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High-throughput Illumina Hiseq2000 seuqencing of small RNAs from Hevea brasiliensis PB260 and PB217 clones

ABSTRACT: MicroRNAs (miRNAs) are non-coding, short, single-stranded RNAs with essential roles in gene regulation in various organisms including higher plants. In contrast to the vast information on miRNAs from many economically important plants, almost nothing has been reported on the identification or analysis of miRNAs from rubber tree (Hevea brasiliensis L.), the most important natural rubber-producing crop. To identify miRNAs and their target genes in rubber tree, high throughput sequencing combined with a computational approach was performed. Four small RNA libraries were constructed for deep sequencing from mature and young leaves of two rubber tree clones, PB 260 and PB 217, which provide high and low latex yield, respectively. 237 miRNAs belonging to 37 known miRNA families were identified, and northern hybridization validated miRNA expression and revealed developmental stage-dependent and clone-specific expression for some miRNAs. We took advantage of the newly released rubber tree genome assembly as well as the genomic databases from leafy spurge and cassava, two species related to rubber tree, and predicted 15 novel miRNAs.

ORGANISM(S): Hevea brasiliensis

PROVIDER: GSE35254 | GEO | 2012/04/03

SECONDARY ACCESSION(S): PRJNA152819

REPOSITORIES: GEO

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Project description:Purpose: de novo sequencing and comparative analysis of the bark transciptomes of Hevea brasiliensis induced without ethephon (C), with ethephon for 8 hours (E8) and 24 hours (E24) to identify the genes and pathways related to the stimulation of rubber production by ethylene. The goals of this study are to reveal the molecular mechanism behind the stimulation of rubber production by ethylene. Methods: Bark RNA was extracted using the TRIzol® Reagent (Invitrogen) and two cDNA libraries, H (healthy rubber trees) and T (TPD-affected trees), were prepared using the mRNA-Seq 8 sample prep Kit (Illumina). The libraries were deep sequenced using Illumina HiSeqTM 2000 (Illumina Inc., San Diego, CA, USA). Raw reads produced from sequencing machines were resorted to de novo assembly and gene annotation. Results: De novo sequencing and assembly of the bark transciptomes of Hevea brasiliensis induced with ethephon for 8 hours (E8) and 24 hours (E24) were performed. 51,965,770, 52,303,714 and 53,177,976 high-quality clean reads from E8, E24 and C (control) samples were assembled into 81,335, 80,048 and 80,800 unigenes respectively, with a total of 84,425 unigenes and an average length of 1,101 bp generated. 10,216 and 9,374 differentially expressed genes (DEGs) in E8 and E24 compared with C were respectively detected. The expression of several enzymes in crucial points of regulation in glycolysis were up-regulated and DEGs were not significantly enriched in isopentenyl diphosphate (IPP) biosynthesis pathway. In addition, up-regulated genes of great regulatory importance in carbon fixation (Calvin cycle) were identified. Conclusions: The rapid acceleration of glycolytic pathway supplying precursors for the biosynthesis of IPP and natural rubber, instead of rubber biosynthesis per se, may be responsible for ethylene stimulation of latex yield in rubber tree. The elevated rate of flux throughout the Calvin cycle may account for some durability of ethylene-induced stimulation. Our finding lays the foundations for molecular diagnostic and genetic engineering for high-yielding improvement of rubber tree.

Project description:Purpose: de novo sequencing and comparative analysis of the bark transciptomes of Hevea brasiliensis induced without ethephon (C), with ethephon for 8 hours (E8) and 24 hours (E24) to identify the genes and pathways related to the stimulation of rubber production by ethylene. The goals of this study are to reveal the molecular mechanism behind the stimulation of rubber production by ethylene. Methods: Bark RNA was extracted using the TRIzolÂ® Reagent (Invitrogen) and two cDNA libraries, H (healthy rubber trees) and T (TPD-affected trees), were prepared using the mRNA-Seq 8 sample prep Kit (Illumina). The libraries were deep sequenced using Illumina HiSeqTM 2000 (Illumina Inc., San Diego, CA, USA). Raw reads produced from sequencing machines were resorted to de novo assembly and gene annotation. Results: De novo sequencing and assembly of the bark transciptomes of Hevea brasiliensis induced with ethephon for 8 hours (E8) and 24 hours (E24) were performed. 51,965,770, 52,303,714 and 53,177,976 high-quality clean reads from E8, E24 and C (control) samples were assembled into 81,335, 80,048 and 80,800 unigenes respectively, with a total of 84,425 unigenes and an average length of 1,101 bp generated. 10,216 and 9,374 differentially expressed genes (DEGs) in E8 and E24 compared with C were respectively detected. The expression of several enzymes in crucial points of regulation in glycolysis were up-regulated and DEGs were not significantly enriched in isopentenyl diphosphate (IPP) biosynthesis pathway. In addition, up-regulated genes of great regulatory importance in carbon fixation (Calvin cycle) were identified. Conclusions: The rapid acceleration of glycolytic pathway supplying precursors for the biosynthesis of IPP and natural rubber, instead of rubber biosynthesis per se, may be responsible for ethylene stimulation of latex yield in rubber tree. The elevated rate of flux throughout the Calvin cycle may account for some durability of ethylene-induced stimulation. Our finding lays the foundations for molecular diagnostic and genetic engineering for high-yielding improvement of rubber tree. De novo sequencing of the transcriptomes of C (bark without ethephon application), E8 (bark with 1.5%-ethephon treatment for 8 hours) and E24 (bark with 1.5%-ethephon treatment for 24 hours) rubber trees was conducted using Illumina HiSeq 2000.

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High-throughput Illumina Hiseq2000 seuqencing of small RNAs from Hevea brasiliensis PB260 and PB217 clones

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