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Tag-based identification of differentially expressed genes under cold and freeze stress in seabuckthorn (Hippophae rhamnoides L.)

ABSTRACT: Purpose: Tag-based approach for global gene expression analysis has been revolutionised with the advent of next generation sequencing (NGS) technology. The aim of the present study is to present comprehensive view of differentially expressed genes under cold and freeze stress in seabuckthorn (Hippophae rhamnoides L.) Methods: DeepSAGE, a tag based approach, was used to identify differentially expressed genes under cold and freeze treatments in seabuckthorn (Hippophae rhamnoides L.). The 30 days old plantlets, at six leaves stage, were subjected to cold stress (CS) at 4°C and freeze stress (FS) at -10°C treatment for 6 hr. The seedlings grown at 28°C were taken as control (CON). Total RNA from all the three samples was isolated. Illumina Gene Expression Sample Prep Kit and Solexa Sequencing Chip (Flowcell) were used for tag preparation and the main instruments used for sequencing included Illumina Cluster Station and Illumina HiSeqTM 2000 System. Bioinformatics analysis resulted in to high number of differentially expressed genes under cold and freeze stress. Results: 36.2 million raw tags including 13.9 million distinct tags were generated from three leaf tissue libraries (control, cold stress and freeze stress). After removing low quality tags, 35.5 million clean tags including 7 million distinct clean tags were obtained. In total, 11922 differentially expressed genes (DEGs) were identified including 6539 up regulated and 5383 down regulated genes. Conclusions: DeepSAGE data of seabuckthorn provided useful resource and reference dataset for further functional genomics analysis in seabuckthorn and other important crops. The present study implicated a large number of genes with different biological functions expressing differentially in response to cold and freeze stress treatment. Isolation and further characterization of these genes will help researchers in understanding their role in cold and freeze tolerance in seabuckthorn and may provide important gene resources to be exploited for the development of stress tolerant crop plants in future.

ORGANISM(S): Hippophae rhamnoides

PROVIDER: GSE62489 | GEO | 2017/02/09

SECONDARY ACCESSION(S): PRJNA264283

REPOSITORIES: GEO

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Project description:Purpose: the technology of Solexa/Illumina (RNA-seq) is an attractive alternative to the transcriptome sequencing. The goals of this study are to investigate transcriptional changes in replanted R. glutinosa leaves and identify genes responding to the disease. Methods:Totally 6.01and 6.15 million raw tags were measured from leaves of the first year planted (L1) and the second year replanted R. glutinosa (L2) at the tuberous root expansion stage, respectively. mRNA profiles of leaves of the first year planted (L1) and the second year replanted R. glutinosa (L2) at the tuberous root expansion stage, were generated by deep sequencing (Illumina HiSeqâ¢ 2000 system). qRTâPCR validation was performed using SYBR Green assays. Results: we obtained a total of 6.01 and 6.15 million total tags including 377,200 and 361,659 distinct tags from the L1 and L2 samples, respectively. Removal of low quality tags, 161,298 and 149,290 distinct clean tags were remained.With reference to the 94,544 non-redundant consensus sequences defined by the RNA-seq transcriptomic procedure, 60,574 genes had the diagnostic CATG site, which were taken as reference distinct sequences for DGE analysis. 47,458 of the L1 tag library and 42,247 of the L2 tag library were perfectly matched the reference sequences. we screened differentially expressed genes in the L1 and L2 libraries by digital gene expression (DGE) technique. Finally, a set of 1,954 genes may be in differential expression of L2. By bioinformatics and qRT-PCR, the 117 most strongly differentially expressed ones were considered to be prime candidates for responsible for replanting disease. Conclusions: The study provides an important resource for further investigating the cause of replanting disease and developing the methods to control or subtract its injury. Leaf mRNA profiles of L1 (The first year planted R. glutinosa leaves ) and L2 (the second year replanted R. glutinosa leaves) root expansion stage were generated by deep sequencing (using Illumina HiSeqâ¢ 2000 system)

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Tag-based identification of differentially expressed genes under cold and freeze stress in seabuckthorn (Hippophae rhamnoides L.)

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