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ThesisItem Open Access Molecular characterization of cluster bean (Cyamopsis tetragonoloba L. Taub.) genotypes(CCSHAU, 2019) Alka Rani; Yadav, Neelam R.Cluster bean (Cyamopsis tetragonoloba L. Taub.) is an annual, diploid legume crop with 14 number of chromosomes and highly valued for presence of galactomannan in its seeds. For commercial use of seeds, their genetic purity determination is very important. The present study was carried out for development of molecular profile of commercial varieties of cluster bean using SSRs and to study molecular diversity in selected genotypes of cluster bean using EST-SSRs. For this study, seeds of 22 commercial varieties and 41 germplasm lines of guar were grown in net house of the department and Farm area of Forage Section, Department of Genetics and Plant Breeding. DNA was extracted using CTAB method. Molecular analysis was carried out using 79 SSRs which included 40 EST-SSRs and 39 miSSRs from Medicago truncatula and cluster bean (developed in our lab). Out of 79 SSRs, 55 SSRs were found polymorphic and employed for varietal identification. Among these polymorphic SSRs, eight SSRs identified selected varieties. Ct-miSSR-17, Ct-miSSR-19, Mt-miRNA-SSR45, Mt-miRNA-SSR47 and Mt-miRNA-SSR164, EST-SSR2, EST-SSR17 and EST-SSR40 identified selected varieties. Among 55 polymorphic SSRs, size of amplified products ranged from 100-1160 bp and PIC value ranged from 0.28 to 0.91. The molecular analysis divided the 22 cluster bean varieties into two major clusters at similarity coefficient of 0.06 and HG 6 existed as an isolated genotype. Cross-transferability of Medicago truncatula miRNA-SSRs showed 31% cross-transferability among cluster bean varieties. Molecular analysis showed that IC 402295-2, IC 522486, IC 113452, IC 415161 and IC 113489 were extreme and diverse group with around 30 % gum content while IC 113472 and IC 370725 were low gum (<20%) genotypes. The molecular analysis of 63 genotypes together showed that all the 22 varieties are in different genetic pool than 41 germplasm lines and can be exploited in cluster bean breeding programme.ThesisItem Open Access Development and characterization of transgenic pigeon pea (Cajanus cajan L. Millspaugh) plants with Psp68 for improvement against salinity stress(CCSHAU, 2019) Chhabra, Neha; Kharb, PushpaPigeon pea (Cajnus cajan L.) is a nutrient rich, sixth most important and second most important legume pulse crop of the world and India respectively after chickpea.Production and productivity of pigeon pea is affected by various abiotic stresses but salt stress is the most deleterious accounting for upto 100% yield losses. In the present study, transgenic pigeon pea plants (var. Manak) carrying Psp68 gene were developed for salt stress tolerance using Agrobacterium tumefaciens strain LBA4404 with pCAMBIA1300-Psp68 (Kharb et al., 2018). Putative transformants were screened at early stage through PCR amplification using gene specific primers and a transformation frequency of 16% was observed. Southern blot and real time PCR analysis revealed stable and single copy insertion of the transgene in pigeon pea genome. Seeds from each of the PCR positive T0 pigeon pea plants were sown to raise T1 generation and a total of 42 plants (out of 100) showed the presence of Psp68 gene. Physio-biochemical analysis of selected T1 transgenic plants subjected to 75 mM salt stress showed that transgenic plants were able to maintain higher chlorophyll content, relative water content, proline content, total soluble sugars, catalase and peroxidase activity compared to the wild type plants. Whereas, electrolytic leakage and lipid peroxidation were reduced as compared to the wild type plants under 75 mM stress. Among all transgenic lines, line 53 performed well with respect to all the parameters studied and can be taken further for the development of transgenic pigeon pea plants for salt stress tolerance.ThesisItem Open Access Genetic diversity analysis in Kiwifruit (Actinidia spp.) using RAPD/SSR markers(CCSHAU, 2019) Kamboj, Aarti; Kharb, PushpaKiwifruit (Actinidia spp.) is a very important plantation crop, generally dioecious, deciduous and scrambling vines with chromosome number 2n=58, belonging to family Actinidiaceae. Dioecy in kiwifruit lays an important constraint for breeding programs. The present study was undertaken to study genetic diversity in