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Title: Use of in silico and semiquantitative RT-PCR approaches to develop nutrient rich rice (Oryza sativa L.)
Authors: Banerjee, Shubha
Sharma, D J
Verulkar, S B
Chandel, G
Keywords: Expression analysis;in silico;iron zinc protein rich rice;MGS;MTS;RT-PCR
Issue Date: Apr-2010
Publisher: CSIR
Abstract: Inspite of striking agricultural progress and adequate food grain production, protein energy and micronutrient malnutrition are widespread among rural and poor population. The pharmaceutical and diet diversification based approaches have achieved little success due to poverty and educational unawareness. Biofortification of staple food crop rice, which is consumed in large amounts daily, will serve as an important vector to combat malnutrition. The possible approach to improve nutritive value of rice involves exploitation of available genetic variability for grain protein and Fe/Zn contents with conventional and modern biotechnological tools. We have analysed variability in grain protein and Fe/Zn levels in rice, and factors affecting them for identification of rice genotypes with higher nutritive value. Wide variation for grain protein and micronutrient levels were recorded among the tested rice genotypes, which ranged from 6.19 to 10.75% for grain protein content, 4.82 to 22.69 μg/g for grain Fe and 13.95 to 41.73 μg/g for grain Zn content. Significant effect of nitrogenous fertilizer dose and native soil properties were observed on grain protein content, whereas grain Fe and Zn levels were more significantly affected by native soil properties and showed non-significant effect of nitrogen applied. Three genotypes, R-RF-31, Lalmati and R 1033-968-2-1 were identified as high protein and Fe/Zn containing rice lines. In view of our findings and previous studies, where significant Gene Environment (G E) effect has been reported on grain nutritive traits, molecular markers are of great use for intensive screening of large populations and identification of environmentally stable outperforming genotypes. The genomic sequence of candidate genes governing micronutrient content in rice were, thus, analysed in silico for identification of novel gene specific markers (SSRs and SNPs) and expression sequence tags (ESTs and MPSS) to understand putative expression pattern. Further, to confirm in silico expression results and functionally characterized Fe and Zn homeostasis related genes, root and shoot transcriptome analysis of a set of 12 diverse rice genotypes was carried out for expression of 21 metal homeostasis related genes belonging to OsYSLs, OsFROs, OsZIPs, OsNRAMPs and OsFERs families, and OsNAAT1, OsVIT1, OsNAC and OsNAS2 genes. A total of 176 novel SNPs and 39 novel SSRs were identified within metal related genes, which can be used for developing gene specific markers. The ESTs and MPSS tag based in silico expression analysis results were in consistency to the semi-quantitative RT-PCR based transcriptome analysis. Expression profiling of rice root and shoot transcriptome at maximum tillering and mid-grain filling stages revealed high level of expression of most of the Fe and Zn uptake and transport related genes in genotypes showing higher grain Fe and Zn concentrations. The rice genotypes with high grain protein and micronutrients, identified in this study, will provide the basis of bioavailability assay and will also serve as potential genetic material for molecular breeding of nutrient rich rice.
Page(s): 203-212
ISSN: 0975-0967 (Online); 0972-5849 (Print)
Appears in Collections:IJBT Vol.09(2) [April 2010]

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