IC4R014-RNA-Seq-2013-23322175

Project Title

Transcriptomic analysis of rice (Oryza sativa) endosperm using the RNA-Seq technique

The endosperm plays an important role in seed formation and germination, especially in rice (Oryza sativa). In this project, the researchers used a high-throughput sequencing technique (RNA-Seq) to reveal the molecular mechanisms involved in rice endosperm development.

The endosperm were derived from rice (O. sativa L.ssp. indica cv.9311), which were grown in a greenhouse at Wuhan University, China. Caryopses were tagged at the initiation of pollination. The samples consisted of three replicates arranged in a randomized complete block design. For each replicate, development seed were collected from 15 different plants at 3, 6, and 10 DAP during the growing season. The three-stage endosperm was taken from caryopses with microdissection needles under a dissection microscope (Olympus, Tokyo, Japan). The materials were immediately frozen in liquid nitrogen and kept at -80℃ until required (for RNA extractions).

Total RNA was prepared using Trizol according to the manufacturer’s protocol (Invitrogen, Burlington, ON, Canada). The yield and purity of each RNA sample was determined by the absorbance (Abs) at 260 and 280 nm and the integrity of all RNA samples was monitored on 1 % agarose gels. The total RNA samples were pooled into three-stage samples and 10 lg of total RNA from each sample was used to isolate poly (A) mRNA and to prepare a nondirectional Illumina RNA-Seq library with an mRNASeq 8 Sample Prep Kit (Illumina). Each library had an insert size of 200 bp, and 42–50 bp sequences were generated via Illumina HiSeqTM 2000.

By comparison with the rice genome and trimming, all 35.9 high-quality million clean reads were assembled into 23,836 genes. Among the three stages, the number of genes found were 21,596 (R1), 20,910 (R2) and 19,459 (R3), providing massive data for endosperm development analysis. From these numbers, there is an apparent decline in quantity during endosperm development. A Venn diagram shows the overlapping relationship between the three stages (Fig. 1).

Figure 1. Venn diagram showing the genes expressed in each of the three stages of rice endosperm development.

To identify clusters with functional enrichment, the researchers performed hierarchical clustering of the expression patterns of all DEGs and found that there was a similarity relationship in gene expression among three stages (Fig. 2). The hierarchical clustering analysis indicates that the majority of genes were downregulated in all three comparisons, and that a minority of genes were up-regulated.

Figure 2. Hierarchical cluster analysis of gene expression based on log ratio RPKM data.

State Key Laboratory of Hybrid Rice, College of Life Sciences, Wuhan University, Wuhan 430072, China