IC4R011-RNA-Seq-2010-21122150

Project Title

• Massive parallel sequencing of mRNA in identification of unannotated salinity stress-inducible transcripts in rice (Oryza sativa L.)

The Background of This Project

• Microarray technology is limited to monitoring the expression of previously annotated genes that have corresponding probes on the array. Computationally annotated genes have not fully been validated, because ESTs and full-length cDNAs cannot cover entire transcribed regions. Here, mRNA-Seq (an Illumina cDNA sequencing application) was used to monitor whole mRNAs of salinity stress-treated rice tissues.

Plant Culture & Treatment

• Seeds of rice (Oryza sativa L.‘Nipponbare’) were germinated in the dark at 28°C on a sterilized germination tray. Germinated seeds were evenly distributed on 96-well PCR plates supported by a plastic container. Seeds were grown in a growth chamber at 28°C, as previously described. After the seedlings had been grown for 7 days, they were transferred on their 96-well plates into containers filled with 150 mM NaCl solution, or with control solution, and placed at 28°C in a growth chamber for 1 h. Four kinds of tissue (normal shoot, normal root, shoot with 1-h salinity stress, or root with 1-h salinity stress) were collected and immediately frozen in liquid nitrogen. For RNA extraction from each treatment group, 10 plants were collected and mixed, to minimize the effect of transcriptome unevenness among plants.

Research Findings

Figure 1 Accumulation of 36-bp reads to cover whole transcripts. (a) Cumulative coverage of rice genome and annotated region. Data from nine technical replicates of reads from roots after salinity stress were accumulated. Cumulative coverage was calculated by using reads uniquely mapped on the rice genome (black) or the RAP2 annotated region (white). As the number of reads increased, the cumulative coverage approached a plateau. (b) Robustness of the measurement of transcripts in four different expression classes. Saturation of sequencing was estimated on the basis of the fraction of RAP2 genes supported by FL-cDNA sequences that had reached their final RPKM (reads per kilobase of exon model per million mapped reads). Vertical axis indicates the fraction of genes for which the RPKM was within 5% of the final value, and horizontal axis indicates the cumulative number of uniquely mapped reads. The fraction of highly expressed genes was almost unchanged, whereas those of genes with relatively low expression converged slowly. N indicates the number of transcripts in each of the four classes.

Labs working on this Project

• National Institute of Agrobiological Sciences (NIAS), Division of Genome and Biodiversity Research, 1-2, Kannondai 2-chome, Tsukuba, Ibaraki 305-8602, Japan.
• Institute of the Society for Techno-innovation of Agriculture, Forestry and Fisheries, 446-1, Ippaizuka, Kamiyokoba, Tsukuba, Ibaraki 305-0854, Japan.
• Hitachi Government & Public Corporation System Engineering Ltd., Solution Division 4, Research & Development Center Bioinformatics Group, 2-4-18 Toyo, Koto-ku, Tokyo 135-8633, Japan.

Corresponding Author

• mat@nias.affrc.go.jp