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ThesisItem Open Access Genome wide association mapping for heat tolerance in maize (Zea mays L.)(Punjab Agricultural University, Ludhiana, 2019) Longmei, Ningthaipuilu; Gill, Gurjit KaurThe present investigation entitled “Genome wide association mapping for heat tolerance in maize (Zea mays L.)” was carried out at Punjab Agricultural University, Ludhiana during spring, 2016 and 2017 under optimal and heat stress conditions. Substantial significant variability was observed in the association mapping panel for heat associated traits under both the conditions. These traits were significantly affected by high temperature stress. Under heat stress, anthesis-silking interval (ASI) prolonged and some genotypes had no silk emergence. Average grain yield (3.46 t/ha) was low compared to that under optimal condition. Broad-sense heritability ranged from low to high in both conditions. The traits viz., days to 50% anthesis (AD) and silking (SD) and anthesis-silking interval (ASI) had significantly negative association with grain yield while plant and ear height and ear position showed significant positive correlation with grain yield. In 2016, 2017 and pooled over both years, total 159, 154 and 130 SNPs, respectively, were found associated with these traits under optimal condition. However, under heat stress, total 182, 109 and 71 SNPs were found associated with these traits during 2016, 2017 and pooled condition, respectively. Out of these, 7 and 9, SNPs was found associated with more than one trait under optimal condition while 23 and 11, SNPs under heat stress condition in 2016 and 2017, respectively. Similarly, total 122 and 123 haplotype blocks were associated with these traits individually while 20 and 6 haplotype blocks with more than one trait under optimal and heat stress conditions, respectively. Eighty one DH lines were found promising in both the conditions and were found associated with favorable alleles of significant SNPs and haplotype blocks for heat associated traits.ThesisItem Open Access Disruption and delineation of the Brassica fruticulosa introgressions in Brassica juncea (l.) coss(Punjab Agricultural University, Ludhiana, 2019) Agrawal, Neha; Banga, S. S.Three B. juncea - B. fruticulosa introgression lines (ILs) AD-64, AD-101 and AD-104, carrying resistance against mustard aphid, were investigated using cytogenetic and genome sequence analysis to physically map the introgressed chromatin fragments into B. juncea genome. For the cytogenetic characterization of lines, four sets of massive oligo probes were developed for the first time in Brassicas. These probes allowed identification of all chromosomes of recipient B. juncea. Subsequently, these probes were used together with B. fruticulosa genomic probe to physically map introgressed segments using modified genomic in situ protocol. Multicolour in situ analysis led to identification of introgressed segments on chromosomes A05, B02, B03 and B04 in the distal regions in ILs AD 104 and AD-64. IL-101 revealed the introgressions on the same set of chromosomes with an additional introgressed segment on chromosome A01 at sub terminal to intercalary position. Introgressions on A01, B02, B03 and B04 were validated through bioinformatic analysis of whole genome sequences from these ILs, B. juncea and B. fruticulosa. We also attempted to disrupt these introgressions through gamma irradiation. A carefully selected set of 35 M3 progenies (out of 160 M3, developed following gamma irradiations (200 kR) were phenotypically characterized for aphid resistance and agronomic traits. These progenies showed varied levels of resistance to mustard aphid under screen house conditions coupled with early flowering, short stature and improved seed and pod size. Bioinformatic analysis confirmed post irradiation, breakdown of large alien chromosome fragments in several progenies. Phylogenetic analysis of 35 M3 lines along with their parental lines and three B. juncea cultivars allowed identification of many progenies that became genetically closer to cultivated B. juncea as compared to the parental ILs. Few of these were resistant to mustard aphid as well. Progenies N-31, N-36, N-38, N-78, N-65 of IL AD-64 were considered most important in that respect.ThesisItem Open Access Genetic and physiological basis of nitrogen uptake and utilization in wheat (Triticum aestivum L.)(Punjab Agricultural University, Ludhiana, 2019) Mohd. Shamshad; Sharma, AchlaNitrogen (N) plays the crucial role for plant growth, and plant canopy photosynthesis drives grain yield and grain quality. However, excessive input of nitrogen ultimately increases the production cost and pollutes the environment. For combating these environmental and health issues, breeders need to improve the nitrogen uptake and utilization efficiency in elite agronomic backgrounds. Therefore, the present investigation was planned to assess genetic and physiological basis of nitrogen uptake and utilization efficiency in structured mapping population derived from the cross between PBW677 and PBW 703. The study also dissected the location of genes/QTLs governing nitrogen uptake and utilization efficiency in the wheat genome using high through-put sequencing (35K Axome® Breeder