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ThesisItem Open Access Molecular Characterization of Fatty Acid Desaturases Genes in BRASSICA JUNCEA L.(Punjab Agricultural University, Ludhiana, 2021) Pawar, Vishlavath Harish; Gurpreet KaurThe present study was undertaken for establishing allelic variation for FAD2 and FAD3 genes and developing primers for low/high oleic acid content for marker-assisted selection in B. juncea. Four orthologous of FAD2, BjuA.FAD2.a1, BjuA.FAD2.a2, BjuB.FAD2.b1 and BjuB.FAD2.b2 is located on the chromosomes A01, A05, B01, and B05 and six orthologs of FAD3, BjuA.FAD3.a1, BjuA.FAD3.a2, BjuA.FAD3.a3, BjuB.FAD3.b1, BjuB.FAD3.b2 and BjuB.FAD3.b3, located on chromosomes A03, A04, A05, B03, B04, and B05, respectively, were identified for genotypes with high, intermediate and low oleic acid content. The coding sequences of BjuA.FAD2.a1 showed premature termination of protein synthesis, whereas BjuA.FAD2.a2, BjuB.FAD2.b1 and BjuB.FAD2.b2 genes indicated a single exon without any intron. The same sequence length in both high and low oleic acid genotypes clearly revealed the role of point or missense mutations for high/low oleic acid. Multiple sequence alignment between sequences of low, intermediate, and high oleic acid for each orthologs indicated nucleotide variations at 18 and 6 positions only for BjuA.FAD2.a2 and BjuA.FAD2.b1, respectively, of these, only one and two SNPs could be translated to amino acid changes. Complete sequence information of FAD3 orthologs indicated exons interrupted with introns. Multiple sequence alignment indicated nucleotide variations only for BjuA.FAD3.a3 and BjuB.FAD3.b2. The inheritance of oleic acid on the F2 population indicated digenic additive control with high oleic acid being recessive in expression. CAPS markers developed for differentiating SNPs (high/low oleic acid) showed co-segregation with oleic acid content in the parents as well as in the F2 population. The agronomic evaluation indicated that some of the high oleic acid genotypes could serve as an excellent donor with minimal linkage drag.ThesisItem Open Access Development and validation of kaspmarkers for heat stress tolerance QTLs in wheat (Triticum aestivum L.)(Punjab Agricultural University, Ludhiana, 2022) Ranjeet Singh; Dhaliwal, InderpreetA panel of 24 wheat genotypes, comprising 19 heat tolerant, 3 heat susceptible and 2 parental lines were evaluated under timely and late sown conditions during rabi 2020 in a Randomized block design with an aim to develop and validate a KASP assay for heat stress tolerance. Eleven major reproductive traits, including grain yield, plant height and days to maturity were evaluated. In general, there was a reduction in grain yield under late sown compared to timely sown conditions. This trait was highly variable among the 24 genotypes as some of them (KSG1188, KSG089, KSG849, KSG1189 and KSG1195) exhibited very low reduction in yield, indicating a tolerance to heat stress for maintaining the desired yield levels. Similar behavior was observed for other plant traits also, viz, days to heading, days to maturity, mean grain weight per main spike, spikelet fertility and tiller number per meter square. There were also some genotypes that displayed increased height and biomass under late sown conditions, which is rare for a heat stressed crop. The stability in performance of the genotypes may be attributed to heat stress tolerance under late sown conditions. Since the QTL for stress tolerance were already described in an earlier study, the identified GBS-SNPs were converted to 4 KASP primers following standard procedures to run the KASP assay on this panel. Of these four primers, 2 were successful in allele calling, thereby giving an efficiency of 50%. Hence, converting these identified GBS-SNPs into KASP assays can successfully be used to differentiate the genotypes or populations for heat stress related QTL.ThesisItem Open Access 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 Open Access Marker assisted development of high yielding wheat combining disease resistance and premium quality(Punjab Agricultural University, Ludhiana, 2021) Pradeep Kumar; Sharma, AchlaDevelopment of end product specific wheat germplasm is the comparatively newer objective in wheat breeding programme. Wheat breeding programme in our country has mainly focused on yield and resistance breeding. There has been no dedicated chapati quality breeding programme in until recent past. The present study aimed to decipher the basis of chapati quality and to develop premium quality wheat germplasm carrying high levels of stripe rust resistance and leaf rust resistance through pyramiding of Lr57/Yr40 and Yr15 rust resistance genes in premium chapati quality background. Analysis of chapati quality traits in specially constituted core set of wheat genotypes at multilocations of the state revealed that the environment in which wheat is grown has significant effect on grain quality. The major components of chapati making quality include various components such as dough handling, chapati colour, texture, flavor, taste, sweetness pliability etc. and should be given due weightage for deriving a composite chapati score. Chapati quality traits protein content (0.143), hectoliter weight (0.278), thousand grain weight (0.474), sedimentation volume (0.194), gluten index (0.260), water absorption (0.359), puffing height (0.302) were found to be positively correlated with overall chapati score. The negative