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ThesisItem Embargo Development and validation of a novel core set of KASP markers for the traits improving grain yield and adaptability of rice under direct-seeded cultivation conditions(Punjab Agricultural University, Ludhiana, 2022) Jasneet Singh; Sandhu, NitikaThe development and utilization of molecular markers play important role in genomics-assisted breeding (GAB) during the pyramiding of valuable genes. The aim of present study was to develop and validate novel core set of KASP (Kompetitive Allele-Specific PCR) markers associated with traits improving grain yield and adaptability of rice under direct seeded cultivation conditions. The 110 phenotypically validated KASP assays out of 171 designed KASP include assays for biotic resistance genes (BPH3, BPH17, Gm4, Pita2, Xa4, xa5, Xa21, xa13, Xa38), anaerobic germination (qAG9.1), root traits (qNR4.1, qNR5.1, qRHD1.1, qRHD5.1, qRHD8.1), grain yield under reproductive stage drought stress (qDTY2.1, qDTY3.1, qDTY12.1), grain yield under direct seeded cultivation conditions (qGY1.1, qGY10.1), lodging resistance (qLDG4.1, qLDG3.1) and early uniform emergence (qEUE1.1, qEUE11.1). The KASP assays were validated for their robustness and reliability at five different levels using diverse germplasm, segregating and advanced population, comparison with PCR-based markers and on F1s. The present research work will provide rice breeders (i) the breeding material in the form of anticipated pre-DSR varieties (ii) single improved breeding line with many useful genes and (iii) the KASP assay information for the useful QTL/genes providing biotic resistance/tolerance, adaptability and grain yield stability under DSR.ThesisItem Embargo Mapping and transfer of higher grain length and multiple rust resistance from T.dicoccoides to cultivated wheat(Punjab Agricultural University, Ludhiana, 2022) Manpreet Kaur; Satinder KaurWild emmer wheat, Triticum dicoccoides, the progenitor of modern tetraploid and hexaploid wheats, is an important resource of new variability for disease resistance genes and grain yield traits. T.dicoccoides acc. pau14723 showed resistant to leaf and stripe rust races and was crossed with T.dicoccoides acc. pau4663 (susceptible to leaf and stripe rust) for studying the inheritance and mapping the genes for leaf and stripe rust resistance. Recombinant inbred lines were developed and screened against highly virulent Pt and Pst pathotypes at the seedling and adult plant stages. Inheritance analyses revealed that both the rust infections were controlled by dominant major single gene. For mapping these genes, the markers showing diagnostic polymorphism in the resistant and susceptible bulks were amplified on all RILs. The molecular characterization identified the genes to be present on 1A chromosome of wheat (short arm). MapDisto version 1.7.5. Beta 4 software was used to determining the linkage present between the genes governing resistance and the SSR markers Xbarc148, Xbarc240, Xwmc93, Xwmc818. A total map size of 9.7cM was obtained showing no segregation between Lr and Yr genes and the marker lying closest to the resistance genes was Xbarc148 at 1 cM distance from the resistance genes. The variation in the T. dicoccoides for the yield related traits was studied by crossing the accessions having longer and wider grains with the accessions having shorter grains resulting in three different crosses: T.dico.14723 and T.dico. 4663, T.dico.5219 x T.dico. 4663 and T.dico. TA1027 x T.dico. 5232. The RIL populations developed from these crosses were screened for different traits affecting the yield which include 100 grain wt. (100 Gwt.), spike length (SpL), spike length with awns (SpLWA), no. of spikelets/spike (Splts/Sp), grain length (GL), grain width (GW) and grain area (GA). The interaction between the traits and their contribution to grain yield was studied. The molecular and the phenotypic data was statistically analyzed for the significant contribution of marker alleles. The evaluation of allelic effects of the polymorphic markers for each trait using Kruskal–Wallis test showed that the phenotypic differences in the mean value of GL, GA and GWT were statistically significant for the three different classes of TaGASR7-A and TaGASR7-D. The study suggests the use of variation existing in T.dicoccoides germplasm for wheat breeding programmes.ThesisItem Embargo Cloning of juvenile hormone esterase gene of whitefly [Bemisia tabaci (Gennadius)] into RNAi vector for Agrobacterium-mediated genetic transformation of upland cotton(Punjab Agricultural University, Ludhiana, 2022) Gagan Jeet; Mohanpuria, PrashantCotton (Gossypium spp.) is one of the most crucial commodities for the textile industry. Whitefly is one of the most blemishing polyphagous pests of cotton which led to great