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ThesisItem Open Access EXPRESSION ANALYSIS OF NUTRITION RESPONSE AND ROOT GROWTH (NRR) AND ACID PHOSPHATASE GENES IN RICE UNDER NUTRIENT DEFICIENCY STRESS(Indira Gandhi Krishi Vishwavidyalaya, Raipur, 2019) Pandey, Shriraj; Banerjee, Shubha; Prasad, Archana S.; Savu, Rama Mohan; Saxena, R.R.Deficiencyof Phosphorusis an importantfactoradversely affectingrice yieldandproductionduetopoorbiomass,delayinmaturityandgrainsterility. Rice has developed the adaptive mechanisms for increasing P uptake and to enhance its utilization underlimitingconditions.Inthis study thevariationamong ricegenotypesat root morphologyandmolecular level. Fieldevaluationofthe selectedlinesindicated significantvariationsinplantheight,leafcolor,No. of tillers/plant, No. of panicle/ plant,No. of leaf/plant,Leaf length,Leaf width,Days to 50% flowering, Root length, Shoot length, Shoot fresh weight, Root fresh weight,Shootdryweightetc.8lineswereselectedfor expressionanalysisonthe basisoffieldevaluation.Significantvariationwasobservedinstudiedgenotypes forphenotypictraitsandinexpressionanalysis.Averagereductionwasrecorded forallthetraitsviz.28.84%forplantheight,6.10%fornooftillersperplant, 51.76 % for biological yield, 49.27 % for grain yield under field condition. Hydroponicstudies revealed that thegenotypesSahbhagiDhan,RRF-78, MTU-1010,R-RF-105,Shenong, SxR-111,SxR-120performedwellunderP deficiency. Analysisof shoottissues forPcontentshowedR-RF-105hasminimum P utilization and SXR-120 has maximum P utilization under P deficiency. Similarly, analysis of root tissue for P content showed that SXR-111 has minimumPutilizationandShenonghasmaximumPutilizationunderP deficiency whereasthe genotypesRRF-78showedminimum Putilizationand SXR-111 showedmaximum P utilizationunder P supplemented condition.The genotypesR- RF-78,SXR111,SXR120can thusbeconsidered tolerantto Pdeficiencystress, based on fieldand hydroponic studies. UnderPdeficient condition the pH of hydroponicssolutionin all the8 genotypewasshowingsimilartrendhavingthevaluerangingbetween4.4to4.9 upto36days,butafter36daysof transplantinginallthegenotypesthepHwas decreasedindicatingthat theexudatesreleasedbyrootunderhydroponicculture systemhas the correlation withchange in pH ofthe solution. ItisreportedthatOryzaSativaH+-ATPase(OSA)genesarepresent in plasmamembraneof plant cellsandthesearecategorizedinto fivedifferent subgroups.Itisestimatedthat themembersof thesubgroup-1(OsA1, OsA2and OsA3)andsubgroup-2 (OsA5andOsA7)are highlyexpressed,whereas members ofthesubgroup-3(OsA9),subgroup-4(OsA4,OsA6andOsA10)andsubgroup- 5(OsA8)areexpressed inrelativelylowerlevelsundernormalconditions.Herein ourstudyweanalyzedtheexpressionof 12genes(RFT-1,Hd3a,Ehd1,OSA1, OSA2, OSA3,OSA7, OSA8,OSA9,OsSPX1,OsMS,OsALMT)under Pdeficient and supplemented conditionsand these genesshowed differential expression. The genes OsPSX-1, OSA-9 and OSA-2 showed two different amplicons in PandN deficiencystress,inspecificgenotypes indicating alternative splicingof thesegenes undernutrientdeficiency stress.Almost,allgeneswereexpressedat higher levelinRRF-78(Pdeficiency tolerant)under P deficiency.Nutrient deficiencystressspecificup-regulationwasobservedinOSA1(RRF-78,R-RF- 105,MTU-1010andSxR-111),OSA-2(RRF-78,R-RF-105,SxR-120andSxR- 111)indicatingcorrelationwithphenotypicobservations. WhereasOSA-8wasup regulatedonlyinonegenotype(SahbhagiDhan)whichis reportedto bedown- regulated under nutrientdeficiency.ThesisItem Open Access IDENTIFICATION AND CHARACTERIZATION OF QTLs RELATED TO GLYCEMIC INDEX IN RICE (Oryza sativa L.)