kiwifruit genotypes using RAPD markers. Molecular polymorphism in 7 kiwifruit genotypes (2 males viz. Tomuri & Allision and 5 females viz. Bruno, Hayward, Monty, Allision & Abbott) was evaluated using 94 RAPD primers, out of which 23 were found to amplify the genomic DNA in all the seven genotypes. Analysis of RAPD data was done using NTSYS-pc software and dendrogram was constructed using UPGMA method. The 7 kiwifruit genotypes grouped in two main clusters comprising of male and female genotypes separately. Similarity matrix indices ranged from 0.521 to 0.719 showing maximum similarity between Allision (M) and Tomuri (M) with a similarity coefficient of 0.719 while Allision (M) and Abbott (F) were found least similar with a similarity coefficient of 0.521. Primers OPA-01, OPB-02, OPB-20 and OPC-05 amplified unique amplicons in Bruno, Hayward, Abbott, Monty and Allision (M), and primers OPA-02 & OPN-02 amplified unique amplicons in Tomuri (M) & Allision (M) and male and female plants of Allision genotype respectively. Thus, these primers can be used to distinguish the kiwifruit genotypes and can also be validated as putative sex linked markers which have the prospective to be used for further characterization in specific kiwifruit genotypes.ThesisItem Open Access Phenotypic and molecular characterization of F5 and backcross generations for salt tolerance in wheat (Triticum aestivum L. em. Thell)(CCSHAU, 2019) Tiwari, Prakash Narayan; Shikha YashveerSoil salinity is one of the major abiotic stress having multifarious adverse effects on all growth stages of wheat. In Haryana, WH 1105 is widely cultivated wheat variety with many agronomically superior characteristics but is affected by soil salinity. In the present investigationNax1 and Nax2 genes were introgressed from Kharchia 65 into WH 1105 for salinity tolerance through marker assisted backcross breeding. F5, BC1F4, BC2F3 and BC3F1 generations of WH 1105 x Kharchia 65, were evaluated under initial salt stress condition for various morphological traits. On the basis of genotypic and phenotypic variations 57 high yielding plants of WH 1105 x Kharchia 65, were selected. Out of total 210 screened SSRs 30 markers were found polymorphic for WH 1105 and Kharchia 65. These polymorphic SSRs were utilized to produce molecular diversity among selected plants. Cluster tree analysis of parents and all the four generations of the cross, portrayed that all the selected plants were inclined toward recurrent parent WH1105 indicating higher similarity with the recurrent parent. Seven best plants were selected from the cross as high grain yielding and salt tolerant plants. These plants could be further backcrossed with the recurrent parent to develop salt tolerant wheat lines.ThesisItem Open Access Phenotyping and molecular marker analysis of selected F3 segregating lines from aerobic x low land indica rice (Oryza sativa L.) crosses(CCSHAU, 2015) Kanika Rani; Jain, R.K.Experiments were conducted to evaluate F3 segregating populations derived from the five crosses (PAU201 x MAS25, MASARB25 x PAU201, PAU201 x MAS26, MASARB25 x HKR47 and MAS25 x HKR47) for various physio-morphological and/or root traits and microsatellite markers linked to the traits promoting aerobic adaptation. MASARB25, MAS25 and MAS26 are aerobic while PAU201 and HKR47 are low-land indica rice varieties. In all the five populations, wide variation was observed for plant height, panicle length, number of panicles per plant, number of effective tillers per plant, root length, root thickness, fresh and dry root weight, 1000 grain weight, grain length-breadth ratio and grain yield per plant. In these populations, significant positive correlation was observed between yield per plant with plant height, effective no. of tillers per plant, length-breadth ratio, 1000 grain weight, root biomass and/or root length. The NTSYS-pc UPGMA tree cluster analysis and 2-D PCA scaling of selected F3 plants derived from the five crosses clearly showed large variation among two parental genotypes and F3 plants were invariably interspersed between them. A number of promising F3 plants have been selected, which had higher grain yield, root length and biomass greater than MAS25, MAS26 and MASARB25 for further progeny analysis. Most of these selected plants had the desired allele for the markers reported earlier to be linked with the aerobic adaptation traits (RM234 and RM547 for root length on