IC4R010-RNA-Seq-2016-27029818

Project Title

Transcriptome analysis in different rice cultivars provides novel insights into desiccation and salinity stress responses.

The Background of This Project

• Drought and salinity are the major environmental factors that affect rice productivity. Comparative transcriptome analysis between tolerant and sensitive rice cultivars can provide insights into the regulatory mechanisms involved in these stress responses. In this study, researchers performed RNA-seq analysis to explore the transcriptional variations among three rice cultivars, including IR64 [stress-sensitive (SS)], Nagina 22[(N22), drought-tolerant (DT)] and Pokkali [salinity-tolerant (ST)] under control and stress conditions.

Table 1. Summary of read data, mapping and reference-based assembly obtained for each rice cultivars.

Plant Culture & Treatment

Figure 1. Categorization of all the transcripts into annotated, un-annotated, and novel transcripts (annotated and un-annotated) in IR64, N22 and Pokkali rice cultivars.

• The seeds of three rice (Oryza sativa L.) cultivars, IR64, N22 and Pokkali, were surface sterilized with 2% sodium hypochlorite for 45 min and grown on reverse osmosis (RO) water saturated cotton in a culture room (14 h light/10 h dark at temperature 28 ± 1 °C) for 14 days. The seedlings were subjected to control, desiccation and salinity stress treatments. For control treatment, seedlings were kept in RO water; for desiccation (water-deficit) treatment, seedlings were kept on filter paper 10 and for salinity treatment, seedlings were kept in 200 mM NaCl solution 73 for 3, 6 and 12 h time points. Three independent biological replicates for each tissue sample were harvested

Illumina RNA-Sequencing

• Total RNA was isolated from each tissue sample (control, desiccation and salinity from each time points) using TRI reagent (Sigma Life Science, St. Louis, MO) according to manufacturer’s instructions. Total RNA (~5 μg) (pooled in equal quantity from three biological replicates of all time points) for each genotype and condition was used for library preparation and sequencing. Libraries were prepared using Illumina TrueSeq RNA library method according to TrueSeq RNA Sample Preparation guide (Illumina Technologies, San Diego, CA) and paired-end sequencing was done using Illumina Genome Analyzer II (Illumina) to obtain reads of 100 bp length.

Figure 1. Distribution of various AS events in IR64, N22 and Pokkali rice cultivars is shown. IR-intron retention, AA-alternate-3′ -acceptor, AD- alternate-5′ -donor and ES-exon skipping.

• TopHat was used for mapping the HQ reads using the options–mate-inner-dist 350,–mate-std-dev 100, on the rice genome downloaded from RGAP (MSU version 7). Assembly was performed via Cufflinks using the TopHat mapping files with default parameters. The final assembly was obtained by merging the individual assemblies with default options using Cuffmerge.

Research Findings

• In total, the researchers obtained more than 180 million raw reads for each rice cultivar with at least 60 million reads for each condition (control, desiccation and salinity). These reads were filtered using NGS QC toolkit 28 to remove the low-quality reads. More than 84% of high quality (HQ) reads with average Phred quality score of ≥ 30 at each base position were obtained and used for downstream analyses.
• The researchers identified a total of 17444, 18424 and 16963 novel transcript isoforms in IR64, N22 and Pokkali rice cultivars, respectively. In addition, a total of 2499 (1832 loci), 3054 (2265 loci) and 2433 (1785 loci) novel transcripts in IR64, N22 and Pokkali, respectively, could also be identified.
• AS results in production of various transcript isoforms from a single gene and enhances the transcriptional complexity and diversity. Our analysis revealed that intron retention (IR) is the most dominant AS event represented by 9016, 9962 and 8852 events in IR64, N22 and Pokkali cultivars, respectively. Other major AS events were identified as alternate 3′ acceptor (AA) (7164 in IR64, 7511 in N22 and 7238 in Pokkali), alternate 5′ donor (AD) (3788 in IR64, 3992 in N22 and 3769 in Pokkali) and exon skipping (ES) (2941 in IR64, 2953 in N22 and 2918 in Pokkali).
• The analysis revealed a total of 3119 and 756 significantly differentially expressed transcripts in IR64 cultivar under desiccation and salinity stress, respectively. Similarly, a total of 2510 and 1809 transcripts in N22, and 3132 and 1036 transcripts in Pokkali were differentially expressed under desiccation and salinity stresses, respectively. In addition, we identified a total of 859 and 587 differentially expressed transcripts in N22 and Pokkali, respectively, as compared to IR64 under control condition.

Labs working on this Project

• Functional and Applied Genomics Laboratory, National Institute of Plant Genome Research (NIPGR), Aruna Asaf Ali Marg, New Delhi - 110067, India
• School of Computational and Integrative Sciences, Jawaharlal Nehru University, New Delhi-110067, India. Correspondence and requests for materials should be addressed to M.J.