(Indira Gandhi Krishi Vishwavidyalaya, Raipur, 2018) Premi, Vinay; Chandel, Girish; Prasad, Archana S.; Khokhar, Dhamendra; Saxena, R.R.Glycemic Index (GI) of food is a measure of the rate of glucose releases upon consumption of a specified quantity of food. GI classifies foods as low GI (55 or <55), medium (56 to 69) and high GI (≥70). It is of vital importance in the management of diabetes. It can be used to assist in the selection of foods which help to control diabetes. Rice is a major staple food and energy source of half of the world’s but due to its energy-richnature, it is removed from the diet of diabetic people. Diabetes especially Type II is becoming one of the major health issues for urban as well as rural population. An attempt has been made to determine the biomolecules potentially affecting the GI value of rice in terms of QTLs in rice. In this study, a mapping population of 159 rice lines of F6 generation derived from a cross between ChaptiGurmatiaX DagadDeshiwasanalyzed for total soluble carbohydrate, amylose content and alkali spreading value. Based on the phenotypic data of the above traits total 38 rice lines were selected from 159 lines by using Power Core software for predicted GI (pGI) estimation. The pGI of rice lines was analyzedin vitro using artificial digestion machine named “GI Analyzer 20”. For QTL identification purposes, total 557 SSR markers were used to detect the polymorphic loci between the parents ChaptiGurmatia and DagadDeshi. Total 102 were SSR were found to be polymorphic among the parents which further were used for the genotyping of the mapping population. Then QTLs were identified for total soluble carbohydrate, amylose content, alkali spreading value and predicted glycemic index by WinQTLCart version 2.5_011. Additionally, some important yield-related traits i.e. plant height, number of tillers per plant, panicle length, days to 50% flowering, grain length and width was also recorded for all 159 lines including parents. Biochemical results showed wide variation for total carbohydrate ranged from 57.64% in CXD-298 to 87.75% in CXD-214 with an average of 77.74%. Amylose content ranged from 8.18% in CXD-26 to 22.73% in CXD-280 with an average of 18.79%. Alkali spreading value ranged from 1 in CXD-84 and CXD-109 to 5 in CXD-22 and CXD-26 with an average of 2.69. Predicted glycemic index value ranged from 45.3 in CXD-106 to 79.21 pGI in CXD-125 with an average of 57.92 pGI among the 38 selected lines. Wide variability was also observed for the yield contributing traits. Early flowering in 80 days was observed in rice line CXD-187 while flowering in CXD-318 took 105 days. The average tiller number per plant varied from 3.6 in CXD-143 to 12.6 in CXD-60. The highest plant height was 225.84 cm in CXD-56 while it was lowest in CXD-22 (105.72). Rice line CXD-178 had the maximum panicle length (34.24 cm) whereas it was minimum for CXD-257 (18.50 cm). The grain length ranged from 7.5 mm in CXD-2 to 9.53 mm in CXD-109. Grain width ranged from 2.3 mm to 4.13 mm in CXD-130 and CXD-308 respectively. The genotypic data generated by polymorphic markers were scored for segregation of ChaptiGurmatialike alleles and DagadDeshilike alleles in the mapping population. The allelic segregation revealed that on an average the parent ChaptiGurmatia contributed 52.35% of total amplified alleles whereas the parent Dagaddeshi contributed about 45.19% alleles indicating a slight departure from the theoretically expected ratio of 1:1 or equal contribution from the parents. Total 8 QTLs; ca8.1, ac1.1, as3.1, gi5.1, gi5.2, gi5.3, gi6.1 and gi7.1 with more than 3.0 LOD score were detected for the 4 traits in the study. The phenotypic variance ranged from 