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Gene expression profiles in the peel and pericarp of pepper fruit

ABSTRACT: The quality of the pepper fruit is significantly influenced by the properties of its surface such as color, glossiness and texture. The fruit surface is composed of a peel containing several layers including the cuticle, epidermis and the hypodermis. The peel acts as a protective barrier against biotic and abiotic stresses and is the most critical tissue affecting water loss during post harvest storage. The peel is composed of an outer epidermis with thick waxy (lipid) cuticle and few cell layers of thick-walled hypodermal cells. Despite its agronomic importance and due to the fact that the majority of studies in fruits have been conducted using flesh and peel tissues as a whole, the biochemical and genetic bases of variation in peel properties are largely unknown. In this proposal we aim to determine peel-specific gene expression in pepper by micro array hybridizations of peel and flesh RNA extracted at different developmental stages of the fruit. The cultivar Celica (Capsicum annuum) that has a large blocky fruit will be used for studying gene expression in the peel and flesh. Plants were grown in the greenhouse during the spring of 2006. Fruits were harvested at three developmental stages: young- 10 days after anthesis, mature green- 30 days after anthesis and ripe red- 45 days after anthesis. These stages were chosen because each represents a distinct phase in fruit development. At each stage, a biological replicate consists of bulked tissue from 3 fruits from each of 3 plants (a total of 9 fruits). We have a total of 4 biological replicates. For each fruit, the peel was separated from the flesh by manual dissection using thin forceps and scalpel blade. Peel and flesh samples were immediately frozen in liquid nitrogen and stored at -800C until RNA extraction. Total RNA was extracted using the GenElute Mammalian Total RNA Miniprep kit (Sigma). Keywords: Reference design

ORGANISM(S): Capsicum annuum Solanum tuberosum

PROVIDER: GSE8282 | GEO | 2007/09/01

SECONDARY ACCESSION(S): PRJNA101241

REPOSITORIES: GEO

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Project description:Gene-to-gene coexpression analysis is a powerful approach to infer function of uncharacterized genes. To perform non-targeted coexpression analysis of tomato genes, we collected a developmental gene expression dataset using various tissues of tomato plant. Expression data are collected from 24 different tissue types including root, hypocotyl, cotyledon, leaf at different stages, and fruit tissues at 4 different ripening stages from 4 different Solanum lycopersicum cultivars. Fruits were separated to the flesh and the peel. These two tissue types indeed showed remarkably different gene expression profiles. We also collected data from 4 different ripening stages (mature green, yellow, orange, and red) to detail the changes during ripening. By using this gene expression dataset, we calculated pair-wise Pearsonâs correlation coefficients, and performed network-based coexpression analysis. The analysis generated a number of coexpression modules, some of which showed an enrichment of genes associated with specific functional categories. This result will be useful in inferring functions of uncharacterized tomato genes, and in prioritizing genes for further experimental analysis. We used Affymetrix GeneChip Tomato genome Arrays to detail the global gene expression change using 24 different tomato tissue types (67 hybridizations). We collected gene expression data from 24 different tomato tissue types using 67 hybridizations. Root, hypocotyl, cotyledon, and leaf were sampled from 3-week-old or 5-weekâold plant of Solanum lycopersicum cultivar Micro-Tom. Fruit tissues were sampled from S. lycopersicum cultivars Micro-Tom, Anthocyanin fruit (Aft, LA1996), Line27859, and Momotaro 8 (Takii, Japan). From Micro-Tom fruit, the peel and the flesh were separately sampled from 4 different ripening stages: mature green (MG, approximately 30 day after anthesis), yellow (Y, approximately 35 days after anthesis), orange (O, approximately 38-40 days after anthesis), and red (R, approximately 45-48 days after anthesis). From fruits of Aft and Line27859, the peel and the flesh were sampled at mature green (MG, approximately 40 days after anthesis) and red (R, approximately 50-55 days after anthesis) stages. From Momotaro 8, the peel and the flesh were sampled at red (R, 50- approximately 50-55 days after anthesis) stages. For each tissue type, 2-4 biological replicates were made in RNA preparation.