chromosome 7 and 8 respectively; RM525 for root thickness and root biomass on chromosome 2).ThesisItem Open Access Micronutrient and SSR marker analysis of pearl millet [Pennisetum glaucam (L.) R. Br.] inbred lines(CCSHAU, 2015) Sangwan, Sonali; Shikha YashveerA set of thirty-six pearl millet inbred lines was used to study genetic variability and correlation analysis for seven morphological characters namely days to 50% flowering, plant height (cm), ear length (cm), ear diameter (cm), 1000 grain weight (g), grain yield per plant (g) and dry fodder yield per plant (g) along with micronutrient (Fe and Zn) status. The pearl millet inbred lines were evaluated in randomized block design with two replications in two environments during 2014. Analysis of variance for all the characters under study showed significant differences indicating the presence of genetic variability among the inbreds. The estimates of variability parameter for grain yield, contributing traits and Fe and Zn content revealed the high PCV value as compared to GCV value that suggests the role of environment in the expression of all these characters. An additive gene action was indicated for dry fodder yield per plant, grain yield per plant, Fe and Zn content by high to moderate heritability and genetic advance as per cent of mean values. Correlation analysis showed highly significant correlation between grain Fe and Zn content which signifies simultaneous improvement in the two traits. Grain yield per plant showed no significant negative correlation with Fe and Zn that suggests improvement in nutrient value without sacrificing yield. A set of 30 SSR primers was also used for molecular diversity assessment in 36 pearl millet inbred lines. Mean allele per locus and PIC obtained was 10.5 and 0.796, respectively. Cluster analysis distributed these genotypes into 9 clusters where 4 genotypes failed to fall in any of these clusters.ThesisItem Open Access Phenotyping and molecular marker analysis in backcross and F4 generations of wheat for salt tolerance(CCSHAU, 2018) Varsha; Shikha YashveerWheat is a major staple food of world population and occupies about 21.8% of total cultivated area accounting for 35.5 percent of total food grain production at global level. In India, approximately 8.5 million hectare of land area is affected by high salinity (EC ≥ 5 dS m-1). In Haryana alone, 2Mha of soil is affected by salinity which contributes to 20% of the total yield loss. Development of salinity tolerant varieties through marker assisted breeding is most efficient and effective strategy for management of soil salinity. In Haryana, HD 2967 and WH 1105 are widely cultivated wheat varieties with many agronomically superior qualities but are affected by soil salinity. In the present study, two genes (Nax1 and Nax2) for salinity tolerance were introgressed from Kharchia 65 into the genetic background of wheat varieties, HD 2967 and WH 1105 through marker assisted backcross breeding and BC1F3, BC2F2 and F4 generations of the two crosses i.e. HD 2967 x Kharchia 65 and WH 1105 x Kharchia 65, were evaluated for various morphological traits under initial salt stress condition. On the basis of phenotypic and genotypic variations 68 and 44 plants of the cross HD 2967 x Kharchia 65 and WH 1105 x Kharchia 65, respectively, high yielding plants were selected. A total of 178 parental polymorphic markers were used for background selection of the foreground selected plants. Out of total 178 SSR markers screened, 31 markers were found polymorphic for the parents HD 2967 and Kharchia 65 and 30 markers were polymorphic for WH 1105 and Kharchia 65. These polymorphic SSR markers were used to produce molecular diversity among selected plants. Cluster tree analysis of parents and all the three generations of both the crosses, showed that all the selected plants were inclined toward recurrent parent HD 2967 or WH1105 indicating higher similarity with the recurrent parent. Six and four best plants were selected from the respective crosses as high grain yielding and salt tolerant plants. These plants could be further backcrossed with the recurrent parent to develop salt tolerant wheat lines.ThesisItem Open Access Marker assisted selection for introgression of yellow rust resistance (Yr) genes in bread wheat (Triticum aestivum L. em. Thell)(CCSHAU, 2018) Reema Rani; Yadav, Neelam R.Wheat is major cereal crop that provides food to half of the world’s