array) methods. A total of 296 wheat genotypes were sown under full nitrogen (Nf), half nitrogen (Nh) and zero nitrogen (Nz) conditions in alpha lattice design in the experimental area of wheat section, Department of Plant Breeding and Genetics, PAU, Ludhiana during rabi season of 2016-17 and 2017-18. Analysis of variance indicated a larger amount of genetic variability for components traits i.e., morpho-physiological traits, nitrogen uptake (NUpE), utilization efficiency (NUtE), grain protein content, biological yield and grain yield. The spikelets per spike and tiller number were found to have the highest heritability and genetic advance whereas straw N percentage in above ground parts was found to be least heritable. Half N regime in comparison to full nitrogen regime was more effective in discriminating the genotypes for various nitrogen uptake and utilization efficiency component traits. Grain yield, biological yield, grain N yield, total N yield, straw N yield, NUpE, NUtE and NHI has strong correlation with NUE whereas N percentage in shoot has no impact, thereby identifying the selection of traits to be used for screening genotypes. Principal component analysis revealed that the PCs contribution were more for grain yield, tiller number, total N yield, biological yield, straw yield, harvest index, NHI, NUtE grain protein content and GNC, So selection for these traits can be helpful for selecting N use efficient physiology. PCA also showed that the NUE, NHI and NUtE were markedly higher when there was no nitrogen apply (Nz regime). Further investigation on roots revealed the importance of root length, root diameter and root shoot ratio for NUpE, indicating the improvement for NUE. Few lines having most consistent performance under low N conditions were also identified and tested under local trials for preliminary yield evaluation. Four lines viz. BWL 6289, BWL 6838, BWL 6300 and BWL 6293 had high yield in local yield trials. These lines may be exploited for improving nitrogen use efficiency in wheat breeding programs. Inclusive composite interval mapping based on 35K SNPs array detected a total of 294 QTLs of which 105 QTL under full N, 93 under half N and 96 under zero N for nitrogen uptake, utilization efficiency and others agronomic traits were identified. The highest QTLs were found on D genome, with 90 QTLs (30.6%); 100 QTLs (34%) and 104 QTLs (35.4%) on A, B and D genomes respectively. The proportion of phenotypic variance ranged from 3.02 to 19.31 percent with LOD score 2.5 to 21.24. The number of QTLs ranged from 2 to 24 for the studied traits. These QTLs were mapped on different clusters. The identified QTLs will be further validated in other populations for their use in marker assisted selection for wheat improvement.ThesisItem Restricted GENETICS OF ROOT AND PLANT IONOMIC TRAITS IN A DIVERSITY SET OF Brassica rapa L.(Punjab Agricultural University, Ludhiana, 2019) Shekhawat, Pooja Kanwar; Banga, S. S.Phenotypic variations were investigated for root morphology, leaf and seed mineral composition (ionome) in a diversity set comprising 195 B. rapa genotypes. This set included 92 natural (NR) and 103 derived (DR) inbred lines. The derived B. rapa lines were earlier extracted from B. juncea. Root architectural traits were measured manually or computed through digital imaging software (DIRT) at maturity using detached roots from the plants grown in PVC pipes. Mineral composition of leaves and seeds was determined by inductively coupled plasma-optical emission spectrometry (ICP-OES). Considerable variations were recorded for most traits. Natural forms appeared superior for the root traits. Compositional analysis of leaf and seed minerals indicated similar accumulation patterns across germplasm groups for P, Mg, Mn and Cu. DR lines had higher buildup of N, K, Zn and Se in leaves. Same was true for K, Cr and Se accumulation in the seeds. DArT genotyping and association mapping led to the identification of many marker trait associations. Annotation of the genomic regions close to the peak SNPs allowed prediction of genes controlling many root traits. Notable among these were: PP2A-4, PXMT1 and WOX11; which are vital for root meristem initiation, root organogenesis, root architecture and growth. We could also predict transporter genes for Zn, K, Fe, P, Ca etc. NLP8, NLP4, NRT1.8, PHT, ACA and MTP family proteins were notable genes predicted for tolerance to Mn, Zn, K and some heavy metal stresses. Our results clearly demonstrated that elemental and root analysis of diversity sets can be used to identify SNPs in linkage disequilibrium with root and ionome associated genes which can be targeted in the future for genetic enhancement of this important oilseed crop.ThesisItem Restricted Pyramiding bacterial blight resistance genes (Xa21, Xa38) in maintainer and cytoplasmic male sterile line of rice (Oryza sativa L.) through marker assisted selection(Punjab Agricultural University, Ludhiana, 2019) Lego, Nancy; Thind, K.S.Hybrid rice has great potential in certain districts of Punjab. However, most of the hybrids are susceptible to bacterial blight (BB). In the present study BB resistance genes (Xa21 and Xa38) have been pyramided through marker assisted selection (MAS) into PCMS 16A/B which has good outcrossing ability. An