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.

Illumina Sequencing

• Total RNA was extracted using TRIzolH reagent according to the manufacturer’s protocol.
• The total RNA samples were pooled and 10 mg of total RNA from each pool was used to isolate poly(A) mRNA and to prepare a nondirectional Illumina RNA-Seq library with an mRNA-Seq 8 Sample Prep Kit (Illumina).Each library had an insert size of 200 bp; 42- to 50-bp sequences were generated via Illumina HiSeqTM 2000.

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)(Table 1).

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

• The distribution of tag expression was used to evaluate the normality of our RNA-Seq data. As shown in Figure 1, the distribution of distinct tags over different tag abundance categories showed similar patterns for all three RNA-Seq libraries. The similarity distribution had a comparable pattern with more than 20% of the sequences having a similarity >80%, while approximately 80% of the hits had a similar range(Figure 1).

Figure 1. Percent of coverage representing the percentage of genes which expressed in each of the three stages mapped in the rice genome.

• A total of 23,971 (R1), 23,732 (R2), and 23,592 (R3) genes, ranging from 100 to ≥2,000 bp, were detected in the samples. As shown in Table 2, the proportion of sequences with matches to rice databases was higher among the longer assembled sequences. The removal of partial overlapping sequences yielded 27,190 genes, providing abundant data for the analysis of rice embryo development. Their expression in the three developmental stages is summarized in Figure 2.

Table 2. Distribution of the gene sequences detected in rice developing embryo via RNA-Seq.

Figure 2. Venn diagram showing the genes expressed in each of the three stages of rice embryo development.

• Of 27,190 detected genes, 18,307 were categorized into 53 functional groups based on sequence homology. In each of the three main categories (biological process, molecular function, and cellular component) of the GO classification, there were 16, 17, and 20 functional groups, respectively (Figure 3).To identify the biological pathways that are active in rice embryos, the researchers mapped the detected genes to reference canonical pathways in the Kyoto Encyclopedia of Genes and Genomes (KEGG). These annotations provide a valuable resource for investigating specific processes, functions, and pathways during rice embryo development.

Figure 3. Histogram presentation of gene ontology (GO) classification.

• A total of 672 significantly changed genes were detected between the R1 (3 DAP) and R2 (7 DAP) rice embryo libraries, with 275 genes up-regulated and 397 genes down-regulated (Figure 4). Between the R2 (7 DAP) and R3 (14 DAP) rice embryo libraries, a total of 504 DEGs were detected, with 128 up-regulated genes and 376 down-regulated genes (Figure 4 and Table S3). This suggests that the differentiation of expressed genes between R1 (3 DAP) and R2 (7 DAP) is larger than that between R2 (7 DAP) and R3 (14 DAP).

Figure 4. Changes in gene expression profile among the different developmental stages.

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