IC4R012-RNA-Seq-2012-22347394

Project Title

• Transcriptomic Analysis of Rice (Oryza sativa) Developing Embryos Using the RNA-Seq Technique

The Background of This Project

• Although embryo development is a major subject in plant growth and development research, there is still a long way to go in order to understand the mechanism of this developmental process. The classification of gene expression patterns associated with specific stages of embryo development and a functional understanding of the encoded genes is critical for comprehending the molecular and biochemical events associated with embryogenesis. Rice (Oryza sativa) is an excellent model monocot with a known genome sequence for studying embryogenesis.
• RNA-Seq technology had not been used to analyze embryogenesis in rice. In this research, the researchers reported the transcriptome profiling analysis of rice developing embryos using RNA-Seq as an attempt to gain insight into the molecular and cellular events associated with rice embryogenesis.

Plant Culture & Treatment

• Rice (O. sativa L.ssp. indica cv.9311) plants were grown in a greenhouse at Wuhan University (30u330N, 114u190E), China. Some spikelets were tagged at the initiation of pollination, and harvested at 3–5, 7, and 14 DAP. Before 3 DAP, the zygote reiterates cell divisions to form a globular embryo with no apparent morphological differentiation. At 5 DAP, the first leaf primordium is visible on the opposite side of the coleoptile, and the shoot apical meristem becomes dome-shaped at 7 DAP [32]. Morphological maturity of the rice embryo was noted at approximately 14 DAP. Since the number of embryos at 3 DAP was extremely low, a mixture of embryos at 3–5 DAP was collected as one sample. The three-staged embryos were taken from spikelets with microdissection needles under a dissection microscope (Olympus, Tokyo, Japan) according to the manual microdissection method and frozen immediately in liquid nitrogen for total RNA extraction. Three embryo clusters were collected from 15 different plants at 3–5, 7, and 14 DAP during the growing season. The embryos were randomly selected from each cluster and pooled with embryos from other plants at the same greenhouse site, resulting in three independent pools for each developmental stage.

Research Findings

• The researchers sequenced three cDNA libraries, R1 (3–5 DAP), R2 (7 DAP), and R3 (14 DAP), and generated 17,755,890 sequence reads, each of which was 42–

50 bp in length, encompassing 2.34 Gb of sequence data (Table 1). Each stage was represented by approximately 6 million reads, a tag density sufficient for the quantitative analysis of gene expression. The sequence reads were aligned to the rice reference genome database using SOAPaligner/soap2 software (set to allow two base mismatches). Of the total reads, 83.28% matched either to a unique (77.53%) or to multiple (5.75%) genomic locations; the remaining 16.72% were unmatched (Table 1), because only reads aligning entirely inside exonic regions will be matched (reads from exon-exon junction regions will not match).

Table 1. Summary of read numbers based on the RNA-Seq data from rice developing embryos.

Labs working on this Project

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

Corresponding Author

• jbwang@whu.edu.cn