Identification of miRNAs and their target genes in developing seeds of soybean by deep sequencing
ABSTRACT: In our study, small RNA library and degradome library were constructed from developing soybean seeds for deep sequencing. We identified 26 new miRNAs in soybean by bioinformatic analysis, and further confirmed their expression by stem-loop RT-PCR. The miRNA star sequences of 38 known miRNAs and 8 new miRNAs were also discovered, providing additional evidence for the existence of miRNAs. Through degradome sequencing, 145 and 25 genes were identified as targets of annotated miRNAs and new miRNAs, respectively. Many identified miRNA targets may perform functions in soybean seed development by GO analysis. Additionally, soybean homolog of Arabidopsis SUPPRESSOR OF GENE SLIENCING 3(AtSGS3) was detected as target of the new identified miRNA Soy_25, suggesting presence of feedback control of miRNA biogenesis Overall design: sample 1: Examination of small RNA in soybean seed sample 2: identification of miRNA targets in soybean seed
INSTRUMENT(S): Illumina Genome Analyzer II (Glycine max)
Project description:In our study, small RNA library and degradome library were constructed from developing soybean seeds for deep sequencing. We identified 26 new miRNAs in soybean by bioinformatic analysis, and further confirmed their expression by stem-loop RT-PCR. The miRNA star sequences of 38 known miRNAs and 8 new miRNAs were also discovered, providing additional evidence for the existence of miRNAs. Through degradome sequencing, 145 and 25 genes were identified as targets of annotated miRNAs and new miRNAs, respectively. Many identified miRNA targets may perform functions in soybean seed development by GO analysis. Additionally, soybean homolog of Arabidopsis SUPPRESSOR OF GENE SLIENCING 3(AtSGS3) was detected as target of the new identified miRNA Soy_25, suggesting presence of feedback control of miRNA biogenesis sample 1: Examination of small RNA in soybean seed sample 2: identification of miRNA targets in soybean seed
Project description:We deep sequenced a degradome library constructed from different soybean tissues. As a result, 19,830,257 represented 5,337,590 distinct signatures were obtained. 70.98% of the signatures were assigned to one soybean cDNA sequence and 24.05% matched with two cDNA sequences. 428 potential targets of small RNAs and 25 novel miRNA families were identified in soybean. A total of 211 potential miRNA targets including 150 conserved miRNA targets and 69 soybean-specific miRNA targets were identified. The signatures distribution on soybean primary miRNAs (pri-miRNAs) showed that most of the pri-miRNAs had the characteristic pattern of Dicer processing. The TAS3 small RNAs (siRNAs) biogenesis was conserved in soybean and nine Auxin Response Factors (ARFs) were identified as the TAS3 siRNA targets. The global identification of miRNAs targets would contribute to the functional research of the miRNA in soybean. one sample, We deep sequenced a degradome library constructed from different soybean tissues.
Project description:Five degradome libraries were constructed from three different seed developmental stages. Separate degradome libraries were constructed for seed coat and cotyledons to identify the tissue specific miRNAs and their potential targets. Sequencing and analysis of degradome libraries gives identification of 183 different targets for 80 known soybean miRNAs. We found 30 cotyledon specific, 18 seed coat specific and 32 miRNAs found in both tissues irrespective of the developmental stages. One interesting observation is that we found more miRNA targets in late seed developmental stages than earlier stages. Additionally, we have validated four different auxin response factor genes as targets for gma-miR160 via RNA ligase mediated 5′ rapid amplification of cDNA ends (RLM-5′RACE). GO analysis indicated the enrichment of miRNA target genes in seed development. Construction of degradome libraries using cotyledons and seed coats from 3 different developmental stages
Project description:MicroRNAs (miRNAs) are important post-transcriptional regulators of plant development. In soybean (Glycine max), an important edible oil crop, valuable lipids are synthesized and stored in the cotyledons during embryogenesis .This storage lipids are used as energy source of the emerging seeds, during the germination procces. Until now, there are no microRNAs related to lipid metabolism in soybean or any other plant. This work aims to describe the miRNAome of germinating seeds of B. napus by identifying plant-conserved and novel miRNAs and comparing miRNA abundance in mature versus germinating seeds. A total of 183 familes were detected through a computational analysis of a large number of reads obtained from deep sequencing from two small RNA libraries of (i) pooled germintaing seeds stages and (ii) mature soybean seeds. We have found 39 new mirna precursors which produce 41 new mature forms. The present work also have identified isomiRNAs and mirnas offset (moRNAs). This work presents a comprehensive study of the miRNA transcriptome of soybean germinating seeds and will provide a basis for future research on more targeted studies of individual miRNAs and their functions in lipid consumption in development soybean seeds. MicroRNA profiles in 2 different seed libraries (mature seeds and a pool of germinating seed stages) of Glycine max by deep sequencing (Illumina GAII).
