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ThesisItem Open Access Identification of pearl millet [Pennisetum glaucum (L.) R. Br.] hybrids and their parents using SSR markers(CCSHAU, 2017) Sanjay Kumar; Shikha YashveerPearl millet [Pennisetum glaucum (L.) R. Br.] (2n=14) is a short life span, highly crosspollinated, diploid, photo-synthetically efficient C4 monocot belonging to family Poaceae. It is primarily grown as a rain fed crop in the low rainfall zones of Sub-Saharan Africa and Indian subcontinent. Since 1986, 67 cultivars based on ICRISAT-bred germplasm (14 OPVs and 53 hybrids) have been released and notified in India. The improved cultivars are grown on more than 4.5 million ha (of about 9 million ha), of which the OPVs cover only 0.6 to 0.8 million ha, with the remaining area cultivated with hybrids. Determining the genetic purity of hybrid seed is an essential requirement for its commercial use. The SSR markers are preferred molecular markers for genetic purity identification due to their high efficiency, co-dominance, reproducibility, simplicity and accessibility to laboratories. Total six pearl millet hybrids along with their parents were used in this study. Genomic DNA of pearl millet hybrids and their parents (HHB67-Improved ,ICMA 843-22, ICMB 843-22, H 77/833-2- 202,HHB146,ICMA 95222, ICMB 95222, HTP 94/54, HHB94,ICMA 89111, ICMB 89111, G 73-107, HB256,ICMA 94222, ICMB 94222, H 1305, HHB216,HMS 37A, HMS 37B, HTP 03/13, HHB117, HMS 7A HMS 7B 77/29-2) was isolated using CTAB method. Out of 84 SSR primer pairs used, 7 SSR primers could distinguish the different hybrids and their parents. The SSR data was also used to determine genetic relationship among these genotypes. This study would a great help for testing purity of these hybrids.ThesisItem Open Access Genetic variability in BC1F2 and F3 wheat genotypes derived from WH 1105 x Kharchia 65 for salinity tolerance(CCSHAU, 2017) Sharma, Kritika; Shikaha YashveerSoil salinity is one of the major abiotic stress having multifarious adverse effects on all growth stages. In present study, WH1105 x Kharchia 65 derived BC1F2 and F3 and BC2F1 generations were evaluated for various morpho-physiological traits under salt stress and field conditions. Marker assisted selection confirmed the presence of Nax1 and Nax2 markers in 98 and 49 plants respectively for salinity tolerance. Twenty nine plants comprising of 16 BC1F2, 6 F3 and 7 BC2F1 were found to have both Nax1 and Nax2 loci. On the basis of phenotypic and genotypic variations fifty-four high yielding plants were selected for genotyping using polymorphic SSR markers. Out of total 149 SSR markers screened, 24 polymorphic SSR markers were used to produce molecular diversity among 54 selected plants. Cluster tree analysis of parents and BC1F2, F3, and BC2F1 plants showed that all the selected plants were inclined toward recurrent parent WH1105 representing higher similarity with WH1105. Six best plants were selected as high grain yielding and salt tolerant plants i.e. plant number 53C from BC1F2, 140B from F3 and 13af, 13bf, 14bf, 15af from BC2F1 and these plants could be further backcrossed with the parent WH1105 to develop salt tolerant wheat lines.ThesisItem Open Access Expression profiling of ADP-glucose pyrophosphorylase gene for thermo-tolerance in developing grains of wheat (Triticum aestivum L. Em. Thell)(CCSHAU, 2017) Mehndiratta, Ravi; Shikha YashveerThe present investigation was conducted to study the expression profile of ADP - glucose pyrophosphorylase (AGPase) large subunit for thermo-tolerance in developing grains of two genotypes (WH 711 and WH 730) of wheat under two different sowing conditions (normal and late sown). Gene expression and AGPase activity was studied at six different stages of grain filling (4, 8, 12, 16, 20 and 24DAA) along with physiomorphological and yield attributes. Under conditions of heat stress, there was a considerable reduction in grain yield/ plant, plant height, number of tillers and 1000 grain weight in thermosensitive genotype (WH 711) as compared to thermotolerant genotype (WH 730). The activity of AGPase was correlated with data obtained through expression profiling; the activity and expression of AGPase large subunit was highest at 20DAA for thermotolerant genotype (WH730) under both NS and LS