GSE70093
Differential gene expression in developing grains of two tropical indica rice genotypes differing in grain iron concentration through RNA-seq
Rice (Oryza sativa L.) is the main staple food for nearly half of the world’s population. Cereals, especially rice is deficient in micronutrients such as Fe. However, rice genotypes differ in grain Fe concentration (Panda B, et.al. Am J Plant Sci. 2014;5:2829-41. doi: 10.4236/ajps.2014.518299). The present study is focused on identification of gene(s) involved in Fe accumulation in developing rice grain through high throughput RNA-seq technology and to understand the basis of differential Fe accumulation in developing rice grain. Two cultivars of rice viz. Sharbati (high Fe) and Lalat (low Fe) differing in grain iron concentration were used in the study. Root and grain transcriptome sequences of these two cultivars (at mid-grain filling stage) were generated using RNA-Seq (Illumina Hiseq 2000 platform). For each genotype, on an average 9.7 and 7 Gb data was generated for grain and root samples, respectively. The short reads were aligned against the Nipponbare reference genome (IRGSP build 5.0), thereby successfully mapping 95% and 67% of the reads from grain and root samples, respectively.  Genes known to be involved in Fe metabolism were analyzed for expression. Among the genes coding for phytosiderophore synthesizing enzymes, OsNAS1 and OsNAAT2 showed higher expression in the grains of Sharbati while OsDMAS1 had higher expression in its root. Of the 18 yellow-stripe like (YSL) genes in rice, only 11 were found to be expressed in the two cultivars. Out of these, 2 (OsYSL2 and OsYSL 8) were up-regulated in the grains of Sharbati highlighting their importance in the uptake of Fe from soil and its accumulation in the developing grain. Two other genes (OsFRO1 and OsIRT1) known to be involved in Fe uptake by the root were also found to be highly expressed in the root of Sharbati.  Our findings suggest that higher grain iron concentration of cv. Sharbati might be due to higher expression of key Fe transporters (viz. OsYSL2, OsYSL8 and OsITR1) and root membrane bound OsFRO1, which give an advantage in terms of absorption, transport and assimilation of Fe by this cultivar as compared to the low iron containing cultivar, Lalat. Overall design: CRD was used to grow rice plants in the net house under ambient condition. Plants Sharbati and Lalat having contarsting grain Fe concentration (24.88 ppm and 7.80 ppm of Fe in the brown rice samples of Sharbati and Lalat, respectively) [Panda et. al., 2014] were grown in the net house. One week old seedlings were transferred to plastic pots filled soil and farm yard manure in 3:1 ratio and grown in the net house under ambient conditions till maturity. Fifteen days after  panicle emergence (mid-grain-filling stage) roots and developing grains were collected, washed in sterile nanopure water, blotted, dipped in RNA stabilizer solution in a falcon tube and stored in refrigerator for 24h. The tubes were sifted to -80oC ultra freezer for until future use. Differences in gene expression between different samples were tested with Cuffdiff package of Cufflinks using FPKM (Fragments Per Kilobase of transcript per Million mapped reads) from reference-guided mapping. Genes expressed at very low levels (read counts < 10 across all six libraries) were not used in analysis of differential gene expression. Significance tests for differential expression were based on a modified exact test. A false discovery rate (FDR) of 0.05 and p-value =0.05 was used for identifying differentially expressed genes.