Project description:Background The set of all mRNA molecules present in a cell constitute the transcriptome. The transcriptome varies depending on cell type as well as in response to internal and external stimuli during development. Chili pepper is an economically and culturally important horticultural crop as well as a good model for the study of secondary metabolism during fruit development. Here we present a study of the changes that occur in the transcriptome of chili pepper fruit during development and ripening. Results RNA-Seq was used to obtain transcriptomes of whole Serrano-type chili pepper fruits (Capsicum annuum L.; 'Tampiqueno 74') collected at 10, 20, 40 and 60 days after anthesis (DAA). 15,550,468 Illumina MiSeq reads were assembled de novo into 34,066 chili genes. We classified the expression patterns of individual genes as well as genes grouped into Biological Process ontologies and Metabolic Pathway categories using statistical criteria. For the analyses of gene groups we added the weighted expression of individual genes. This method was effective in interpreting general patterns of expression changes and increased the statistical power of the analyses. Subsets of genes were expressed only at a single time point sampled (1,278, 1,596, 1,519 and 1,583 genes at 10, 20, 40 and 60 DAA, respectively). We also estimated the variation in diversity and specialization of the transcriptome during chili pepper development. Approximately 17% of genes exhibited a significant change of expression in at least one of the intervals sampled. In contrast, significant differences in approximately 63% of the Biological Processes and 80% of the Metabolic Pathways studied were detected in at least one interval. Confirming previous reports, genes related to capsaicinoid and ascorbic acid biosynthesis were significantly upregulated at 20 DAA while those related to carotenoid biosynthesis were highly expressed in the last period of fruit maturation (40-60 DAA). Our RNA-Seq data was validated by examining the expression of nine genes involved in carotenoid biosynthesis by qRT-PCR. Conclusions In general, more profound changes in the chili fruit transcriptome were observed in the intervals between 10 to 20 and 40 to 60 DAA. The last interval, between 40 to 60 DAA, included 49% of all significant changes detected, and was characterized predominantly by a global decrease in gene expression. This period signals the end of maturation and the beginning of senescence of chili pepper fruit. The transcriptome at 60 DAA was the most specialized and least diverse of the four states sampled. RNA-seq libraries of whole chili pepper (Capsicum annuum Serrano 'Tampiqueno 74' landrace) were prepared from fruits at 10, 20, 40 and 60 days after anthesis (DAA). Two biological replicates of each fruit state were prepared (8 libraries in total). Libraries were sequenced in the Illumina MiSeq platform. The title of the libraries are: <DAA>repbio<#> where <DAA> are the days after anthesis (10, 20, 40 or 60) and <#> is the number of biological replicate, 1 or 2. Details follow. Biological material and RNA extraction Capsicum annuum Serrano 'Tampiqueno 74' seeds were germinated and the seedlings were cultivated until maturity under greenhouse conditions in a completely randomized experimental design at Cinvestav-Unidad Irapuato (Guanajuato, Mexico). The plants were grown during the spring and summer, and individual flowers were tagged at anthesis. Chili pepper fruits were randomly collected from different plants at 10, 20, 40 and 60 DAA. After sampling, the fruits were cleaned with ethanol immediately frozen in liquid nitrogen and stored at -80C until further use. For total RNA extractions, 10 fruits at the 10 DAA developmental stage and 5 fruits from each of the 20, 40 and 60 DAA stages were randomly selected from the pool of all harvested fruits. Whole fruits (pericarp, placenta and seeds) were ground in liquid nitrogen with a mortar and pestle to form a fine uniform powder. Samples were mixed vigorously and 100 mg aliquots were measured for each RNA extraction. The process was repeated with different sets of fruits in order to obtain two independent biological replicates at each developmental stage. A NucleoSpin RNA Plant kit (Macherey-Nagel) was used for total RNA extraction and contaminating genomic DNA was removed by DNase I (Macherey-Nagel) treatment following the manufacturers' protocols. Total RNA concentration was quantified using a NanoDrop ND-1000 spectrophotometer (Nano-Drop, Wilmington, DE, USA) and RNA quality was evaluated by gel electrophoresis on 1.0% denaturing agarose gels. In addition, aliquots of RNA were run on an Agilent 2100 Bioanalyzer using RNA 6000 chips (Agilent, Santa Clara, CA, USA) to test the RNA integrity number (RIN). All eight samples had RIN values higher than 8.7. Thirty ?g of total RNA from each of the eight samples (two biological replicates of four fruit developmental stages) was used for cDNA library preparation. Library construction and sequencing The eight total RNA samples (two biological replicates from chili pepper fruits at 10, 20, 40 and 60 DAA) were prepared for RNA-Seq using the Illumina TruSeq RNA Sample Preparation v2 Guide following the manufacturer's instructions. Briefly, mRNA was purified from 20?g of total RNA using poly-T oligo- attached magnetic beads using two rounds of purification. During the second elution of the poly-A RNA, mRNA was fragmented using divalent cations under elevated temperature and primed for cDNA synthesis. The cleaved RNA fragments were primed with random hexamers and reverse transcribed into single-stranded cDNA using reverse transcriptase. In the next step, the RNA template was removed and the complementary cDNA strand was synthesized using RNAse H and DNA polymerase I, respectively. The overhangs that resulted from fragmentation were polished into blunt ends using an End Repair Mix (consisting of T4 DNA polymerase, Klenow fragment and T4 polynucleotide kinase). A single T nucleotide was added on the 3' end of the adapter for ligating the adapter to the cDNA fragments. Indexing adapters were ligated to the ends of the cDNAs using T4 DNA ligase, preparing them for hybridization onto a flow cell. Finally, the DNA fragments with adapter molecules at both ends were amplified by PCR to enrich the amount of DNA in the library. PCR was performed with a PCR primer cocktail that anneals to the ends of the adapters. Quantity and quality of the DNA libraries was assessed using Agilent DNA-1000 chips on an Agilent 2100 Bioanalyzer. The 8 cDNA libraries were pooled and simultaneously sequenced from both 5’ and 3’ ends using the Illumina MiSeq System platform according to the manufacturer's instructions. We performed three sequencing runs (technical replicates) with the aim of increase the sequencing depth. 150 bp paired-end reads were obtained in each sequencing run. Fluorescent image processing, base-calling and quality value calculations for each of the three runs were performed using Illumina MiSeq Control Software. Data were deposited at the NCBI (GEO database under series record GSE54123).

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Gene expression profiles in the peel and pericarp of pepper fruit

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