correlation was found for phenol score (-0.149), water loss after baking (-0.198) and water loss after 4 hour (-0.380) with chapati score. The farinographic traits showed genotypic difference among genotypes. Identification and selection based on quick and reliable traits in segregating populations of targeted crosses is very important to develop germplasm with specific quality traits. In the segregating population, the F2 and F3 homozygous pyramided plants were identified for both the genes Lr57/Yr40 and Yr15 using KASP and SSR markers, respectively. The plants homozygous for both of the genes were further analyzed for agronomic and quality traits.ThesisItem Open Access Characterization of celery (Apium graveolens L.) genotypes for economic traits(Punjab Agricultural University, Ludhiana, 2021) Mandeep Singh; Nara, UshaCelery (Apium graveolens L.) is one of the important crops in Apiaceae family. It is cultivated worldwide for food as well as for use in pharmaceutics as it is an excellent source of vitamins, phenolic compounds, volatile oils and other nutrients. Identification of superior genotypes with improved biochemical and agronomic traits is essential to develop varieties for commercial cultivation. The present study focused on characterization of celery genotypes for economic traits. The performance of 20 celery genotypes over two locations during rabi 2019-20 and 2020-21 was investigated using GGE biplot analysis. The major factors influencing traits were genotypes, locations, as well as their interactions. Correlation analysis revealed a positive and significant association of seed yield with secondary branches and the number of umbels per plant. Biochemical profiling of microgreens, leaves, stalks and seeds of celery genotypes was carried out. Antioxidant activity (40.95 to 60.49%) and crude protein (28.81 to 39.94%) contents were highest in microgreens. The study revealed high content of total phenols (7.59 to 13.93 mg/g FW), total soluble sugars (20.49 to 27.77 mg/g FW), reducing sugars (10.24 to 14.83 mg/g FW), phosphorus (5.2 to 6.8 mg/g DW), iron (0.40 to 0.54 mg/g DW), copper (0.015 to 0.026 mg/g DW), ascorbic acid (0.70 to 1.77 mg/g DW), total soluble proteins (173.54 to 201.54 mg/g FW) and tocopherol (10.45 to 14.89 mg/g FW) in seeds. The concentration of total chlorophyll (1.03 to 1.54 mg/g FW), Chl a (0.80 to 1.45 mg/g FW), carotenoids (0.29 to 0.43 mg/g FW), non-reducing sugars (14.41 to 19.42 mg/g FW), and potassium (20.3 to 26.1 mg/g DW) were found highest in leaves harvested 60 DAT while that of Chl b (0.28 to 0.61 mg/g FW), zinc (0.11 to 0.19 mg/g DW), manganese (0.017 to 0.028 mg/g DW) were observed highest in leaves harvested 90 DAT. Highest contents of sodium (5.2 to 8.0 mg/g DW) and calcium (41.5 to 51.3 mg/g DW) were found in stalks harvested 60 DAT whereas crude fibre content (16.39 to 20.86%) was highest in stalks harvested 90 DAT. The study revealed the presence of total oil (17.30 to 24.05%) and essential oil (1.27 to 2.47%) in seeds of celery genotypes. Based on biochemical profiling and agronomic traits, four genotypes, namely, PAU3, PAU8, PAU16 and PAU18 were found superior in terms of seed yield and biochemical composition. Characterization of celery genotypes based on biochemical composition and yield components clustered them into four different groups.ThesisItem Embargo Optimization of protocol for scaling up double haploid production and DNA-free editing of gene controlling haploid induction in maize(Punjab Agricultural University, Ludhiana, 2022) Harjot Kaur; Sandhu, Surinder KIn maize, doubled haploid (DH) technology is an excellent approach to accelerate line development and hence, to fasten the delivery of end products. Several haploid inducers stocks were available but they lacked good agronomic adaptation to tropical regions accompanied with low haploid induction rate (HIR) and survival rate (SR). The low efficiency of chromosome doubling also poses a major challenge towards DH production. In this study, efforts were made to optimize the protocol for DH production in tropical maize. The second generation haploid inducer stocks (CIM2GTAIL P1 and CIM2GTAIL P2) obtained from CIMMYT, Mexico were used to generate induction crosses with F1s , generated from lines harbouring key traits for amenability to high plant density (HPD) stress tolerance. A mean HIR of 10.19% was obtained from crosses with CIM2GTAIL P2 as compared to 7.13% in CIM2GTAIL P1. Two stages of haploid seedlings i.e. 3-4 days old germinating seedling stage (T1) and two leaf stage/V2 stage (T2) and different concentrations of colchicine solution were used for chromosome doubling. The results showed a higher survival rate (50.86%) and overall success rate (19.02%) in case of T2 at 0.07% colchicine and 0.1% DMSO. The DH lines so obtained were evaluated under HPD, using reduced plant to plant spacing from 20 cm to 15 cm. Out of 218 DH lines, 22 donor lines harbouring key agronomic traits such as upright plant architecture, sparse tassel, narrow leaf angle and moderate plant height, etc. were identified. Efforts were made to develop a haploid inducer stock by knocking out the ZmMTL gene via biolistic gene gun mediated delivery of ribonucleoprotein (RNP) complex into immature embryos of maize line, LM-13. Negative results were seen showing no generation of mutants. However, the standardization of media compositions and different RNP complexes formed during the experiment will be useful for determining the efficiency of regeneration and particle delivery into the target cells. The optimized protocol will be highly useful for production of DH lines as a regular process in hybrid breeding for target traits.ThesisItem Embargo Allele mining of genes for grain related traits in rice (Oryza sativa L.)