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.)

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.

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.

As the number of reads increased, the fraction of highly expressed genes(RPKM ≥ 300) close to their final RPKM was almost unchanged, whereas those of genes with relatively low expression (RPKM 3-30) converged more slowly (Figure 1b). With four technical replicates (corresponding to about 27 to 35 million reads), 81.2% of genes with relatively low expression levels (RPKM 3-30) reached to within ± 5% of their final RPKM (Figure 1b).

Figure 1 Accumulation of 36-bp reads to cover whole transcripts.

Rice transcriptome analysis was based on response to salinity stress. mRNAs were prepared from the tissues of normal rice shoots and roots and from those subjected to 1 h of salinity stress. Of the 27 to 35 million quality-evaluated reads (Table 1; Total filtered reads), 72.0% to 75.2% were mapped uniquely to the rice genome (Table 1; Unique-genome); 5.0% to 5.7% of the reads bridged flanking exons (Table 1; Unique-bridged); 6.0% to 11.2% of the reads were repetitive sequences (Table 1; Multiple); and 10.1% to 16.7% had no match in the genome (Table 1; Unmapped). Thus, a total of 76.9% to 80.9% of the reads were mapped uniquely to the rice genome or to exon-exon junctions (Table 1; Unique-total).

Tabel 1 Numbers of mapped reads.

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.