population. Yellow or stripe rust, caused by Puccinia striiformis f. sp. tritici (Pst) is a devastating foliar disease that affects bread wheat (Triticum aestivum L.) in the major wheat growing regions of India. Development of host plant resistance is the most effective and efficient strategy for management of the disease. In the present study, 68 wheat genotypes were evaluated for Yr genes using tightly linked markers. Promising genotypes carrying multiple combinations of Yr genes were identified. Lassik, hard red spring wheat carries Yr36, Yr18 and Yr17 genes while WH711 and PBW343 are high yielding and yellow rust susceptible varieties. Therefore, Yr resistance genes (Yr36, Yr18 & Yr17) were introgressed from donor parent (Lassik) into the susceptible varieties (WH711 & PBW343) through marker assisted backcrossing approach. Foreground selection was carried out in the BC2F7 and BC2F8 plants derived from the crosses of (WH711 x Lassik) x WH711 and (PBW343 x Lassik) x PBW343 using markers linked to Yr genes Gpc/B1/Yr36, Yr18 and Yr17. In both the BC2F7 and BC2F8 generations of the cross (WH711 x Lassik) x WH711, five lines showed the presence of all the three yellow rust resistance genes. Two lines were found to be carrying Yr36 and Yr18 genes. One line had Yr36 and Yr17 genes and one line was identified with Yr36 only. In the cross, (PBW343 x Lassik) x PBW343, one line confirmed the presence of all the three Yr resistance genes. Two lines were found positive for Yr36 and Yr18 genes. One line had Yr36 and Yr17 genes and one line was identified with Yr36 gene only. These Yr gene positive plants were subjected to background selection using polymorphic SSR markers. A total of 575 SSR markers spanning throughout the genome were used to study the polymorphism between parental genotypes out of which 100 and 101 SSR markers produced polymorphic alleles between parents i.e. WH711 and Lassik and PBW343 and Lassik, respectively. Finally, the maximum genome recovery for pyramided genotypes was analysed by NTSYS and GGT softwares. The percentage recovery of recurrent parent genome in the BC2F7 plants of WH711 x Lassik and PBW343 x Lassik ranged from 94.0 % to 96.1 % and 93.6 % to 96.6 %, respectively. Similarly, the percentage recovery of recurrent parent genome in the BC2F8 plants of both crosses ranged from 94.0 % to 96.1 % and 95.5 % to 97.4 %. Based on agronomic evaluation, yellow rust reaction, and percentage recovery of recurrent parent genome, promising lines from both the crosses were identified. The present work demonstrates the successful pyramiding of yellow rust resistance gene i.e. Yr36, Yr18 and Yr17 into WH711 and PBW343 varieties using the marker assisted backcross breeding.ThesisItem Open Access Development and characterization of transgenic chickpea (Cicer arietinum L.) plants with OsRuvB gene for salt stress tolerance(CCSHAU, 2018) Preeti; Kharb, PushpaChickpea (Cicer arietinum L.) is a nutrient rich, second most important legume crop after soybean. Production and productivity of chickpea is affected by various abiotic stresses but salt stress is the most deleterious accounting for upto 100% yield losses. In the present study, transgenic chickpea plants (var. HC-1) carrying OsRuvB gene were developed for salt stress tolerance using Agrobacterium tumefaciens strain LBA4404 with pCAMBIA1301 (Kharb et al., 2012). Putative transformants were screened at early stage through PCR amplification using gene specific primers and a transformation frequency of 17% was observed. Southern blot and real time PCR analysis revealed stable and single copy insertion of the transgene in chickpea genome. Seeds from each of the PCR positive T0 chickpea plants were sown to raise T1 generation and a total of 74 plants (out of 170) showed the presence of OsRuvB gene. Physio-biochemical analysis of selected T1 transgenic plants subjected to 100 mM salt stress showed that transgenic plants were able to maintain higher chlorophyll content, relative water content, proline content, total soluble sugars, catalase and peroxidase activity compared to the wild type plants. Whereas, electrolytic leakage and lipid peroxidation were relatively less as compared to the wild type plants under 100 mM stress. Among all transgenic lines, line 8 performed well with respect to all the parameters studied and can be taken further for the development of transgenic chickpea plants for salt stress tolerance.