conditions. In case of thermo sensitive genotype (WH711), the activity peaked at 20DAA under normal sown conditions and 16 DAA under late sown conditions; the activity was seized at 24 DAA after anthesis under NS and LS conditions. Putative cDNA obtained were sequenced for WH 711(1270bp) and WH 730 (1247bp). Cladogram generated using different AGPase large subunit sequences clustered WH 711 and WH 730 into different groups; WH 730 sequence was phylogenitically more similar to the sequence a cquired from AGPase large subunit of barley. This study supported the role of AGPase for thermotolerance in wheat by getting insights of the expression and enzyme activity at different stages of grain filling in LS as well under NS conditions; thus providing a deep understanding for the biologists working with ADP-glucose pyrophosphorylase in order to understand the functionality of AGPase .ThesisItem Open Access Marker-assisted selection of water-efficient aromatic lines from aerobic x lowland indica rice (Oryza sativa L.) crosses(CCSHAU, 2017) Meena, Rahul Kumar; Jain, R.K.Aerobic (MAS25, MAS26 and MAS-ARB25) x lowland Basmati (PUSA1121 and IB370) rice derived F3, F4, BC1F2 and BC1F3 populations displayed enormous variation for various physio-morphological (plant height, effective number of tillers/plant, panicle length, 1000-grain weight and grain yield per plant), quality (grain length/breadth ratio) and root (root length, thickness and biomass) traits under direct-seeded water-limited aerobic conditions in the field and net house. Frequency distribution curves for various physio-morphological and root traits were parabolic and in some cases, curves were partially inclined towards the respective aerobic rice parent. Phenotypic correlation coefficient analysis showed significant positive correlation between grain yield per plant and yield-related attributes. Positive correlation (r = 0.223 and 0.763) was observed between root length and dry root biomass in three populations and PUSA1121 x MAS26 F3 and F4 populations showed positive correlation (r = 0.429) between grain yield and root length/dry biomass. In these populations, plants had the allelic profile at BAD2A locus similar to the Basmati (585 and 257 bp) or non-Basmati (585 and 355 bp) parents or were heterozygous (585, 355 and 257 bp). A total of 221 of 604 SSR markers well distributed on 12 rice chromosomes, showed polymorphism in five parental rice genotypes; 51-71 polymorphic markers were used to assess the diversity in these populations. The NTSYS-pc UPGMA tree cluster analysis and twodimensional PCA scaling showed that Basmati and aerobic rice genotypes were quite divergent and segregating populations were interspersed between the two parents. Composite Interval Mapping analysis (WinQTL Cartographer 2.5) using SSR databases of various populations led to the mapping of 14 large-effect QTL for grain yield on chromosome 1, 2, 3, 5, 6, 7, 8, 9 and 12 (LOD: 2.5 - 8.4 and R2 0.8 – 72.2%). In addition 22, 13, 10, 7 and 2 QTL for grain L/B ratio, panicle length, effective number of tillers per plant, plant height and 1000 grain weight, were mapped, respectively. Notably, five QTL (qLB9.1, qLB11.1, qTGW10.1, qPL4.1 and qPL2.2) accounted for >70% variation for the respective traits. As many as 68 and 80 plants having higher or comparable grain yield, length/breadth ratio, root length/thickness/biomass than the parental genotypes with intact Basmati specific BAD2A allele in homozygous and heterozygous conditions, were selected from the four crosses in 2014 and 2015 Kharif seasons, respectively. These genotypes will serve as the novel material for the selection of stable aerobic Basmati rice varieties. It will also be interesting to investigate and confirm the role of QTL identified in this study towards improving the grain yield and quality under aerobic conditions.ThesisItem Open Access Marker-assisted selection for mineral (Iron and zinc) rich lines in Indica rice (Oryza sativa L.)