7.2% to 47%. One QTL ca8.1 was obtained for carbohydrate on chromosome 8 between RM408 – RM137 and showed the high additive effect 2.85, with 9% PVE and 3.18 LOD score. One QTL ac1.1 was identified for amylose content on chromosomes 1, showed 7.2 % PV, mapped to the region between RGNMS20 to Hvssr1_52 with 3.14 LOD score. One QTL as3.1 for alkali spreading value was detected on chromosome 3 mapped on the region between RM293 – RM85 explained 8% phenotypic variance with 3.28 LOD Score. Five QTLs were identified for pGI. Three QTLs; gi5.1, gi5.2 and gi5.3 on chromosome 5, one QTL; gi6.1 on chromosome 6 and one QTL; gi7.1 on chromosome 7. Highest phenotypic variance 47% explained by the QTL gi5.3 for the glycemic index mapped to the region from RM274 to RM480 on chromosome 5 also had highest LOD score of 6.86. QTL gi5.1 mapped to the region RM249 - Hvssr5_34 obtained on chromosome 5 had explained 17.1% phenotypic variance and least LOD score of 2.53, similarly QTL gi5.2 also obtained on chromosome 5 with 3.95 LOD score and explained 27.4% phenotypic variance. The QTL, gi6.1 for glycemic index mapped on the region RM217 - RM549 had the highest additive effect 5.61 with LOD score of 5.30. QTL gi6.1 identified on chromosome 6 revealed strong correlations with the waxy gene. Amylose content of rice is inversely correlated with GI and amylose synthesis in developing seeds is primarily regulated by the Waxy (Wx) gene. The newly identified QTL gi6.1 for GI explained 34.3% variation and allowed the identification of GI-associated markers. In silico approaches offers great opportunity to search the specific, tightly linked SSRs in the region underlying a QTL governing important traits as well as to design specific markers from the sequences in the vicinity of candidate genes. This also enables us to look for and target the expressed regions in the genome for marker development. Thus In-silicocharacterization of reported QTLs related to starch on the different chromosome was performed. The genomic region underlying between the flanking markers of QTLs were scanned at 200 bp scanning range for putative/expressed genes and other important loci related to starch biosynthesis. A total of 22 genes with Locus ID, nucleotide sequence length, nucleotide CDS length, amino acid protein length and putative function were identified within the genomic region of different chromosomes related to starch property traits. Four starch biosynthetic genes: OsSSI, OsSSIIa, OsSSIIbandOsSSIII were also identified on chromosome 6. Identified QTLs for all the traits analyzed including PGI will be further used to saturate the marker map and mining of useful alleles, governing the GI and related traits. The results of the study are also very important as QTL mapping for the glycemic index is yet not done earlier Keywords: Glycemic index, diabetes, carbohydrates, amylose, blood glucose.ThesisItem Open Access SCREENING OF PHOPHORUS USE EFFICINCY (PUE) OF RICE GENOTYPES UNDER PHOSPHORUS DEFICINCY AND EXPRESSION ANALYSIS OF RELATED GENES(Indira Gandhi Krishi Vishwavidhyalaya, Raipur, 2018) Paul, Madhav; Banerjee, Shubha; Verulkar, Satish B.; Guhey, Arti; Saxsena, R.R.Soil phosphorus (P) deficiency is one of the major factors that adversely affecting plant growth causing early maturity in rice and significant reduction in grain yield. Recent studies on mitigation of P deficiency stress suggests that rice plant exhibits adaptive mechanisms to increase “P” uptake and enhance its utilization under deficient condition and several genes and signaling cascades involved in this response have been reported. As evident from these studies, increase in P uptake