losses in recent years and till now there is no published report of whitefly resistance in available cotton germplasm. Also, no Bt gene effective against whitefly so far, and thus, there is need to focus on down regulation of novel and potential target genes regulating vital processes important for survival of whitefly. In present study, 202bp cDNA fragment of Juvenile hormone esterase gene (JHE) of whitefly was used to prepare an RNAi construct. The cloning of RNAi cassette was confirmed through PCR with vector specific primer sets. Agrobacterium (GV3101) harboring prepared pFGC1008-BtJHE RNAi construct was used to transform mature embryo containing hypocotyledonary region of geminated seeds of cotton var. PAU Bt3 as per previously standardized protocol. Agro incubation for 120 minutes at 28 ºC under dark condition had resulted in 27 PCR positive RNAi cotton plants with 3.0% overall cotton transformation efficiency after 5-6 weeks of transformation. PCR was also attempted in DNA isolated from all confirmed RNAi cotton plants with Vir C gene specific primers to confirm the absence of Agro contamination in these plants. These RNAi cotton plants will be further characterized in future for stable integration and expression of RNAi cassette in next generations.ThesisItem Open Access Introgression profiling of F4 population derived from the cross of Zea mays × Teosinte spp. mexicana using SSR markers(Punjab Agricultural University, Ludhiana, 2022) Ashmita; Sharma, PritiTeosinte is the wild ancestor of maize also known as God of grains. Teosinte populations could serve as a genetic bridge for transferring genes from one maize population to the next. The present investigation was carried out using 109 recombinant inbred lines (RILs) derived from the cross of Popcorn and Teosinte. RILs along with parental lines were grown in three replications and data was recorded for morphological traits like days to maturity, number of cobs, number of tillers, seed rows per cob and cob length. Analysis of variance showed significant variation among all traits. Highest positive correlation was observed between number of rows per cob and cob length. Genotyping was done using SSR markers from maize database spanning all the maize chromosomes. Out of 250 markers used, 35 were polymorphic. Introgression profiling of all the lines along with parents was done to analyze the introgressed regions of Popcorn and Teosinte in each RIL. Introgression profile of different lines were inferred from consensus of genotypic and morphological data which revealed that marker bnlg1297 was found to be common among the lines having more number of tillers and cobs. For lesser number of maturity days marker bnlg1131 was present in most of the lines. Therefore, these genomic regions might be associated with trait. Further, more number of markers can be used to identify the regions associated with different traits.ThesisItem Open Access Mapping of leaf rust and powdery mildew resistance from Aegilops triuncialis derived disomic substitution line in wheat(Punjab Agricultural University, Ludhiana, 2022) Dhillon, Gurmanpuneet Singh; Satinder KaurWheat (Triticum aestivum) is the leading cereal crop supplying 20% share of the proteins and energy in the human diet. However, it faces significant yield constraints due to losses caused by various diseases, especially rusts and powdery mildew. To manage these losses, incorporation of genetic resistance is the best strategy. In present study a disomic substitution line [DS5Ut(5A)] having adult plat durable resistance to leaf rust (LR) and complete resistance to powdery mildew (PM) was used for mapping of LR and PM resistance. A set 95 chromosome segment substitution lines (CSSLs) generated from a cross of disomic substitution line [DS5Ut(5A)] with Ph1b suppressor Pavon Ph1b followed by crossing with LR and PM susceptible cultivar WL711. The CSSLs were screened against LR and PM for two years in which 25 lines showed resistance against LR (≤ 20MR) and 2 lines showed resistance against PM (≤ 3). Four CSSLs were identified showing resistance against both leaf rusts and powdery mildew which will serve as a useful resource to transfer the respective resistance to susceptible cultivars to develop all stage resistant elite cultivars. Genotyping of CSSLs through genotyping by sequencing (GBS) identified 693 single nucleotide polymorphisms (SNPs) loci specific to chromosome 5A-5U. Introgression profiling detected presence of 26.84% to 55.70% genome of 5U chromosome of Aegilops triuncialis in CSSLs. Genomic region providing resistance against LR races Puccinia triticina (Pt) pathotypes 77-5 (THTTS) and 77-9 (MHTTS) were found to be linked with SNP named S5A_582102813. Similarly genomic region providing resistance against mixture of Blumeria graminis f sp tritici (Bgt) pathotypes found to