Project description:In the present study we have generated a parallel analysis of the 5' RNA ends (PARE) referred to polyadenylated RNAs in somatic embryos from in vitro culture of immature anthers of Vitis vinifera. PARE analysis include also a shortRNA analysis of same material. We have submitted the sRNAseq to mirPROOF and miRCAT (srna_workbench software at http://srna-workbench.cmp.uea.ac.uk) in order to recognize known and putative novel miRNAs, respectively that could be present in somatic embryos. In addition, aRNAs and PARE libraries were integrated in order to identify those 5' RNA ends that could be explained by sRNAs (i.e. identify transcripts that could be cleaved by sRNAs). We have used publicly available annotations and we gained a deep insight into the gene families and the transcriptional regulation mediated by miRNAs in V. vinifera somatic embryos. Gene expression is finely regulated to specific paralogues in gene families such as the NADPH-dependent diflavin oxidoreductase, Phosphoserine aminotransferase, Ethylene-responsive transcription factors, glutathione S-transferase par C, just to name a few. We have indeed characterized at least 4,000 mRNA targets. Size fractionated small RNA from total RNA extracts of somatic Embryos cv. "Brachetto" were ligated to adapters, purified again and reverse transcribed. After PCR amplification the sample was subjected to Illumina high throughput pyrosequencing. The kit used is TrueSeq Small RNA kit, Illumina. Please see www.illumina.com for details of the sequencing technology. PARE libraries were generated starting from Poly(A) fraction. The protocol used have been previously described in German et al., 2009, Nature protocols. Please see www.illumina.com for details of the sequencing technology.
Project description:The objective of the study was to uncover the developmental dynamics in the artificially induced somatic embryogenesis of Vitis vinifera on the transcriptome level.
Project description:gnp07_regeneome_embryogenesis - embryogenesis col0 - Identify genes involved in somatic embryogenesis - compare embryogenic areas of a callus with undifferenciate area in the same callus
Project description:gnp07_regeneome_embryogenesis - embryogenesis ws - Identify genes involved in somatic embryogenesis - To compare embryogenic areas of a callus with undifferenciate area in the same callus
Project description:gnp07_regeneome_embryogenesis - embryogenesis col0 - Identify genes involved in somatic embryogenesis - compare embryogenic areas of a callus with undifferenciate area in the same callus 4 dye-swap - tissue comparison
Project description:gnp07_regeneome_embryogenesis - embryogenesis ws - Identify genes involved in somatic embryogenesis - To compare embryogenic areas of a callus with undifferenciate area in the same callus 3 dye-swap - tissue comparison
Project description:gnp07_regeneome_embryogenesis - embryogenesis ws - Identify genes involved in somatic embryogenesis - To compare embryogenic areas of a callus with undifferenciate area in the same callus 3 dye-swap - tissue comparison
Project description:To uncover the involvement of miRNAs and siRNAs in somatic embryogenesis of the perennial woody crop citrus, we carried out high-throughput (Illumina) sequencing (HTS) of sRNAs and RNA degradome tags in non-embryogenic and embryogenic tissue of Valencia sweet orange. A total of 191 stem-loop structures were identified, emanating 50 known and 45 novel miRNAs, 130 miniature inverted-repeat transposable elements (MITEs) derived small interfering RNAs (siRNAs) and 94 other siRNAs. Combining with the result of degradome sequencing, a total of 235 phased siRNAs (phasiRNAs), and 195 cleaved target genes were identified for miRNAs/siRNAs.
Project description:Identification of differentially expressed genes from RNA-seq data of non-embryogenic and embryogenic ortets. Selected ortets were previously cloned, thereby somatic embryogenesis rates are known for these ortets. Ortets fitting the study criteria were supplied by two agencies, namely L1 and L2. Principal Component Analysis indicated that variance between agencies were higher than the variance between embryogenesis groups within the agency. Therefore, differential analysis was conducted separately for each agency. Differential expression analysis using DESeq2 package suggested the L2 transcriptomes of zero and low embryogenesis groups were more similar compared to the high embryogenesis group. The L1 transcriptomes consisting of zero and low embryogenesis groups similarly showed overlapping clusters. Differential expression analysis was conducted on the L1 samples (low vs. zero embryogenesis) using DESeq2 R package and the identified differentially expressed genes (DEGs) was used for clustering analysis of the L2 samples. The clustering profiles suggested that expression of these DEGs in L2 samples were able to differentiate high embryogenesis from zero-low embryogenesis L2 groups.
Project description:There are several issues complicating somatic embryogenesis in conifers, for instance the low initiation frequency, the weak maturation capacity, and the loss of an embryogenic potential after a prolonged cultivation. The aim of our study was to clarify molecular details of the this process in the tree Pinus nigra Arn. We performed comparative proteomic analysis based on the two-dimensional gel electrophoresis in 2 genotypes coded 362 and 366 of: 1) proliferating embryogenic tissues (E) initiated from immature zygotic embryos, 2) non-embryogenic calli (NEC) started from cotyledons of somatic seedlings, 3) embryogenic tissues that lost the maturation capacity (E-L). Pine tissues showed distinct morphologic features: a) E362 and E366 were characterized by the presence of early bipolar structures capable of maturation and plantlet regeneration; b) NEC362 and NEC366 were composed of round shaped cells without any organisation; c) for E-L362 and E-L366 the long-term culture of initially embryogenic tissues resulted in the disorganisation of early bipolar structures. We found 24 differentially accumulated protein spots common for both genotypes after comparison of E versus NEC.