alone doesn’t provide an advantage over P deficient condition, in terms of higher grain yield which is reflected by P utilization efficiency (PUE). With this view the present study was conducted to study the response of rice roots in relation to grain yield under P deficiency stress in F4 segregating lines derived from cross between Sahbhagi Dhan and RRF-78 and Diverse rice genotypes . Field evaluation of the 148 F4 lines indicated significant variations in plant height, total no. of tillers, biological weight and grain yield. For analysis of root trait 2 lines (SDXRRF-78(77) and SDXRRF-78[9]) were selected from 148 segregating lines and 6 lines (SWARNA, MTU-1010, RRF-78, SAHBHAGI, SHENONG, G-38) were selected from Diverse rice genotypes on the basis of different traits (plant height, no. of tillers, grain yield) and grown in hydroponics. Almost all of the 8 genotypes showed increase in root length (RL) and higher root volume (RV) and surface area (SA) was observed in SDXRRF-78(77), SDXRRF-78(9), SWARNA, MTU-1010, RRF-78, SAHBHAGI, SHENONG and G-38 genotypes, whereas Sahbhagi showed a decrease in RV in both condition (short term and long term phosphorus deficiency) as compare to Phosphorus sufficient condition. The Phosphorus estimation of all genotypes (SDXRRF-78(77), SDXRRF-78(9), SWARNA, MTU-1010, RRF-78, SAHBHAGI, SHENONG, G-38) phosphorus content high in swarna and Sahbhagi genotypes in both tissue (shoot and root) but phosphorus uptake and utilization high in Shenong, SDXRRF-78(9) and G-38 genotypes. The Expression analysis of 9 genes including phosphorus starvation signaling (OsLTN1), Pi deficiency induced transcription factors encoding genes (OsPTF1), Pi transporter (OsPT1), transporters encoding gene (OsPT1CD) and 3 NRR locus related genes (Ehd1, Hd3a, RFT1) showed differential expression of 5 genes (OsLTN1, OsPT1CD, OsPTF1, Osk04 and OsPTI) in shoot and root tissues under P deficiency stress. Up regulation of these of OsPT1CD, OsK04 genes was recorded in the 2 high yielding genotypes (Shenong and SXRRF-78[9]).On the basis of field evaluation, root trait study and expression analysis, Shenong and SDXRRF-78(9)were identified as promising P deficient tolerant genotypes.ThesisItem Open Access SATURATION MAPPING IN-SILICO AND MAPPING OF MOLECULAR MARKERS WITH QTLs ASSOCIATED WITH DROUGHT TOLERANCE IN RICE(Indira Gandhi Krishi Vishwavidyalaya, Raipur, 2011-01) Sinha, Sweta; KOTASTHANE, A.S.; Sharma, D.K.; Verulkar, S.B.; Jha, Zenu; Lakhera, M.L.SATURATION MAPPING IN-SILICO AND MAPPING OF MOLECULAR MARKERS WITH QTLs ASSOCIATED WITH DROUGHT TOLERANCE IN RICEThesisItem Open Access MOLECULAR CHARACTERIZATION OF PLEUROTUS spp. USING PCR BASED RAPD MARKERS AND ANALYSIS OF MATING TYPE GENES USEFUL FOR STRAIN IMPROVEMENT(Indira Gandhi Krishi Vishwavidyalaya, Raipur, 2007-01) Gupta, Bhavna; KOTASTHANE, A.S.; Katiyar, S.K.; Thakur, M.P.; Jha, Zenu; Saxena, R.R.MOLECULAR CHARACTERIZATION OF PLEUROTUS spp. USING PCR BASED RAPD MARKERS AND ANALYSIS OF MATING TYPE GENES USEFUL FOR STRAIN IMPROVEMENTThesisItem Open Access MAPPING QTLs AND VALIDATION OF CANDIDATE GENES FOR IRON AND ZINC CONCENTRATION IN RICE (Oryza sativa L.)