Dataset Information

Comprehensive transcriptome analysis of reference genes for fruit development of Euscaphis konishii.

ABSTRACT: Background:Quantitativereal-time reverse transcriptase polymerase chain reaction is the common method to quantify relative gene expression. Normalizating using reliable genes is critical in correctly interpreting expression data from qRT-PCR. Euscaphis konishii is a medicinal plant with a long history in China, which has various chemical compounds in fruit. However, there is no report describing the selection of reference genes in fruit development of Euscaphis konishii. Methods:We selected eight candidate reference genes based on RNA-seq database analysis, and ranked expression stability using statistical algorithms GeNorm, NormFinder, BestKeeper and ReFinder. Finally, The nine genes related to the anthocyanin synthesis pathway of Euscaphis konishii were used to verify the suitability of reference gene. Results:The results showed that the stability of EkUBC23, EkCYP38 and EkGAPDH2 was better, and the low expression reference genes (EkUBC23 and EkCYP38) were favourable for quantifying low expression target genes, while the high expression reference gene (EkGAPDH2) was beneficial for quantifying high expression genes. In this study, we present the suitable reference genes for fruit development of Euscaphis konishii based on transcriptome data, our study will contribute to further studies in molecular biology and gene function on Euscaphis konishii and other closely related species.

SUBMITTER: Yang CL

PROVIDER: S-EPMC7020815 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Comprehensive transcriptome analysis of reference genes for fruit development of <i>Euscaphis konishii</i>.

Yang Cheng-Long CL Yuan Xue-Yan XY Zhang Jie J Sun Wei-Hong WH Liu Zhong-Jian ZJ Zou Shuang-Quan SQ

PeerJ 20200211

<h4>Background</h4>Quantitativereal-time reverse transcriptase polymerase chain reaction is the common method to quantify relative gene expression. Normalizating using reliable genes is critical in correctly interpreting expression data from qRT-PCR. <i>Euscaphis konishii</i> is a medicinal plant with a long history in China, which has various chemical compounds in fruit. However, there is no report describing the selection of reference genes in fruit development of <i>Euscaphis konishii</i>.<h4 ...[more]

PMID: 32095336

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Project description:BachgroundEuscaphis konishii Hayata, a member of the Staphyleaceae Family, is a plant that has been widely used in Traditional Chinese Medicine and it has been the source for several types of flavonoids. To identify candidate genes involved in flavonoid biosynthesis and accumulation, we analyzed transcriptome data from three E. konishii tissues (leaf, branch and capsule) using Illumina Hiseq 2000 platform.ResultsA total of 91.7, 100.3 and 100.1million clean reads were acquired for the leaf, branch and capsule, respectively; and 85,342 unigenes with a mean length of 893.60?bp and N50 length of 1307?nt were assembled using Trinity program. BLASTx analysis allowed to annotate 40,218 unigenes using public protein databases, including NR, KOG/COG/eggNOG, Swiss-Prot, KEGG and GO. A total of 14,291 (16.75%) unigenes were assigned to 128 KEGG pathways, and 900 unigenes were annotated into 22 KEGG secondary metabolites, including flavonoid biosynthesis. The structure enzymes involved in flavonoid biosynthesis, such as phenylalanine ammonia lyase, cinnamate 4-hydroxylase, 4-coumarate CoA ligase, shikimate O-hydroxycinnamoyltransferase, coumaroylquinate 3'-monooxygenase, caffeoyl-CoA O-methyltransferase, chalcone synthase, chalcone isomerase, flavanone 3-hydroxylase, flavonoid 3'-hydroxylase, flavonoid 3',5'-hydroxylase, flavonolsynthese, dihydroflavonol 4-reductase, anthocyanidinreductase, leucoanthocyanidin dioxygenase, leucoanthocyanidin reductase, were identified in the transcriptome data, 40 UDP-glycosyltransferase (UGT), 122 Cytochrome P450 (CYP) and 25 O-methyltransferase (OMT) unigenes were also found. A total of 295 unigenes involved in flavonoid transport and 220 transcription factors (97 MYB, 84 bHLH and 39 WD40) were identified. Furthermore, their expression patterns among different tissues were analyzed by DESeq, the differentially expressed genes may play important roles in tissues-specific synthesis, accumulation and modification of flavonoids.ConclusionWe present here the de novo transcriptome analysis of E. konishii and the identification of candidate genes involved in biosynthesis and accumulation of flavonoid. In general, these results are an important resource for further research on gene expression, genomic and functional genomics in E. konishii and other related species.

Project description:Due to its high sensitivity and reproducibility, quantitative real-time PCR (qPCR) is practiced as a useful research tool for targeted gene expression analysis. For qPCR operations, the normalization with suitable reference genes (RGs) is a crucial step that eventually determines the reliability of the obtained results. Although pepper is considered an ideal model plant for the study of non-climacteric fruit development, at present no specific RG have been developed or validated for the qPCR analyses of pepper fruit. Therefore, this study aimed to identify stably expressed genes for their potential use as RGs in pepper fruit studies. Initially, a total of 35 putative RGs were selected by mining the pepper transcriptome data sets derived from the PGP (Pepper Genome Platform) and PGD (Pepper Genome Database). Their expression stabilities were further measured in a set of pepper (Capsicum annuum L. var. 007e) fruit samples, which represented four different fruit developmental stages (IM: Immature; MG: Mature green; B: Break; MR: Mature red) using the qPCR analysis. Then, based on the qPCR results, three different statistical algorithms, namely geNorm, Normfinder, and boxplot, were chosen to evaluate the expression stabilities of these putative RGs. It should be noted that nine genes were proven to be qualified as RGs during pepper fruit development, namely CaREV05 (CA00g79660); CaREV08 (CA06g02180); CaREV09 (CA06g05650); CaREV16 (Capana12g002666); CaREV21 (Capana10g001439); CaREV23 (Capana05g000680); CaREV26 (Capana01g002973); CaREV27 (Capana11g000123); CaREV31 (Capana04g002411); and CaREV33 (Capana08g001826). Further analysis based on geNorm suggested that the application of the two most stably expressed genes (CaREV05 and CaREV08) would provide optimal transcript normalization in the qPCR experiments. Therefore, a new and comprehensive strategy for the identification of optimal RGs was developed. This strategy allowed for the effective normalization of the qPCR analysis of the pepper fruit development at the whole pepper genome level. This study not only explored the optimal RGs specific for studying pepper fruit development, but also introduced a referable strategy of RG mining which could potentially be implicated in other plant species.

Dataset Information

Comprehensive transcriptome analysis of reference genes for fruit development of Euscaphis konishii.

Publications

Comprehensive transcriptome analysis of reference genes for fruit development of <i>Euscaphis konishii</i>.

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