(Punjab Agricultural University, Ludhiana, 2022) Gurjeet Singh; Khanna, RenuThe present study was undertaken to evaluate diverse germplasm accessions of rice for phenotypic characterization of yield and related traits as three different experiments. In experiment-I, a set of 180 germplasm accessions, including cultivars, landraces and advance breeding lines along with three replicated checks (PR 113, PR 121 and PR 126), were grown in augmented design consequentially for two years i.e. kharif 2020 at Ludhiana and kharif 2021 at Ludhiana and Rauni. The genotypes showed significant variation for various yield contributing traits viz., days to 50% flowering, plant height, lodging score, panicle length, grain number per panicle, spikelet fertility, grain yield per plant, and thousand seed weight at both locations. Pooled ANOVA revealed were significant genotypic variation different based on the multi-location data. Desirable donors were identified for various morphological, agronomic and quality traits based on the mean performance of genotypes over years and locations. Results of quality parameters revealed that 61 genotypes had higher HRR (>55%) while 68 possessed intermediate amylose content. Based on Bacterial blight (BB) screening and molecular profiling, 49 genotypes showed resistant reaction against four pathotypes (PbXo-1, PbXo-7, PbXo-8, and PbXo-10) and were found to carry three known BB resistance genes (Xa4 + xa13 + Xa21). In experiment-II, high as well as low grain number and grain weight genotypes were selected from first experiment and were amplified in vitro for both gene-specific traits (grain number and grain weight). Based on the results of Sanger sequencing, the SNP was found at the 190th position (A/G) for grain number. Based on the results of multiple sequencing alignments, SNPs were identified at the 65th position (A/G) and at the 115th position (T/G) for grain weight. An identified allele-specific SNP was used for designing KASP marker, which was further used for validation in BC1F1 generation. In experiment-III, ten genotypes were selected from experiment-I based on high grain number and grain weight were crossed with PR 126 (high yielding cultivar with short duration). The ten different backcross (BC1F1) populations (six for grain number and four for grain weight) were developed in the background of PR 126 and a wide range of variation was observed for targeted trait. Based on the allelic variation, three KASP primers were designed for most of the associated genomic regions with grain weight. Among the three KASP primers, TGW_KASP_1 showed polymorphism, but rest were monomorphic. Testing on the BC1F1-25 population did not show the expected segregation ratio, so there is a need to explore more robust markers. The promising lines can be further utilized in rice breeding programme to develop high-yielding, BB resistant cultivars with desirable grain quality.ThesisItem Embargo 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 Embargo Characterization of introgressed Gossypium hirsutum lines for cotton leaf curl disease resistance(Punjab Agricultural University, Ludhiana, 2022) Gurcharn Singh; Paramjit SinghThe plant material consisted of BC2F4 progenies obtained from G. hirsutum cv. F 1861/G. armourianum acc. PAU 1/G. hirsutum cv. LH 1556 cross. This material was evaluated for CLCuD response and several other economic traits. For inheritance of CLCuD resistance and identification of SSR markers linked to CLCuD resistance, a BC5F2 population derived from the cross: G. hirsutum cv. F1861/ G. armourianum acc. PAU 1/*5G. hirsutum cv. F 1861 was used. A set of 121 backcross progenies including F 1861, LH 1556 and F 2228 were planted during kharif 2020 in a simple lattice design (11 x 11) with two replications. A set of 85 cotton specific SSR markers were used for documenting polymorphism between the parents and genotyping the mapping population. Six progenies were observed to be completely free from the disease, 76 progenies were segregating for CLCuD reaction, whereas, 36 backcross progenies were found to be completely susceptible to CLCuD. A simple genetic control for CLCuD resistance (monogenic dominant) has been observed in this material. Significant differences among the cotton genotypes were observed for seed cotton yield, upper half mean length, uniformity index, tenacity and micronaire indicating substantial variation for these characters in the experimental plant material. Mean seed cotton yield plant-1 ranged from 46 g (Progeny 53) through 129.5 g (progeny 298). None of the 118 backcross progenies could significantly out yield standard check F 2228 (126 g), though one progeny 298 produced numerically higher (129.5 g) seed cotton yield than that of the check. Mean fibre strength ranged from 24.9 g/tex (Progeny 567) through 32 g/tex (progeny 113). Sixty four progenies significantly outperformed the check F 2228 (26.65 g/tex). Mean micronaire value ranged from 3.85 (Progeny 108) through 5.4 (progeny 412). Most of the backcross progenies significantly outperformed the standard check F 2228 (5.3) for this trait. One SSR marker (NAU 3903) was found to be polymorphic between the resistant and susceptible F2 bulks.
