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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 Embargo Genome wide association studies and genomic prediction for maydis leaf blight resistance in tropical maize (Zea mays L.)(Punjab Agricultural University, 2023) Wajhat-Un-Nisa; Sandhu, SurinderThe present study was undertaken to identify marker-trait associations (MTA) and candidate genes associated with maydis leaf blight (MLB) in maize (Zea mays L.). A diverse panel of 359 genotypes, known as the CIMMYT Asia Associated Mapping Panel (CAAM), was used in this study. The evaluation of MLB resistance was conducted through artificial inoculation across four different environments (two years and two locations). Disease severity was measured on the scale of 1-9. Significant variation was observed for genotypes. Heritability was high (>70%) for the trait. GGE-biplot depicted the stability and performance with lower disease score. A set of 10 genotypes was reported as resistant across environments with average disease score of 3.0. Best linear unbiased predictor (BLUPs) values were used for GWAS with 128,490 SNPs. Linkage disequilibrium (LD) decay in panel was estimated to be ~ 0.9 kb at r2 =0.1. Based on cumulative analysis of data, a total of 26 significant SNPs, distributed across all ten chromosomes were associated with MLB resistance. Notably, the highest number (6) of SNPs was reported on chromosome 8, 5 SNPs within bin 8.06 and 1 SNP in bin 8.01. This suggests that chromosome 8 plays a crucial role in MLB resistance. Seven novel SNPs were reported from chromosomal bins viz., 9.06, 6.05, 5.01, 1.04, 7.04, 9.01, and 4.06. Gene mining and in silico expression analysis led to the identification of 22 putative candidate genes (on the basis of significant SNPs) with the functional relevance for resistance against necrotrophic pathogen. The expression values were above 2 fragments per kilobase of transcript per million mapped reads (FPKM). The important candidate genes were protein suppressor of PHYA-105 1/SPA, ATP-dependent helicase, MYB DNA-Binding domain, PR5-like receptor kinase, bZIP transcription factor 23, Aspartic proteinase nepenthesin-2, Cdpk7, acetyltransferase NAGS1, Ubiquitin protein ligase binding, B3 domain containing protein, and ABC transporter B family member 4. These genes play pivotal roles in promoting basal defence and effector-triggered immunity (ETI) within the plant through phyto-hormone signaling, oxidative burst, hypersensitivity cell death response, transport of secondary metabolites, mitogen activated protein kinase (MAPK) mechanism, and serine/threonine protein kinase activity against necrotrophic fungi. The co-expression and physical interaction among candidate genes were 88.70%, and 8.98%, respectively. All the candidate genes were co-expressing, indicating potential functional relationships. However, physical interaction was observed between the genes SPA1/ GRMZM2G061602 and ABI3/GRMZM2G313737, associated with single SNP (S8_155841067). The sub-cellular localization predicted signals in nucleus, chloroplast, vacuole, extracellular space, and cell membrane for candidate genes. Genomic Prediction (GP) was performed using a subset of 9223 SNPs including the significant markers with genomic-BLUP model, as five-fold cross-validation. Prediction accuracy of 0.35 was observed across four environments with the training and testing set from the CAAM panel in the ratio of 90:10. The accuracy depicted higher genetic diversity in association mapping panel and potential use of GP strategies to enhance selection efficiency. The study enriched allelic diversity, along with the understanding of underlying resistance mechanism against MLB in maize.ThesisItem Embargo Understanding the effect of combined drought and early heat stress in wheat (Triticum aestivum L.)