44 Rice Genetics Newsletter Vol. 25 18. Expression profiling of metal homeostasis related candidate genes in rice (Oryza spp.) using semi quantitative RT-PCR analysis. G. CHANDEL 1)* , S. BANERJEE 1) and S. B. VERULKAR 2) 1)Department of Biotechnology 2)Department of Plant Breeding and Genetics College of Agriculture Krishak Nager Indira Gandhi Agricultural University Raipur 492 06 C. G. INDIA Tel: 91-771-2442069, 094063-82169 (m) Fax: 91-771-2442131 Email: girishchandel@yahoo.com * Author for correspondence Improving grain iron and zinc content in rice, the major staple food crop is the most recent and promising approach to combat micronutrient malnutrition widespread among the rice eating populations in Asia. Rice absorbs metal ions from soil by roots in the form of divalent metal ions chelated with phytosiderophores which is transported to leaves along with xylem sap and remobilized to sink tissues with phloem sap (developing seeds). Grain micronutrient content is thus a function of plethora of processes governed by genes encoding metal-chelate transporter proteins as well as phytosiderophore biosynthesis pathway related enzymes (Grusak, 2002 Narayanan et al., 2007). Several genes responsible for metal transport, sequestration and phytosiderophore biosynthesis have been identified in rice, Arabidopsis, Barley, Wheat and Maize (Gross et al., 2003 Bughio et al., 2002 Guerinot et al., 2000 Koike et al., 2004 Inoue et al., 2003). Characterization of expression of metal homeostasis related genes in rice leaves and root tissues and analysis of any correlation between level of expression of gene in a tissue with grain Fe and Zn content will help identify the role of candidate genes in iron and zinc uptake, transport and accumulation in sink i.e. seed. Expression of 21 rice genes belonging to 5 gene families (OsNRAMPs, OsFROs, OsZIPs, OsFERs and OsYSLs) and 4 non rice gene homologues (OsNAAT1, OsNAS2, OsNAC, OsVIT1) was analyzed in root and leaf tissue of 12 diverse rice genotypes at maximum tillering and mid grain fill stages by semi quantitative RT-PCR analysis. Since flag leaf is considered as major source of remobilized photo-assimilates to developing grains flag leaf transcriptome was analyzed at mid-grain fill stage. For expression analysis twelve rice genotypes belonging to six categories of cultivated rice described by Garris et al., (2005) as temperate japonica, tropical japonica, indica, aus, aromatic, submerged and three wild rice (O. nivara, O. officinalis, O. latifolia) were grown in controlled environment in pots with coco-peat as potting substrate and nutrient supplied in solution (as described by Grusak, 2002), with 10µM iron as Fe (III)-HEDTA. Grain iron and zinc contents were estimated using Atomic Absorption Spectrophotometer (AAS200) as per the methods described in HP protocol (HarvestPlus, 2006). Differential level of expression was recorded for the 25 candidate genes among the tissue type, stages and genotypes. Root transcriptome analysis revealed that 5 genes (OsFER2, OsNAS2, OsNRAMP6, OsZIP5 and OsZIP10) showed lower or negligible expression, 3 genes (OsFER1, OsVIT1 and OsFRO2) expressed at maximum tillering stage only, 7 genes (OsIRT1, OsFER2, OsZIP7, OsZIP8, OsZIP9, OsNRAMP4, OsNRAMP6 and OsYSL12) expressed at mid-grain filling stage only and 10 genes (OsZIP4, OsZIP11, OsNRAMP5, OsNRAMP7, OsYSL2, OsYSL4, OsYSL6, OsYSL9, OsNAAT1 and OsNAC) expressed at both the stages. Leaf transcriptome analysis at maximum tillering stage indicated that 5 genes (OsFER2, OsNAS2, OsZIP10, OsYSL7 and OsYSL12) showed no expression while 4 genes (OsNRAMP4, OsNRAMP6, OsZIP5, and OsZIP9) expressed at very low level in all 12 rice genotypes while remaining 16 genes showed variable level of expression among genotypes. In flag leaf tissue all 25 genes showed expression in at least one genotype, with uniform high level of expression of 5 genes (OsFER1, OsZIP5, OsYSL2, OsYSL6 and OsYSL12) in all 12 rice genotypes. To pursue role of metal homeostasis related genes in influencing grain iron and zinc contents (Table 1), the level of expression of genes was compared with the level of Fe/Zn in grains. The correlation analysis suggested that 10 genes (OsFER1, OsFRO2, OsNRAMP5, OsIRT1, OsZIP7, OsZIP8, OsYSL2, OsYSL6, OsYSL9, OsVIT1 and OsNAAT1) showed higher expression in leaf tissue of high zinc rice genotypes (R-RF-31, R 1033-968-2-1, BAS370, Nagina22, wild rice lines and Jaldoobi) while 3 genes (OsYSL6, OsFRO2 and OsNAC) expressed at high level in high iron rice lines at maximum tillering stage (lane 7, 8 in Fig. 1c). Root transcriptome analysis revealed higher level of expression of 6 genes (OsFRO2, OsZIP8,