MAPPING QTLs FOR NH4 + AND NO3 - USE EFFICIENCY UNDER WATER STRESS AND NON-STRESS CONDITIONS AND EXPRESSION ANALYSIS OF GLUTAMINE SYNTHETASE AND NITRATE REDUCTASE IN RICE (Oryza Sativa L.)

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Date
2017
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Indira Gandhi Krishi Vishwavidhyalaya, Raipur
Abstract
Rice is the most important staple crop in Asia, where it provides 35–60% of total calorie intake. Faced with scarcity of water resources, water deficit condition/drought depresses the rice production by reducing water and nutrient availability to the plant. Nitrogen (N) is one of the most critical inputs and the current average nitrogen use efficiency (NUE) in the rice field is approximately 33%, poorest among cereals. Predominant form of N in aerobic soils is nitrate (NO3-) while ammonium (NH4+) exists in anaerobic soils. Rice prefers to utilize NH4+ over NO3- as rice is pertained to waterlogged conditions. Development of cultivars with improved NH4+ or NO3- use efficiency by harnessing inherent significant variability for NUE can be an important approach. Considering these facts, the present study was undertaken with one hundred twenty two and selected thirty two recombinant inbred lines (RILs) of two indica genotypes, Danteshwari × Dagad deshi which were evaluated in factorial RCBD design with two replications each under three nitrogen forms and three environments along with root studies under soil filled glass rhizotrons at research cum instructional farm of COA, IGKV, Raipur, for generation of phenotypic data. The previously generated genotypic data of 162 SSR and HvSSR based markers were used for identifying QTLs for agronomically complex trait i.e. NUE. The study of NH4+ & NO3- dynamics in soil, enzyme assay and expression analysis of key known genes was accomplished at Molecular Marker Laboratory, Department of Genetics and Plant Breeding, IGKV, Raipur. The trend analysis of NH4+-N & NO3--N dynamics revealed that NH4+-N concentration persisted more under anaerobic condition and NO3--N concentration under aerobic conditions. Three way-ANOVA showed high level of significance for variance components (G, N, E) and their interactions effects (GXN, GXE, NXE, EXNXG) for yield & NUE and their component traits. Mean performance of genotypes depicted higher values for agronomically important traits i.e. yield and NUE under NH4+ treatment as compared to NO3--N and N0 treatment under all environments. Genotype G-31 manifested the highest NUE values under irrigated condition in all treatments (17.2 gg-1N, 12.72 gg-1N, 16.98 gg-1N) while Genotype G-27 has the maximum NUE values under rainfed condition in all treatments (8.4 gg-1N, 11.6 gg-1N, 6.2 gg-1N). Grain yield showed significant and positive association with NUE indices under all sets of environments. Furthermore, significant and positive correlation existed between total root length and NUE parameters irrespective of NH4+, NO3- and N0 treatment. The phenotypic and genotypic data was statistically analyzed for QTLs identification for yield & NUE traits using QTL Cartalographer and results were projected graphically using Graphical Genotypes (GGT). At logarithmic odds of 2.5, a total of 58 QTLs conferring the corresponding five traits were detected under three N forms and two environments; that as a matter of fact included 14, 15 & 11 QTLs under NH4+, NO3- and N0 level of irrigated condition and 5, 10 & 1 QTLs under NH4+, NO3- and N0 level of rainfed conditions respectively. QTLs for GY under irrigated condition, had positive additive effect indicating allele from female parent (Danteshwari) while under rainfed condition, had negative additive effect revealing contributing allele from tolerant parent genotype (Dagaddeshi). A presence of QTL clusters between HvSSR 1-87(38.9cM) to HvSSR 1-89 (40.6cM) on chromosome 6, RM 449 (81.9cM) to RM5 (9.4cM) on chromosome 5, HvSSR 9-25 (14.6cM) to HvSSR 9-27 (15.5cM) on chromosome 9 and RM 434 (57.7cM) to RM 410 (64.4cM) on chromosome 5 signifies important genomic regions associated with evaluated traits under different conditions and will be useful in marker assisted breeding for NUE in rice. Furthermore, the graphical genotypes (GGT) of NH4+, NO3- and N0 responsive lines under irrigated and rainfed condition with GY and GYR QTLs regions flourish us with the information of novel regions influencing the grain yield. We also investigated the different members AMT (Ammonium transporters), NRT (Nitrate transporters), GS (Glutamine Synthetase) & GOGAT (Glutamate Synthase) genes, involved in NUE and analyzed the expression pattern of each gene using gene-specific primer in young rice seedlings by quantitative real time PCR, revealing a distinct expression pattern of these genes. Collectively, OsGln1;1, OsGln1;2, OsGln1;3 , OsGln2, OsGlt1 and OsGlt2 manifested different and reciprocal responses to nitrate and ammonium supply. Overall, OsGln2 isoform of Glutamine Synthetase showed strong upregulation in shoot under NH4+ (125.8) and NO3- (186.7) treatment in Genotype G-4 which falls in dark green spectrum in colour classes suggesting OsGln2 is major assimilatory form in green tissues. Systemic expression patterns for the AMT and NRT gene families revealed that, OsAMT1;2 exhibited significant strong expression in shoot of genotype G-4 (133.4) also having high OsGln activity thus there is strong induction by endogenous glutamine while OsAMT1;3 exhibited strong expression in root of genotype G-9 (42.1) also having low OsGln activity which signifies suppression by endogenous glutamine. With regard to NRT 7.8 gene, genotype G-10 showed significant upregulation under NH4+ treatment in root while NRT 2.4 gene was significantly upregulated in genotype G-3 (10.3) under NO3- treatment in shoot. The activity of enzymes NR, NiR, GS & GOGAT was significantly affected by NH4+ and NO3- treatment. The NR and NiR activity was highest in NO3- treatment in shoot (1.49 & 0.31) as compared to root (0.006 & 0.017) indicating NR & NiR is nitrate inducible enzyme while GS & GOGAT activity decreases with application of N forms with N0 having highest value for GS and GOGAT activity in shoot (0.15 & 0.29) as well as in root (0.038 & 0.068). Among all genotypes, genotype G-4 which is high yielding and relatively nitrogen efficient showed highest activity values for GS and GOGAT under NH4+, NO3- and N0 treatment suggesting correlation between grain yield and GS activity. These results assist us to identify NH4+ & NO3- responsive cultivars which could be used for cultivation and/or used as parent’s in future breeding program to produce better nitrogen use efficiency varieties under water stress and non-stress conditions.
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MAPPING QTLs FOR NH4 + AND NO3 - USE EFFICIENCY UNDER WATER STRESS AND NON-STRESS CONDITIONS AND EXPRESSION ANALYSIS OF GLUTAMINE SYNTHETASE AND NITRATE REDUCTASE IN RICE (Oryza Sativa L.)
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