be associated with S5A_173251584 and S5A_180049756. The linked SNPs will be helpful in marker assisted transfer of these two important gene into elite wheat background.ThesisItem Open Access Genome wide expression analysis of Multidrug and toxin compound extrusion(MATE) gene in rice for understanding its role in resistance/susceptibility to Rhizoctonia solani AG1-IA(Punjab Agricultural University, Ludhiana, 2021) Rupnaz Kaur; Vikal, YogeshRice (Oryza sativa) is one of the most important staple food crop for approximately 50% of human population across the world and is constantly hampered by sheath blight disease caused by Rhizoctonia solani AG1-IA pathogen. Several cultural, chemical and breeding approaches were applied to combat this disease but till date no major success was achieved. Study of hostpathogens interactions may find a new possible way to understand its molecular mechanism to overcome this disease. Various transporters are well characterized that plays significant role in various stress conditions. MATE gene family is one of them that belongs to secondary transporters group and help in extrusion, transportation of products across membrane via electrochemical gradient way. The current study is aimed to understand role of MATE transporter family towards the resistance/susceptibility in rice caused by R. solani AG1-IA. The commonly grown PR114 (susceptible) variety and ShB-8 (IET-21299) (moderate resistant) line were selected for present study. The R. solani fungus strain was cultured on PDA media and infection was done on targeted rice lines. The RNA was extracted from both infective lines at 0 hr, 24 and 48 hpi and preceded for transcriptome analysis (RNA Seq). The data of RNA Seq dissipated that a set of six differentially expressed MATE genes were identified on the basis of their log2 fold change value. The selected six genes were further validated through RT-PCR. Differential gene expression at different treatments showed that LOC_Os02g45380.1 MATE gene is one of the potential candidate gene involved in ShB resistance/susceptibility. Further in-silico analysis was performed for candidate MATE gene LOC_Os02g45380.1. The evolutionary phylogenetic analysis depicted that candidate MATE gene showed homology to various dicots and monocots. However, in monocots it is highly similar to Oryza nivara which means this gene might be transferred from wild species during natural selection. Various MATE genes are reported in different plant species genome, clustering analysis also suggested that a set of 52 MATE genes were present in rice genome that is supported by previous studies. Clustering of these 52 MATE genes showed that they can be further sub-grouped into four clades on the basis of function as previously reported. The LOC_Os02g45380.1 was present in clade III which may have role in citrate and metal transport. Cis regulating (promoter) analysis of LOC_Os02g45380.1 depicted various cis regulatory elements that are present in UTR which may play significant role in various abiotic and biotic stresses. Protein modelling of LOC_Os02g45380.1 revealed the presence of 11 transmembrane helix which helps to understand its topology. The PPI interaction analysis showed that various interaction molecules and proteins are present that aid in regulation of candidate MATE gene. To identify the variation in genic region of LOC_Os02g45380.1, the gene was amplified and cloned. For functional validation and raising of cis-genic lines, the tissue culture media was standardized for ShB-8 variety.ThesisItem Open Access Cloning of vitellogenin gene of whitefly, Bemisia tabaci (Gennadius) into RNAi vector for genetic transformation of upland cotton(Punjab Agricultural University, Ludhiana, 2022) Ravinder Kaur; Mohanpuria, PrashantCotton is India's important cash crop, and it plays a significant part in the country's industrial and agricultural economy. Whitefly (Bemisia tabaci) has been one of the most threatening polyphagous pests of cotton and no whitefly resistance germplasm is reported in India. Also, there are no Bacillus thuringiensis (Bt) toxins known to be effective against whitefly. Generating RNAi cotton plants expressing dsRNA of vital gene of whitefly can be a novel approach to produce whitefly resistance in available Bt cotton variety. In present work, 208bp cDNA fragment of vitellogenin gene (VG) gene of whitefly was used to prepare an RNAi construct. The cloning of RNAi cassette was confirmed through PCR with vector specific primer sets. Agrobacterium harboring prepared RNAi construct was used to transform cotton variety, PAU Bt3. The germinated cotton seeds containing hypocotyledonary region of mature embryo was used as an explant (pricking and without pricking) for Agro-inoculation for different incubation periods (90 and 120 minutes) and thus, a cultivar-independent, in