Dataset Information

Identification of two putative reference genes from grapevine suitable for gene expression analysis in berry and related tissues derived from RNA-Seq data.

ABSTRACT: BACKGROUND: Data normalization is a key step in gene expression analysis by qPCR. Endogenous control genes are used to estimate variations and experimental errors occurring during sample preparation and expression measurements. However, the transcription level of the most commonly used reference genes can vary considerably in samples obtained from different individuals, tissues, developmental stages and under variable physiological conditions, resulting in a misinterpretation of the performance of the target gene(s). This issue has been scarcely approached in woody species such as grapevine. RESULTS: A statistical criterion was applied to select a sub-set of 19 candidate reference genes from a total of 242 non-differentially expressed (NDE) genes derived from a RNA-Seq experiment comprising ca. 500 million reads obtained from 14 table-grape genotypes sampled at four phenological stages. From the 19 candidate reference genes, VvAIG1 (AvrRpt2-induced gene) and VvTCPB (T-complex 1 beta-like protein) were found to be the most stable ones after comparing the complete set of genotypes and phenological stages studied. This result was further validated by qPCR and geNorm analyses. CONCLUSIONS: Based on the evidence presented in this work, we propose to use the grapevine genes VvAIG1 or VvTCPB or both as a reference tool to normalize RNA expression in qPCR assays or other quantitative method intended to measure gene expression in berries and other tissues of this fruit crop, sampled at different developmental stages and physiological conditions.

SUBMITTER: Gonzalez-Aguero M

PROVIDER: S-EPMC3878734 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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Identification of two putative reference genes from grapevine suitable for gene expression analysis in berry and related tissues derived from RNA-Seq data.

González-Agüero Mauricio M García-Rojas Miguel M Di Genova Alex A Correa José J Maass Alejandro A Orellana Ariel A Hinrichsen Patricio P

BMC genomics 20131213

<h4>Background</h4>Data normalization is a key step in gene expression analysis by qPCR. Endogenous control genes are used to estimate variations and experimental errors occurring during sample preparation and expression measurements. However, the transcription level of the most commonly used reference genes can vary considerably in samples obtained from different individuals, tissues, developmental stages and under variable physiological conditions, resulting in a misinterpretation of the perfo ...[more]

PMID: 24330674

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Project description:BACKGROUND: Accuracy in quantitative real-time RT-PCR is dependent on high quality RNA, consistent cDNA synthesis, and validated stable reference genes for data normalization. Reference genes used for normalization impact the results generated from expression studies and, hence, should be evaluated prior to use across samples and treatments. Few statistically validated reference genes have been reported in grapevine. Moreover, success in isolating high quality RNA from grapevine tissues is typically limiting due to low pH, and high polyphenolic and polysaccharide contents. RESULTS: We describe optimization of an RNA isolation procedure that compensates for the low pH found in grape berries and improves the ability of the RNA to precipitate. This procedure was tested on pericarp and seed developmental series, as well as steady-state leaf, root, and flower tissues. Additionally, the expression stability of actin, AP47 (clathrin-associated protein), cyclophilin, EF1-alpha (elongation factor 1-alpha), GAPDH (glyceraldehyde 3-phosphate dehydrogenase), MDH (malate dehydrogenase), PP2A (protein phosphatase), SAND, TIP41, alpha-tubulin, beta-tubulin, UBC (ubiquitin conjugating enzyme), UBQ-L40 (ubiquitin L40) and UBQ10 (polyubiquitin) were evaluated on Vitis vinifera cv. Cabernet Sauvignon pericarp using three different statistical approaches. Although several of the genes proved to be relatively stable, no single gene outperformed all other genes in each of the three evaluation methods tested. Furthermore, the effect of using one reference gene versus normalizing to the geometric mean of several genes is presented for the expression of an aquaporin and a sucrose transporter over a developmental series. CONCLUSION: In order to quantify relative transcript abundances accurately using real-time RT-PCR, we recommend that combinations of several genes be used for normalization in grape berry development studies. Our data support GAPDH, actin, EF1-alpha and SAND as the most relevant reference genes for this purpose.

Project description:BACKGROUND:Dendrobium moniliforme (Linnaeus) Swartz is a well-known plant used in traditional Chinese medicine due to bioactive constituents. Polysaccharides are the main medicinal ingredients, yet no studies have been published on polysaccharide biosynthesis in D. moniliforme. To comprehensively investigate the polysaccharide at the transcription level, we performed de novo transcriptome sequencing for the first time to produce a comprehensive transcriptome of D. moniliforme. RESULTS:In our study, a database of 562,580 unigenes (average length = 1115.67 bases) was generated by performing transcriptome sequencing. Based on the gene annotation of the transcriptome, we identified 1204 carbohydrate-active related unigenes against CAZy database, including 417 glycosyltransferase genes (GTs), 780 glycoside hydrolases (GHs), 19 carbohydrate esterases (CEs), 75 carbohydrate-binding modules (CBMs), and 44 polysaccharide lyases (PLs). In the cellulose synthase family, 21 differential expression genes (DEGs) related to polysaccharide were identified. Subsequently, the tissue-specific expression patterns of the genes involved in polysaccharide pathway were investigated, which provide understanding of the biosynthesis and regulation of DMP at the molecular level. The two key enzyme genes (Susy and SPS) involved in the polysaccharide pathway were identified, and their expression patterns in different tissues were further analyzed using quantitative real-time PCR. CONCLUSIONS:We determined the content of polysaccharides from Dendrobium moniliforme under different tissues, and we obtained a large number of differential genes by transcriptome sequencing. This database provides a pool of candidate genes involved in biosynthesis of polysaccharides in D. moniliforme. Furthermore, the comprehensive analysis and characterization of the significant pathways are expected to give a better insight regarding the diversity of chemical composition, synthetic characteristics, and the regulatory mechanism which operate in this medical herb.