(Indira Gandhi Krishi Vishwavidhyalaya, Raipur, 2018) Maurya, Shrinkhla; Chandel, Girish; Prasad, Archana; Nair, Sunil; Saxena, R.R.Rice is a major staple food for half of the world’s populations including India. The green revolution research of 1960’s have led India to become food surplus country but the recent data on malnutrition especially micronutrient related malnutrition has been identified as one of major concerns on human nutrition. An effort of genetic improvement of rice for grain micronutrients (rice bio-fortification) over decade is becoming a reality with the release of few zinc rich rice varieties in India. However, the understanding of physiological, genetic and molecular basis of micronutrient uptake, translocation, and loading into grain are still major bottle-neck in breeding micronutrient rice rich varieties. The proportionate quantity of iron and zinc present in edible part is a complex quantitative trait and governed by many genes/ QTLs distributed to different regions of chromosome. Rice breeding program needs to be re-focussed for the development more high yielding micro-nutrient rich rice varieties suitable for different region of rice growing states and provide food based solution for reducing malnutrition. In the present study, an attempt has been made to identify QTLs for enhancing iron and zinc in milled grains of rice and develop molecular markers for the various genes involved in the micro-nutrient uptake, mobilization, redistributions and grain loading. Plants deliver minerals absorbed from the root to the shoot, and also re-mobilize them from senescing leaves to young developing tissues via transporter gene families. Members of the ZIP transporters are involved in transporting metal ions by shifting metal ions such as Fe2+, Zn2+ into the cytoplasm. Out of 43 metal homeostasis related genes, the OsZIP10 gene (Plasma Membrane-Localized Zinc Transporter) reported to have up-regulation in flag leaves tissue of high grain zinc containing rice genotypes at mid grain filling stage compare to that of low grain zinc containing genotypes were selected for development of DNA markers. The 804bp upstream region of OsZIP10 gene of four rice genotypes, two having high grain Zn and two with low grain Zn concentration was sequenced. Multiple Sequence alignment analysis revealed 10 bp deletion in high grain zinc lines and 10 bp insertion in that of low zinc lines. Further to develop the gene tagged size based DNA markers for use in MAS five new markers; ZP1, ZP2, ZP3, ZP4 and ZP5 were designed and tested in 68 diverse rice landraces possessing high and low grain levels for validation. Further, to study the association between allelic variations and grain zinc concentration we have performed the single marker analysis using SPSS 16.0 (SPSS Inc.). Single marker analysis for allelic variation revealed that the gene tagged markers were significantly associated with the phenotypic data showing 39% of phenotypic variance at 1% level of significance. Thus, the developed in the present study can be directly used as a tool for marker-aided selection (MAS) in rice breeding techniques for grain zinc concentration in rice. Elemental analysis in milled rice grains were performed in 3 different RIL populations derived from cross MTU1010 x IR94033 (F6 seed), Swarna x IR681444B (F10 seed), HMT x IR681444B (F10 seed). Fe concentration in F5 166 RIL populations derived from cross MTU1010 x IR94033 varies from 6.05 µg/gm to 17.75 µg/gm and for Zn concentration varies from 16.25 µg/gm to 33.50 µg/gm. Iron concentration in F9 72 RIL populations derived from cross Swarna x IR681444B ranges from 4.2 µg/gm to 16.9 µg/gm and zinc concentration ranges from 12.9 µg/gm to 29 µg/gm. Average iron concentration in RIL lines derived from cross HMT x IR681444B is 11.63 µg/gm and zinc concentration is 21.23 µg/gm. In the study, we have also calculated advantage over control (AOC) for Fe/Zn concentration, to get the consistency in the micronutrient data. Further, we have selected the top eleven lines which possess zinc concentration more than 26 µg/gm in milled rice grains and having more zinc advantage over