(CCSHAU, 2017) Pippal, Amit; Jain, R.K.Molecular markers provide valuable tools to identify and quantify genetic diversity within and between species, population and available germplasm to accelerate the efficacy of breeding programs to improve mineral (iron and zinc) content in rice grains.F5, BC1F4 , F6, BC1F5 and RIL F6 populations derived from the cross between high-yielding (PAU201) and iron-rich (Palman 579) indica rice varietiesdisplayed large variation for various physio-morphological traits (plant height, effective number of tillers/plant, panicle length, grain yield per plant and 1000-grain weight) and mineral (iron and zinc) contents. Iron and zinc content varied between 4.6-312 μg/g and 2.2-117.5 μg/g (F5); 3.9-86.4 μg/g and 1.51-140.6 μg/g (BC1F4); 53-117.2 μg/g and 9.7-112.2 μg/g (F6);37.8-259 μg/g and 20-56 μg/g (BC1F5) and 23.6-383.4 μg/g and 13.6- 101 μg/g (RIL F6) respectively. Transgressive segregation for grain iron content was noticed in one RIL F6 plant (plant no.214A3-3) which had exceptionally higher iron (383.4 μg/g; Palman 579 326.5 μg/g. Phenotypic correlation analysis showed positive correlation between grain iron and zinc content in F5, BC1F4, F6 BC1F5 and RIL F6 populations. In F5 and BC1F4 populations, grain iron and zinc content showed positive correlation with grain yield.DNA fingerprint databases of PAU201 × Palman 579 F6 andRIL populations (30 plants each) were prepared using 62 and 61 polymorphic SSR markers. The NTSYS-pc UPGMA tree cluster analysis and two dimensional PCA scaling exhibited that two parental genotypes were quiet distinct and diverse whereas 30 F6 and RIL plants interspersed between the parental rice genotypes. QTL mapping using two SSR databases by Composite Interval Mapping (CIM) programme of WinQTL Cartographer 2.5, led to the identification of twelve QTL (six each in F6 and RIL population) for iron (8 QTL) and zinc (4 QTL) content in rice grain; ten of these QTL accounted for >30% variation. Out of 8 QTL for iron content, 6(qFE2.1, qFE2.2, qFE7.1, qFE9.1in F6 population; qFE2.1, qFE2.2from RIL population) were from “Palman 579” and rest of the two QTL (qFE10.1and qFE12.1) were from “PAU201”. Out of four QTL mapped for zinc content, one QTL, qZN7.1(F6)was from “PAU201” and three QTL, qZN2.1(F6), qZN2.1(RIL)and qZN10.1(RIL), werefrom Palman 579. In addition, eight QTL (qYP10.1, qYP10.2, qYP12.1, qYP12.2, qYP2.1, qYP2.2, qYP10.1 and qYP12.1) were mapped for grain yield; 7 of these QTL were from “Palman 579” and two QTL (qYP10.2 and qYP12.1) contributed >70% of phenotypic variation. As many as 30 F6 plants and 30 BC1F5 plants were selected on the basis of grain yield and mineral content for further progeny analysis and RIL population was advanced to F6 generation with 93.7% homozygosity.ThesisItem Open Access Molecular diversity and micronutrient content analysis in pearl millet [Pennisetum glaucum (L.) R. Br.](CCSHAU, 2017) Chanchal, Divya; Kharb, PushpaPearl millet is a highly cross pollinated C4 monocot plant belonging to family, Poaceae. In this study, seventy five pearl millet inbred lines were evaluated for six morphological traits viz., days to 50 per cent flowering, plant height, ear length, ear diameter, 1000 grain weight and grain yield per plant along with grain Fe, Zn and crude protein content in randomized block design (RBD) with two replications during kharif 2016. Eighteen polymorphic SSR primer pairs, selected after initial screening of 40 SSR primers were used for molecular diversity analysis among these inbred lines. The analysis of variance for all the traits revealed significant differences indicating the presence of substantial genetic variation among the inbred lines. There was a close resemblance between the corresponding estimates of PCV and GCV indicating the influence of environment on the expression of these traits. The PCV and GCV were maximum for grain yield per plant. There was a positive correlation between Fe and Zn content but no correlation with grain yield suggesting that these traits may be improved simultaneously without any penalty on grain yield. Molecular marker analysis revealed that the 18 SSR primers amplified a total of 43 alleles, ranging between 2-4, with an average of 2.38 alleles per locus and average PIC of 0.30. The size of PCR amplified products ranged from 140 bp to 1200 bp. UPGMA cluster analysis grouped the 75 inbred lines into two major clusters at a similarity coefficient of 0.42. Inbred lines namely; EBL12/237, TPBL-11-105, CPBL-11-103, HBL1113 and HFI-10-140 exhibited higher grain Fe and Zn content as compared to HBL-11, male progenitor of many elite pearl millet hybrids, giving a