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Project description:We explored the effects of ultraviolet B radiation (UV-B) on the developmental dynamics of microRNAs and phased small-interfering-RNA (phasi-RNAs)-producing loci by sequencing small RNAs in vegetative and reproductive organs of grapevine (Vitis vinifera L.). In particular, we tested different UV-B conditions in in vitro-grown plantlets (high-fluence exposition) and in berries from field-grown (radiation filtering) and greenhouse-grown (low- and high-fluence expositions) adult plants throughout fruit development and ripening. The functional significance of the observed UV-coordinated miRNA responses was supported by degradome evidences of ARGONAUTE (AGO)-programmed slicing of mRNAs. Co-expression patterns of the up-regulated miRNAs miR156, miR482, miR530, and miR828 with cognate target gene expressions in response to high-fluence UV-B was tested by q-RT-PCR. The observed UV-response relationships were also interrogated against two published UV-stress and developmental transcriptome datasets. Together, the dynamics observed between miRNAs and targets suggest that changes in target abundance are mediated transcriptionally and, in some cases, modulated post-transcriptionally by miRNAs. Despite the major changes in target abundance are being controlled primarily by those developmental effects that are similar between treatments, we show evidence for novel miRNA-regulatory networks in grape. A model is proposed where high-fluence UV-B increases miR168 and miR530 that target ARGONAUTE 1 (AGO1) and a Plus-3 domain mRNA, respectively, while decreasing miR403 that targets AGO2, thereby coordinating post-transcriptional gene silencing activities by different AGOs. Up-regulation of miR3627/4376 could facilitate anthocyanin accumulation by antagonizing a calcium effector, whereas miR395 and miR399, induced by micronutrient deficiencies known to trigger anthocyanin accumulation, respond positively to UV-B radiation. Finally, increases in the abundance of an anthocyanin-regulatory MYB-bHLH-WD40 complex elucidated in Arabidopsis, mediated by UV-B-induced changes in miR156/miR535, could contribute to the observed up-regulation of miR828. In turn, miR828 would regulate the AtMYB113-ortologues MYBA5, A6 and A7 (and thereby anthocyanins) via a widely conserved and previously validated auto-regulatory loop involving miR828 and phasi TAS4abc RNAs.

Project description:BackgroundThe majority of nitrogen accumulating in cereal grains originates from proteins remobilised from vegetative organs. However, interactions between grain filling and remobilisation are poorly understood. We used transcriptome large-scale pyrosequencing of flag leaves, glumes and developing grains to identify cysteine peptidase and N transporter genes playing a role in remobilisation and accumulation of nitrogen in barley.ResultsCombination of already known and newly derived sequence information reduced redundancy, increased contig length and identified new members of cysteine peptidase and N transporter gene families. The dataset for N transporter genes was aligned with N transporter amino acid sequences of rice and Arabidopsis derived from Aramemnon database. 57 AAT, 45 NRT1/PTR and 22 OPT unigenes identified by this approach cluster to defined subgroups in the respective phylogenetic trees, among them 25 AAT, 8 NRT1/PTR and 5 OPT full-length sequences. Besides, 59 unigenes encoding cysteine peptidases were identified and subdivided into different families of the papain cysteine peptidase clade. Expression profiling of full-length AAT genes highlighted amino acid permeases as the group showing highest transcriptional activity. HvAAP2 and HvAAP6 are highly expressed in vegetative organs whereas HvAAP3 is grain-specific. Sequence similarities cluster HvAAP2 and the putative transporter HvAAP6 together with Arabidopsis transporters, which are involved in long-distance transfer of amino acids. HvAAP3 is closely related to AtAAP1 and AtAAP8 playing a role in supplying N to developing seeds. An important role in amino acid re-translocation can be considered for HvLHT1 and HvLHT2 which are specifically expressed in glumes and flag leaves, respectively. PCA and K-means clustering of AAT transcript data revealed coordinate developmental stages in flag leaves, glumes and grains. Phloem-specific metabolic compounds are proposed that might signal high grain demands for N to distantly located plant organs.ConclusionsThe approach identified cysteine peptidases and specific N transporters of the AAT family as obviously relevant for grain filling and thus, grain yield and quality in barley. Up to now, information is based only on transcript data. To make it relevant for application, the role of identified candidates in sink-source communication has to be analysed in more detail.

Dataset Information

Identification of two putative reference genes from grapevine suitable for gene expression analysis in berry and related tissues derived from RNA-Seq data.

Publications

Identification of two putative reference genes from grapevine suitable for gene expression analysis in berry and related tissues derived from RNA-Seq data.

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