elite breeding line, PAU 5942-1-1-1-1-1-1 with good agronomic and quality characters and possessing Xa21 and Xa38 genes was used as donor parent. Marker assisted foreground selection was carried out using the gene specific markers pTA248 for Xa21 and Os04g53050-1 for Xa38 and background selection was done using 64 polymorphic SSR markers between donor and recipient parent. The recipient parent genome (RPG) recovery ranged from 80.5 to 92.9% in BC2F1 plants. A set of 254 BC2F2 progenies carrying different combinations of Xa21/Xa38 genes along with parents were screened for BB resistance using pathotype X of BB prevalent in Punjab. Based on the RPG% and BB screening, 11 BC2F2 plants were found to be desirable. Agronomically superior lines of improved PCMS 16B line are being converted into CMS line through backcrossing. The improved BB resistant CMS lines can be further used in developing hybrid varieties resistant against all the 10 pathotypes of BB in Punjab. Further, a set of 109 testcross hybrids were generated using 79 diverse rice genotypes and 21 Wild abortive (WA) based-CMS lines to identify new maintainers and restorers from the pool of locally adapted germplasm for the development of component lines in a hybrid breeding program. Molecular markers DRRM-RF3-10 and DRCG-RF4-14 were used to screen for the presence of restorer genes Rf-3 and Rf-4 in these 79 genotypes. Among these genotypes, 56% carried Rf4Rf4/rf3rf3, 10% carried Rf3Rf3/rf4rf4, 10% carried Rf3Rf3/Rf4Rf4 and 24% carried rf3rf3/rf4rf4 allelic combination. Rf-4 was found to be predominant than Rf-3. Based of spikelet fertility 17 maintainers, 17 partial maintainers, 18 partial restorers and 37 restorers were identified. The molecular and testcross results revealed differential fertility behaviour of genotypes with different WA based-CMS lines.ThesisItem Open Access Production of doubled haploids in rice (Oryza sativa L.) through anther culture and wide crosses and their molecular characterization(Punjab Agricultural University, Ludhiana, 2019) Saundarya Kumari; Gill, R.S.The doubled haploidy is one of the fundamental tools of plant breeding which provides the fastest way to accelerate plant breeding and develop homozygous lines in the shortest possible time. The F4/F5 population of japonica x indica derived cross (Dubraj x IR 75862-206-2-8-3-B.B.B.) was used for the production of doubled haploids through anther culture for two crop seasons. Approximately 74,834 and 88,100 anthers from 87 and 21 lines respectively were cultured during kharif 2016 and 2017 crop seasons. Standard N6 medium supplemented with 2, 4-D (2.0 mgL-1) + BAP (0.5 mgL-1) +Sucrose (3 %) was used. Callus induction frequencies (CIF) varying from 0.07405 to 8.235 and 0 to 8.835 respectively were obtained during kharif 2016 and 2017 and upon transfer of 2-3 mm calli on regeneration media with varying compositions the regeneration frequencies varying from 0 to 100 and 0-35 per cent respectively were obtained. Delayed callus response initiating 90 days after culturing was observed during both the culturing seasons. All the regenerated shoots were albinos and no green plantlets could be obtained, which may be due to genotypic specificity leading to delayed callusing. The parental lines also had poor response towards anther culturability with CIF of 0.00, 0.020 and 0.064, 0.04 for Dubraj and IR 75862-206-2-8-3-B.B.B. respectively during kharif 2016 and 2017. Anther culture of a different set of material belonging to 41 crosses combinations of Basmati and non basmati genotypes during kharif 2018 though in general, gave low callus induction frequencies but high frequency of plant regeneration varying from 2.56 to 100 per cent was observed. Contrary to previous seasons results the callus initiation started 30 days after culturing and about 16 putative doubled haploid plants were generated. Studies on identification of genomic regions associated with anther culture response by using twenty two polymorphic SSR markers revealed random distribution of indica and japonica type alleles in callus as well as callus responsive and callus non-responsive lines. From the present set, no marker could clearly differentiate its association with calli and callus responding lines. RM228 and RM23662 markers had heterozygous alleles in the callus indicating their origin from the callus wall. Studies on wide crosses of rice x maize and rice x pearl millet indicated strong pre-fertilization barrier. Pollen tube of maize failed to penetrate the stigma of rice even up to 48 hours of observations. The pollen tube of pearl millet penetrated the stigma in some samples of rice but only one pollen tube of pearl millet out of 400 microscopic fields was found to reach near the micropylar end at 24 hours of cross-pollination. Hence, pearl millet could be a better candidate for further study of wide crosses of rice for induction of haploids. Pseudo seed formation was observed in the wide crosses of rice due to application of 2, 4-D in low frequencies. Further, studies on media manipulation, identification of genes/QTLs for anther culturability, overcoming barrier of wide crosses in rice x pearl millet and exploration of new alternate techniques like use of CENH3, OSMATL and BBM1 are suggested.ThesisItem Restricted