scope for developing superior hybrids. Based on the mean performance, HBL 1107 and HPT-1-12-189-2 were found to be micronutrient and protein and with high grain yield. The line HMS 18B shared 89% similarity with HBL 11 at genetic level suggesting that HMS 18B may be used as male parent in hybrid development programmes.ThesisItem Open Access Simple sequence repeat analysis and micronutrient content in pearl millet [Pennisetum glaucum (L.) R. Br.](CCSHAU, 2017) Chaudhary, Rinku; Kharb, PushpaPearl millet [Pennisetum glaucum (L.) R. Br.] is highly cross-pollinated, diploid, photo-synthetically efficient C4 monocot plant belonging to family Poaceae. In present investigation, seventy three pearl millet inbred lines were evaluated for genetic variability in yield and its contributing traits, micronutrients (Fe and Zn) and crude protein content. Molecular diversity analysis was assessed using SSR primers. The analysis of variance indicated significant differences among all inbred lines for all the traits. A narrow difference was observed between PCV and GCV for all the traits suggesting the little role of environment in the expression of all these traits. A highly significant and positive correlation between grain Fe and Zn content was observed. Grain yield per plant showed no significant correlation with Fe, Zn and protein content suggesting improvement in nutrient value can be attempted without compromising on yield. Three inbred lines, H2302, HMP 808 and HBL 1121 were found to have high Fe, Zn and protein content as well as high yield. Inbred lines HMS 53B, HPT-2-12-59, H12/009, HMS 14B and H72-2-2 contained higher Fe and Zn than HBL 11, male progenitor of elite hybrids HHB 197, HHB 223, HHB 226, and thus may be used in developing superior hybrids. Out of 40 SSR primers used for screening, 19 were found to be polymorphic and used for molecular diversity assessment. A total of 46 alleles were amplified with 2.42 average allele per locus and 0.38 average PIC. At a similarity coefficient of 45%, seventy three inbred lines were grouped into two major clusters. HBL 0551 shared a very high genetic similarity with HBL 11 thus may be used as male parent in hybrid development programmes.ThesisItem Open Access Molecular marker analysis for QTL identification in Indian mustard (Brassica juncea L. Czern & Coss.) for salinity tolerance(CCSHAU, 2017) Patel, Rekha; Yadav, Neelam R.Salinity tolerance is quantitative and complex phenomenon controlled by numerous interacting quantitative trait loci (QTLs). To find out genomic regions associated with terminal salinity tolerance a study entitled Molecular marker analysis for QTL identification in Indian mustard (Brassica juncea L. Czern & Coss.) for salinity tolerance was conducted with the objective to identify QTLs for salinity tolerance in RH 30 × CS 52 derived F2 and F2:3 populations of Brassica juncea (RH 30-Salinity sensitive and CS 52-Salinity tolerant) and to evaluate both generations for yield attributes such as plant height (cm), physiological maturity, main shoot length (cm), number of primary branches/plant, number of secondary branches/plant, number of siliquae/plant, number of seeds/siliqua, 1000-seed weight (g), seed yield/plant (g) and biological yield/plant (g); physiological traits such as relative water content (%), osmotic potential (-MPa), proline content (µmol/g) and membrane injury (%) under field and salinity treatments i.e. control, 6 dS/m and 8 dS/m. Out of 358 SSR markers 42 were found polymorphic and 153 were monomorphic. Linkage map was prepared using 41 SSR markers covering 8 linkage groups and 1 marker did not map to any of the linkage groups. A total of 91 alleles were identified in the CS 52 and RH 30 genotypes. The total length of the genetic linkage map was 2298.5 cM and PIC ranged from 0.427-0.730. Composite interval mapping revealed a total of 23 QTLs were identified for yield attributes and physiological traits in F2 population. Five QTLs identified for salinity tolerance on two linkage groups 4 and 8 under field and salinity treatments. QTLs identified in the present study firstly needs to be validated in other populations and then fine mapping of these salinity related QTLs have to be carried with them in marker assisted selection and breeding for salinity tolerant genotype in Brassica juncea.