(Punjab Agricultural University, 2023) Gudi, Santosh; Sharma, AchlaWheat is an important cereal crop constrained by several abiotic stresses, including heat and drought. Heat and drought can occur either independently or together, and in either case, they significantly affect wheat productivity. The present investigation was carried out to understand the effect of heat and drought stress on agronomic, physiological, and grain quality parameters in the wheat core-set consisting of 198 germplasm lines. Huge genetic variation for the studied traits under all the environmental conditions was observed. Late sowing reduced grain yield by 45.9 and 67.3 percent, respectively, during 2020-21 and 2021-22. Combined heat and drought stresses had a more severe effect on seedling characteristics than the individual stresses. Late sowing significantly increased the phenol reaction score, grain hardness, grain protein content (GPC), and grain moisture, whereas it reduced the grain appearance score, hectoliter weight (HW), and solvent retention capacity (SRC). Grain quality parameters are significantly reduced in modern cultivars as compared to land races or old varieties. Genome-wide association study (GWAS) using 12,511 single nucleotide polymorphisms (SNPs) identified total 204 QTNs for agronomic traits, of which 25 were high-confidence MTAs, 205 for chlorophyll fluorescence parameters, of which 11 were high-confidence MTAs, 25 for seedling vigour, of which 3 were high-confidence MTAs, 27 for coleoptile length, of which 7 were high-confidence MTAs, 4 for grain hardness, of which one was high-confidence MTA, 13 for HW, of which 3 were high-confidence MTAs, 13 for GPC, of which 5 were high-confidence MTAs, 4 for sedimentation volume, of which 3 were high-confidence MTAs, and 55 for SRC, of which 17 were high-confidence MTAs. The "field-based speed breeding" protocol developed in-house aids in achieving F5 generation in two years. Drought and heat-tolerant genotypes (viz., BWL-7504 and PN-317) identified in this study may become the starting material for breeding stress-resilient cultivars. High-confidence QTNs identified from this study may become the basis for marker-assisted breeding of heat and drought resilient wheat cultivars. The advanced breeding lines (F5 generation) with enhanced heat and drought tolerance identified in this study can be evaluated in multi-locational trials before being released as a new variety.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 Morpho-physiological and molecular characterization of wheat-rye lines carrying 5R chromosome and its transfer into wheat background for nutrient use efficiency(Punjab Agricultural University, Ludhiana, 2022) Ayoubi, Habiburahman; Sohu, V.S.The aim of present study is to understand and confirm the role of 5R chromosome in micronutrient deficiency tolerance in triticale/wheat derivatives and to explore the genes related to micronutrient deficiency tolerance, transfer of 5R into wheat backgrounds known for NUE and the interaction of micronutrient uptake efficiency with nitrogen uptake. Therefore, in this investigation we evaluated a set of 316 lines derived from TRITICALE X WHEAT crosses and these were evaluated for field traits for characterization. The traits like plant height, presence of awns, number of grains, grain weight etc was observed. A total of 32 molecular markers specific to a particular arm of rye chromosome were used. The result showed that different lines carried different arms of a particular rye chromosome. The number of lines found for 1RS (54 lines) and 1RL (76 lines) to carry to short and long arms of rye chromosomes respectively. For the second chromosome of rye, 2RS and 2RL (34 lines) were carried for 2RS and (93 lines) were identified for 2RL, respectively. The number of lines identified for the rye chromosome of 3RS was (131 lines) and (51 lines) were carried out for 3RL respectively. The number of lines observed for the rye chromosome of 4RS was (36 lines) to carry the short arm of the fourth chromosome of rye and (124 lines) were found to carry the long arm of the fourth chromosome. For the short arm of the fifth chromosome of rye we identified (27 lines) while we found (82 lines) for the long arm of the fifth chromosome of rye respectively. The number of lines found for the rye chromosome of 6RS was (84 lines) to carry the short arm of the sixth chromosome of rye and (76 lines) were found to carry the long arm of the sixth chromosome of rye. Finally the number of lines found for the rye chromosome of 7RS was (46 lines) to carry the short arm of seventh chromosome of rye and (24 lines) were identified for 7RL respectively. 5R lines are found to be good in phytosiderophore content. The mean PSs of 15 5R including checks was in barely followed by rye, MT-8-SNO.38 and MT-8-SNO.45. Further the 5R chromosome was transferred to wheat background PBW677 and the F2 generation plants positive for 5R from the same was subjected to estimation of PS content. We estimated 28 5R positive and 11, 5R negative plants. In positive lines the mean of plant number 14 was found to be highest and the lowest mean vale for plant number 22.