control. High AOC for zinc were observed in MTIR-156 (+14.1µg/gm) followed by MTIR-127 (+13.5) and MTIR-150 (+11.6). Thus, the RIL lines identified with higher grain zinc concentration can serve as donor lines for the improvement of elite and popular rice cultivar. F5 population of 166 recombinant inbred lines (RILs) derived from the cross MTU1010 x IR94033, was used to identify and map QTLs for average Fe/Zn concentration and AOC (advantage over control) for Fe/Zn concentration in milled rice grains. QTL analysis for average Fe/Zn concentration led to the identification of 6 QTLs. Out of 6 QTLs, 1 QTL for iron concentration was located in chromosome 8 (qFE8.1) and 5 QTLs (qZN1.2, qZN2.1, qZN5.2, qZN7.1, qZN10.1) for zinc concentration were located on chromosomes 1, 2, 5, 7, 10 with LOD scores ranging from 3.12 to 4.68. The phenotypic variation explained by qFE8.1 for iron concentration was observed to be 7.4%. QTLs together for grain zinc concentration explained 7.8% phenotypic variance. Further QTLs have also been identified using (advantage over control) AOC grain Fe/Zn concentration. Out of 13 QTLs, 1 QTL (qFE8.1) for iron concentration was located in chromosome 8 and 12 QTLs for zinc concentration were located on chromosomes 1, 2, 5, 7, 10, 11 with LOD scores ranging from 3.27 (qZN1.3) to 5.56 (qZN2.1). QTLs together explained 17.15% phenotypic variance for grain zinc concentration and 7.4% phenotypic variance for iron. Among all QTLs observed for AOC Fe/Zn concentration, were found to be co-localized with the average Fe/Zn concentration in milled rice grains. For grain Zn concentration LOD value of AOC found to be more than average Zn concentration for the same QTL, indicating more consistency in the data. One QTL hotspot region for grain zinc concentration was detected in chromosome 7, qZN7.1 underlying within the flanking markers RM234-RM248 (1.75 Mb QTL size). 7 new QTLs were identified present in chromosome 1 (qZN1.1), 2 (qZN2.2, qZN2.3), 5 (qZN5.2, qZN5.3), 10 (qFE8.1) and 11 (qZN11.1). Among all, 7 candidate genes viz., OsYSL1, HMA, OsZIP14, OsNAC5, OsYSL3, OsNAS3, OsDMAS1 known for iron and zinc homeostasis underlying within QTLs were identified based on our genetic mapping studies. The region between flanking markers identified in the study will be further used to saturate the marker map and mining of useful alleles governing micronutrient related trait. The concept of QTL identification for micronutrient concentration for the advantage over control has the potential to give more consistent result particularly for complex and low phenotypic variance trait.ThesisItem Open Access MORPHOGENETICAL AND MOLECULAR CHARACTERIZATION OF ELITE RICE LANDRACES OF CG COLLECTION USING MICROSATELLITE MARKERS(INDIRA GANDHI KRISHI VISHWAVIDYALAYA, RAIPUR (C.G.), 2012) UGALE, VINOD VITTHAL; Chandel, Girish; Sharma, D.K.; Jha, Zenu; Verma, Ravindra; Saxena, R.R.ThesisItem Open Access MORPHOGENETICAL AND MOLECULAR CHARACTERIZATION OF ELITE RICE LANDRACES OF CG COLLECTION USING MICROSATELLITE MARKERS(Indira Gandhi Krishi Vishwavidyalaya Raipur(c.g), 2012-06) VITTHAL, UGALE VINOD; CHANDEL, GIRISH; SHARMA, D.K.; JHA, ZENU; VERMA, RAVINDRA; SAXENA, R.R.MORPHOGENETICAL AND MOLECULAR CHARACTERIZATION OF ELITE RICE LANDRACES OF CG COLLECTION USING MICROSATELLITE MARKERSThesisItem Open Access IDENTIFICATION OF QTLs FOR YIELD AND YIELD CONTRIBUTING TRAITS IN RICE(Oryza sativa L.)(INDIRA GANDHI KRISHI VISHWAVIDYALAYA RAIPUR(C.G), 2015-01) Sahu, Vikrant Kumar; Chandel, Girish; Kotasthane, A.S.; Nair, S.K.; Saxena, R.R.; Verulkar, S.B.IDENTIFICATION OF QTLs FOR YIELD AND YIELD CONTRIBUTING TRAITS IN RICE(Oryza sativa L.)