planta genetic transformation protocol has been standardized. In transformation one cotyledon was removed followed by pricking with needle on hypocotyledonary region resulted in 29 PCR (with RNAi cassette specific primers in isolated DNA) positive RNAi cotton plants with overall cotton transformation efficiency was 3.29%. PCR was also attempted in DNA isolated from all confirmed RNAi cotton plants with Vir C gene specific primers which showed absence of Agro contamination in these plants even after 5-6 months of transformation. These RNAi cotton plants will be further characterized in future.ThesisItem Open Access Identification and Molecular mapping of leaf rust and stripe rust resistance genes introgressed from wild progenitor species in cultivated wheat.(Punjab Agricultural University, Ludhiana, 2021) Jose, Alin Maria; Chhuneja, ParveenWild emmer wheat, Triticum dicoccoides, the tetraploid progenitor of wheat and Einkorn wheat, Triticum monococcum, diploid progenitor of modern hexaploid wheat, are important sources of novel disease resistance genes. T. dicoccoides acc pau 14716, which showed resistance to most common leaf rust and stripe rust races in India, was used to develop introgression line (ILdic) in the background of T. aestivum cultivar WL711(NN). An F4 mapping population was developed by crossing ILdic with T. aestivum cultivar HD2967 and the population was screened against highly virulent Pt leaf rust pathotypes at seedling and adult plant stage and Pst stripe rust pathotypes at the adult plant stages. The population segregated for a single all-stage resistance gene (ASR) against leaf rust and single gene for stripe rust resistance, according to inheritance analyses. Similarly, T. monococcum G1277 showed resistance to the most prevalent Pst stripe rust pathotypes in India. Therefore it was used to create a stable introgression line ILmono into hexaploid wheat background. T. aestivum cultivar HD2967 was used to develop F6 mapping population. ILmono mapping population upon screening against Pst stripe rust pathotypes at adult plant stage segregated for a single gene for stripe rust resistance. For mapping, leaf rust and stripe rust resistance genes in the F4 mapping population derived from HD2967-ILdic, bulk segregant analysis in combination with whole-genome resequencing (BSA-seq) was conducted. Two resistant bulks (RB) and two susceptible bulks (SB) were prepared separately for performing BSA-seq for mapping leaf rust and stripe rust resistance genes. A 10Mb candidate region on chromosome 7A with three highly significant SNPs linked to leaf rust resistance and 61 annotated genes was identified. It was observed that out of 61 annotated genes, 12 genes were involved in the defense mechanism against diseases and considered as candidate putative genes for leaf rust resistance gene in ILdic. Similarly, a 20Mb region with two highly significant SNPs was identified on chromosome 3A to be associated with stripe rust resistance and was identified to carry 49 annotated genes. Out of 49 annotated genes, four genes were found to be involved in defense mechanisms against diseases in which one gene had an NBS-LRR domain and therefore considered as candidate putative genes. The current study discovered useful leaf rust and stripe rust resistance genes that are effective against several rust races and can be deployed in the wheat breeding program.ThesisItem Open Access Agrobacterium mediated transformation of sugarcane variety CO 0238 with chitinase gene for resistance against red rot(Punjab Agricultural University, Ludhiana, 2022) Sharma, Amandeep Sahil; Sandhu, Jagdeep SinghEndochitinase gene cloned from Trichoderma spp. was introduced into the sugarcane variety CO 0238 using Agrobacterium-mediated genetic transformation. The gene construct carrying endochitinase which was earlier mobilised into two Agrobacterium tumefaciens strains, LBA4404 and EHA105 independently, were used to agro-infect sugarcane spindle leaf roll segments. When spindle leaf rolls were agro-infected with LBA4404 strain a total of 45 plants were obtained from 326 agro-infected spindles and similarly 534 plantlets were obtained from 1911 agro-infected spindles with EHA105. The presence of transgene was confirmed by PCR, revealing amplicon size of 1275 bp corresponding to the endochitinase with transformation efficiency of 0.52% only with EHA105. RT-PCR revealed the differential expression of transgene in all PCR positive plants followed by quantitative relative expression analysis using qRT-PCR revealing up to 6-fold increase in endochitinase expression in seven sugarcane plants as compared to non-transgenic controls. This suggested that plant expressing higher expression can impart resistance against fungal pathogens, indicating the role of endochitinase in providing resistance against red rot.