Characterization of interspecific hybrids and BC1F1 plants derived from Gossypium hirsutum and G. arboreum crosses(Punjab Agricultural University, Ludhiana, 2019) Mondal, Saptarshi; Pathak, DharminderInterspecific F1 hybrids (G. hirsutum/G. arboreum) and BC1F1 plants (G. hirsutum/G. arboreum/ G. hirsutum) were characterized under the current research project. The hybrids were evaluated at cytological, morphological, and molecular levels. F1 plants were subjected to air layering for induction of roots that were used for their cytological analysis. Of the four hybrid plants, two were found to be triploid with 2n = 39 and the other two were tetraploids (2n = 52), the latter are probably formed due to the fusion of normal n (AD) gametes from female parent (G. hirsutum) and unreduced gamete 2n (AA) from male parent (G. arboreum). Hybridity of the interspecific F1 hybrids was confirmed using simple sequence repeat markers. Morphological traits used for characterization included stem colouration, leaf shape, leaf lobe number, nectaries on mid-rib, average leaf length, average leaf width, number of sepals and petals, petiole length, number of serrations on bracts, petal colour, petal spotting, anther number, anther colour, pollen fertility, average pollen size and CLCuD symptoms. Hybrids were found to be intermediate for some traits and resembled either parent for the other characters. Variation among the interspecific hybrids was observed for some traits such as pollen fertility, petal colour etc. Of the 80 microsatellite markers, 29 (36.25%) were observed to be polymorphic between the parents and five primers (CGR5717, NAU3717, BNL3479, DPL0533, and MGHES31) detected introgression from G. arboreum parent in the BC1F1 (G. hirsutum/G. arboreum/ G. hirsutum) plants. American cotton parents namely, F 846, LH 2108, LH 2107 two of the F1s, and all the BC1F1 plants amplified viral DNA. However, desi cotton parents (LD 949 and LD 491), the remaining two F1s did not show any amplification of viral DNA.ThesisItem Open Access Anatomical and genetic analysis of damaged grains in rice (Oryza sativa L.)(Punjab Agricultural University, Ludhiana, 2019) Batra, Ritika; Mangat, Gurjit SinghRice grain discoloration or rice black pinhead damage reduces the yield of milled rice and directly reflects on consumer acceptability. The black pinhead damage in grains of PR125 and F3 population of cross PR121 X PR125 and PR121 X PR126 were studied for its percentage damage intensity, anatomical evaluation and marker analysis. The range of percentage damage intensity for both the crosses was 0.5-7.2 and 0.5- 4.9 per cent respectively. The anatomical evaluation was done using scanning electron microscopy (SEM) and the results showed that there was malformation of starch granules at cellular level. The starch granules of damaged grains were loosely packed while they were densely packed in undamaged ones. 200 SSR markers were applied to check parental polymorphism and 15% (30 out of 200) polymorphism was recorded between parents. Two bulks were prepared for bulked segregant analysis, in which one was with maximum percentage damage and other with no damage. From the above 29 polymorphic markers, only 10% polymorphism was observed in the bulks. Chi square analysis was done to check the inheritance of black pin head damage. Since the statistical analysis followed normal distribution, the damage was attributed to be controlled by two or more genes.ThesisItem Restricted Marker assisted selection in wheat for high carotenoid and protein content in grains(Punjab Agricultural University, Ludhiana, 2019) Padhy, Asish Kumar; Sharma, AchlaBiofortification in wheat has been always a neglected facet in the breeding programmes. The research was planned to biofortify the wheat grain with higher carotenoid and protein with low polyphenol oxidase activity and rust resistance. 7E translocated segment containing Y gene/Lr19/Sr25 from Lophopyrum ponticum, GpcB1/Yr36 and Yr15 were the genes to be pyramided in elite agronomic background for high carotenoid content, high grain protein content and stripe rust resistance respectively. 160 lines having Y gene in HD2967 background with the gene in 7A and 7D separately were screened for the presence of Y gene and their carotenoid content and other yield related traits. Biochemical observations showed the differential expression of the gene in different genotypes and increased carotenoid content up to 14ppm where probably, two doses of gene were pyramided in a same genotype (7A and 7D) in F2 population while the commercial hexaploid wheat varieties have carotenoid in the range of 3-4 ppm. UCW8 and BARC8 were used to screen the genes GpcB1 and Yr15 and phenol reaction was carried out for testing the poly phenol oxidase activity. In the F2 population, out of seven crosses synthesized, only one gave good results for carotenoid content while three crosses died in F1 generation due to necrosis. A total of 16 lines were identified which showed high carotenoid and protein content in grains with low PPO activity. The lines having GpcB1 gene were also screened for their yield characteristics and related traits, out of which 3 lines have been identified with high grain protein content and relatively equivalent yield with the commercial checks.