Project description:In this study, two small RNA libraries and two degradome libraries were constructed from roots of Al-treated and Al-free Glycine soja seedlings. For miRNA, a total of 7,287,655 and 7,035,914 clean reads in Al-treated and Al-free small RNAs libraries were generated, and 105 known miRNAs ,51 p3/p5 strands of known miRNA and 80 novel miRNAs were identified. Among them, expression of 34 miRNAs was responsive to Al stress. Through degradome sequencing, 82 and 11 genes were identified as tagerts of known and novel miRNAs obtained from this study, respectively. Gene Ontology (GO) annotations of target transcripts indicated that 52 out of 66 targets cleaved by conserved miRNA families may play role in regulation of transcription. sample 1: Examination of small RNA in Al-free wild soybean roots; sampple 2: Examination of small RNA in Al-treated wild soybena roots; sample 3: identification of miRNA targets in Al-free wild soybean roots; sample 4: identification of miRNA targerts in Al-treated wild soybean roots
Project description:To identity the targets of miRNAs, we bundled 12 samples from different developing satages into four mixture samples. These samples were used to cosntruct degradome libraries and preform degradome sequencing on Illumina Hi-seq 2000 analyzer. More than 44.98 millions clean reads were obtained and 33.52 million reads were mapped to the soybean cDNA. The mapped reads were used to identity miRNA targets by CleaveLand4 pipeline. 4 degradome mixed samples, no replicates, but every degradome data consists of two parts data. Please note that every degradome sample has two processed and two raw data files. To have enough data, additional sequencing was performed from each sample library. And each sample raw data was processed separately (tissue_name*degradome.txt) and also combined (all_degradome*.txt).
Project description:Chilling stress is a major factor limiting the yield and quality of vegetable soybean (Glycine max L.) on a global scale. Systematic identification and function analysis of miRNA under chilling stress could be helpful to clarify the molecular mechanism of chilling resistance. In the present study, two independent small RNA libraries from leaves of vegetable soybean were constructed, and sequenced with the high-throughput Illumina Solexa system. A total of 434 known miRNAs and three novel miRNAs were identified. Moreover, the expression patterns of these miRNAs have been verified by qRT-PCR analysis. Furthermore, we identified their gene targets by high-throughput degradome sequencing and validated using 5'-RACE. A total of 898 transcripts were targeted by 54 miRNA families attributed to five categories. More importantly, we identified 55 miRNAs that differentially expressed between chilling stress and the control. The targets of these miRNAs were enriched in oxidation-reduction, signal transduction, and metabolic process functional categories. The qRT-PCR confirmed that there was a negative relationship among the miRNAs and their targets under chilling stress. Our work provides comprehensive molecular evidence for the possible involvement of miRNAs in the process of chilling-stress responses in vegetable soybean. Overall design: 24h chilling treatment and control were collected to extract total RNA to do small RNA sequencing and pooled to do degradome sequencing.
Project description:MicroRNAs or miRNAs are endogenously encoded small RNAs that play a key role in diverse plant biological processes. Jatropha curcas L. has received much attention as a potential oilseed crop for the production of renewable oil. Here, a sRNA library from mature seeds and three mRNA libraries, from three different stages of seed development, were generated by deep sequencing in order to identify and characterize miRNAs and pre-miRNAs of J. curcas. Computation analysis allowed the identification of 180 conserved miRNAs and 41 precursors (pre-miRNAs), as well as 16 novel pre-miRNAs. The predicted miRNA target genes are involved in a broad range of physiological functions, including cellular structure, nuclear function, translation, transport, hormone synthesis, defense and also lipid metabolism. Some of pre-miRNA and miRNA targets have different abundance among the three stages of seed development. A search for sequences that produce siRNA was performed indicating that J. curcas siRNAs play a role in nuclear functions, transport, catalytic process and diseases resistance. This study presents the first large scale identification of J. curcas miRNAs and their targets in mature seeds based on deep sequencing and contributes to understand the function of these miRNAs. microRNA identification in a mature seed library of Jatropha curcas by deep sequencing (Illumina HiSeq2000).
Project description:To identify more targets in soybean, particularly specific targets of Cd-stress-responsive miRNAs, high-throughput degradome sequencing was used. In total, we obtained 8913111 raw reads from the library which was constructed from a mixture of four samples (HX3-CK, HX3-Cd-treatment, ZH24-CK and ZH24-Cd-treatment). After removing the reads without the CAGAG adaptor, 5430126 unique raw-reads were obtained. The unique sequences were aligned to the G. max genome database, and 6516276 reads were mapped to the genome. The mapped reads from the libraries represented 51481 annotated G. max genes. Identification of miRNA targerts in soybean roots
Project description:MicroRNAs play critical roles in various biological and metabolic processes. The function of miRNAs has been widely studied in model plants such as Arabidopsis and rice. However, the number of identified miRNAs and related miRNA targets in peach (Prunus persica) is limited. To understand further the relationship between miRNAs and their target genes during tissue development in peach, a small RNA library and three degradome libraries were constructed from three tissues for deep sequencing. We identified 117 conserved miRNAs and 186 novel miRNA candidates in peach by deep sequencing and 19 conserved miRNAs and 13 novel miRNAs were further evaluated for their expression by RT-qPCR. The number of gene targets that were identified for 26 conserved miRNA families and 38 novel miRNA candidates, were 172 and 87, respectively. Some of the identified miRNA targets were abundantly represented as conserved miRNA targets in plant. However, some of them were first identified and showed important roles in peach development. Our study provides information concerning the regulatory network of miRNAs in peach and advances our understanding of miRNA functions during tissue development. To identify more conserved and peach-speciﬁc miRNAs and their target genes and to understand further the mechanism of miRNA-regulated target genes during tissue development in peach, a small RNA library and three degradome libraries were constructed from three different tissues for deep sequencing.