IC4R007-miRNA-2014-24315823

Project Title

Identification and characterization of salt responsive miRNA-SSR markers in rice (Oryza sativa)

The Background of This Project

[[File:|700px|thumb|right|Figure 1. GWA Analysis of Al Tolerance within and across Rice Subpopulations.]]

• Salinity is an important abiotic stress that affects agricultural production and productivity. It is a complex trait that is regulated by different molecular mechanisms. miRNAs are non-coding RNAs which are highly conserved andregulategene expression.Simplesequencerepeats(SSRs) are robustmolecular markers for studyinggenetic diversity. Although several SSR markers are available now, challenge remains to identify the trait-specific SSRs which can be used for marker assisted breeding.
• High salinity causes multifarious effects on plant growth and development in the form of disturbed cell expansion, impaired metabolism, necrosis and limited protein synthesis that eventually leads to the enhancement of cell death. Soil salinity is one of the major limitation factors that alone causes 5% yield loss. To counteract salinity stresses, plants utilize number of defence mechanisms, ultimately leading to stress tolerance. This comprises a range of physiological and biochemical adjustments in plants including leafwilting,leafarea reduction,leafabscission,rootgrowthstimulation, and alterations in relative water content. Molecular responses to abiotic stress on the other hand include perception of the particular stress, signal transduction, gene expression and ultimately metabolic changes in the plant thus providing stress tolerance. Among the different regulatory elements, miRNAs are wide-spread class of newly discovered, about21 bp long, non-coding RNAs that silence the gene expression at post-transcriptional level in plants. More specifically, miRNAs cleave the target genes to prevent gene expression in plants . Several recent findings are reminiscent of the fact that miRNAs contribute significantly to the plant adaptation to salt stress. Developingsalt tolerant plant through marker assisted breeding has added advantage over either conventional breeding or through transgenic technology. Due to their dense distribution throughout the genome, high reproducibility, co-dominant alleles and highly variable nature, microsatellites or simple sequence repeats (SSRs) meet the requirements of an ideal genetic marker. Subsequently, they have become markers of choice for genome mapping, finger-printing and population analysis as well as in evolutionary studies.

Plant Culture & Treatment

• Twenty four rice genotypes comprised of one salt tolerant and one saltsusceptiblepanel,eachwith12genotypes, wereusedin thepresent study.DNA wasextracted from 100 mggreenleaftissueusing cetyltrimethyl ammonium bromide (CTAB). Briefly, tissues were homogenized with a pestle in a CTAB buffer (CTAB 2%; 0.1 M Tris, pH 8; 0.02 M EDTA pH 8; 1.4 M NaCl) and incu-batedat60 °Cfor45 min.TheDNAwasextractedfromthelysismixture with chloroform/isoamylalcohol (24/1;v/v)andprecipitatedbyadding isopropanol (v/v) to the DNA containing phase. After centrifugation (10,000 ×g, 15 min), the pellet was rinsed with 70% ethanol, air-dried, re-suspended in 1X TE (10 mM Tris Base and 1 mM EDTA) buffer. The DNA samples were stored at −20 °C until PCR amplification. The quality and purity of RNAase-treated DNA were checked in 0.8% agarose 1X TAE (40 mM Tris–acetate and 1 mM EDTA) gel. Isolated purified DNA was quantified using spectrophotometer (Thermo Scientific Nano drop 1000) and diluted with double distilled water to the final concentration of 25 ng/μl.

Research Findings

[[File:|700px|thumb|right|Figure 2. Haplotype analysis of the Nrat1 gene region.]]

Labs working on this Project

• Division of Genomic Resource, National Bureau of Plant Genetic Resource, Pusa, New Delhi 110012, India

Corresponding Author

• Tapan Kumar Mondal(mondaltk@yahoo.com)