 and 09. Three polymorphic KASP markers for traits viz., grain iron content, number of primary branches and yield per plot were validated and showed that parents have homozygous alleles for the traits and alleles were found to be segregating in the F2:3 population. Promising genotypes for grain iron, grain zinc content and yield attributing traits were identified which can be used as donor parents in pigeonpea breeding programme and developed markers can be used in marker assisted selection (MAS) for identifying desirable genotypes having allele of interest.ThesisItem Restricted Mapping of QTLs for grain zinc and exploitation of protein variability in rice (Oryza sativa L.)(Punjab Agricultural University, Ludhiana, 2022) Rajvir Kaur; Rupinder KaurThe present study was conducted to map QTLs for high grain zinc (Zn) in 119 F9 RILs derived from the cross of Dubraj and high Zn donor, IR 75862-206-2-8-3- B-B-B at Punjab Agricultural University from 2018-2020 along with checks. Significant genotypic variance was observed for grain Zn content in brown rice of the RIL population ranging from 16.8 to 40.8 ppm. QTL analysis revealed two QTLs for high grain Zn in brown rice viz., qZn4.1 and qZn4.1 with phenotypic variance 11 % and 10.9 % respectively on chromosome 4. Another QTL, qZn2 with phenotypic variance of 6% was identified on chromosome 2. Protein variability was also studied in a set of 90 wild rice accessions belonging to AA and CC genome, 19 non-basmati genotypes and 9 Basmati cultivars. The wild rice accessions had significantly high protein levels than cultivated ones. For wild rice accessions, the protein content ranged from 7.5 % to 20.8 % with a mean of 14.2%. For non-basmati cultivars and Basmati it ranged from 7.3 % to 11.5 % and 10 % to 11 %, respectively. Fractionation of storage proteins and SDS gel electrophoresis revealed differences in the protein content between the wild rice species and cultivated rice genotypes was mainly due to differences in their glutelin contents and high molecular weight glutelin polypeptides. Total tocopherol content in wild rice accessions along with non-basmati and Basmati cultivars was also evaluated and significant differences were found among them. The high Zn lines along with lines that have high protein and tocopherol content has potential to improve the nutritional quality of rice.
