IC4R007-RNA-Seq-2016-27216147

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Project Title

Comparative Leaf and Root Transcriptomic Analysis of two Rice Japonica Cultivars Reveals Major Differences in the Root Early Response to Osmotic Stress

The Background of This Project

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  • Drought is one of the most important environmental constraints affecting plant growth and development and ultimately leads to yield loss. Water deficiency is a global concern because even the most productive agricultural regions can occasionally experience short periods or years of severe drought. The development of drought-tolerant varieties still represents a challenging task, being hampered by the occurrence of genotype × environment interactions, the difficulty of effective drought tolerance screening and a still partial understanding of the molecular mechanisms of plant drought tolerance.
  • In this study, researchers donducted a parallel transcriptomic analysis on two Italian rice genotypes characterized by a contrasting phenotype in response to osmotic stress. RNA-Sequencing was performed separately on leaves and roots to characterize the specific response of these organs in the considered genotypes. The results of this study may contribute to elucidating the mechanisms involved in the rice response to osmotic stress and to identify genes that are putatively responsible for the stress-tolerant phenotype.

Plant Culture & Treatment

  • Seventeen Italian rice cultivars (O. sativa L. ssp japonica) were subjected to PEG-mediated osmotic stress, namely Arborio, Asia, Augusto, Baldo, Carnaroli, Eurosis, Gigante Vercelli, Gladio, Koral, Loto, Maratelli, Salvo, SISR215, Thaibonnet, Venere, Vialone Nano and Volano. The rice seeds were kindly provided by the Rice Research Unit (CREA-RIS, Vercelli, Italy).
  • After 20 days of growth (third-leaf stage), half of the plants were transferred to a nutrient solution with 20% (w/v) PEG6000 (Duchefa Biochemistry, Harlem, The Netherlands) at 11.00 am (3 h after the onset of illumination), to impose an osmotic stress treatment. For the gene expression analysis, the leaves from control and treated plants were collected after 3 (14.00 pm) and 24 h (11.00 am) of PEG treatment.

Illumina RNA-Sequencing

  • Four micrograms of total RNA was subjected to library preparation using the TruSeq mRNA Sample Prep Kit from Illumina (Illumina, Inc., CA, USA) following the manufacturer’s instructions. Library concentration and size were assayed on a 2100 Bioanalyzer (Agilent). Single-end sequencing (50 bases) was conducted on an Illumina HiSeq2000 with samples run in 6-plex. Illumina sequencing was performed at IGA Technology Services Srl Service Provider (Udine, Italy).
  • A preliminary read trimming (from 50 to 43 bases) step was performed to discard poor quality bases that were abundantly detected in the 3′ terminal region. The resulting trimmed reads were checked for contaminants, and low quality bases and contaminants were removed using the cutadapt software. TopHat version 1.4.1 was used to map reads to rice O. sativa Nipponbare genome (MSU release 6.16). The DESeq Bioconductor package version 1.10.1 was used to call DEGs under R release 2.15.2.
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Research Findings

  • Gene expression changes for 10 selected genes, that were identified as DEGs during the RNA-Seq experiments and were reported to be involved in the osmotic stress response, were validated using qRT-PCR. The comparison between RNA-Seq and qRT-PCR fold change data, which was obtained by a regression analysis, revealed a substantial agreement in the extent of the osmotic stress-induced variations in transcript accumulation for the 10 tested genes.
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  • The RNA-Seq analysis showed that osmotic stress caused significant changes in gene expression in the roots and leaves of both genotypes. In particular, 3 h of osmotic stress led to a substantial modulation of gene expression in the roots of the tolerant genotype Eurosis (6007 genes), whereas fewer DEGs were called in the roots of the susceptible genotype Loto (3962 genes). At 24 h of treatment, a similar number of DEGs was found in Eurosis and Loto roots (3065 and 3102 genes, respectively). In the leaves, a similar number of DEGs was found in the two genotypes, both at 3 and 24 h of osmotic stress treatment (2977 and 4223 in Eurosis, and 3088 and 4813 in Loto, respectively)

Labs working on this Project

  • Institute of Agricultural Biology and Biotechnology - National Research Council, via Bassini 15, 20133, Milan, Italy
  • Dipartimento di Scienze Agrarie e Ambientali - Produzione, Territorio, Agroenergia, Università degli Studi di Milano, Via Celoria 2, 20133, Milan, Italy
  • Consiglio per la ricerca in agricoltura e l'analisi dell'economia agraria, Genomics Research Centre, Fiorenzuola d'Arda, Piacenza, Italy