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Identification of early fruit development reference genes in plum.

ABSTRACT: An RNAseq study of early fruit development and stone development in plum, Prunus domestica, was mined to identify sets of genes that could be used to normalize expression studies in early fruit development. The expression values of genes previously identified from Prunus as reference genes were first extracted and found to vary considerably in endocarp tissue relative to whole fruit tissue. Nine other genes were chosen that varied less than 2-fold amongst the 20 RNAseq libraries of early fruit development and endocarp tissues. These gene were tested on a series of developmental plum fruit samples to determine if any could be used as a reference gene in the analyses of fruit-based tissues in plum. The three most stable genes as determined using RefFinder were IPGD (imidazole glycerol-phosphate dehydratase), HAM1 (histone acetyltransferase) and SNX1 (sorting nexin 1). These were further tested to analyze genes expressed differentially in endocarp tissue between normal and minimal endocarp cultivars. To determine the universality of those nine genes as fruit development reference genes, three other data sets of RNAseq from peach and apple were analyzed to determine the reference gene expression. Multiple genes exhibited tissue specific patterns of expression while one gene, the SNX1, emerged as possessing a universal pattern between the Rosaceae species, at all developmental stages, and tissue types tested. The results suggest that the use of existing RNAseq data to identify standard genes can provide stable reference genes for a specific tissues or experimental conditions under exploration.

SUBMITTER: Galimba K

PROVIDER: S-EPMC7164607 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Identification of early fruit development reference genes in plum.

Galimba Kelsey K Tosetti Roberta R Loerich Karen K Michael Leann L Pabhakar Savita S Dove Cynthia C Dardick Chris C Callahan Ann A

PloS one 20200417 4

An RNAseq study of early fruit development and stone development in plum, Prunus domestica, was mined to identify sets of genes that could be used to normalize expression studies in early fruit development. The expression values of genes previously identified from Prunus as reference genes were first extracted and found to vary considerably in endocarp tissue relative to whole fruit tissue. Nine other genes were chosen that varied less than 2-fold amongst the 20 RNAseq libraries of early fruit d ...[more]

PMID: 32302301

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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

Identification of early fruit development reference genes in plum.

Publications

Identification of early fruit development reference genes in plum.

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