Dataset Information

Genome-wide identification of ubiquitin proteasome subunits as superior reference genes for transcript normalization during receptacle development in strawberry cultivars.

ABSTRACT:

Background

Gene transcripts that show invariant abundance during development are ideal as reference genes (RGs) for accurate gene expression analyses, such as RNA blot analysis and reverse transcription-quantitative real time PCR (RT-qPCR) analyses. In a genome-wide analysis, we selected three "Commonly used" housekeeping genes (HKGs), fifteen "Traditional" HKGs, and nine novel genes as candidate RGs based on 80 publicly available transcriptome libraries that include data for receptacle development in eight strawberry cultivars.

Results

The results of the multifaceted assessment consistently revealed that expression of the novel RGs showed greater stability compared with that of the "Commonly used" and "Traditional" HKGs in transcriptome and RT-qPCR analyses. Notably, the majority of stably expressed genes were associated with the ubiquitin proteasome system. Among these, two 26?s proteasome subunits, RPT6A and RPN5A, showed superior expression stability and abundance, and are recommended as the optimal RGs combination for normalization of gene expression during strawberry receptacle development.

Conclusion

These findings provide additional useful and reliable RGs as resources for the accurate study of gene expression during receptacle development in strawberry cultivars.

SUBMITTER: Chen J

PROVIDER: S-EPMC7845027 | biostudies-literature | 2021 Jan

REPOSITORIES: biostudies-literature

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Publications

Genome-wide identification of ubiquitin proteasome subunits as superior reference genes for transcript normalization during receptacle development in strawberry cultivars.

Chen Jianqing J Zhou Jinyu J Hong Yanhong Y Li Zekun Z Cheng Xiangyu X Zheng Aiying A Zhang Yilin Y Song Juanjuan J Xie Guifeng G Chen Changmei C Yuan Meng M Wang Tengyun T Chen Qingxi Q

BMC genomics 20210128 1

<h4>Background</h4>Gene transcripts that show invariant abundance during development are ideal as reference genes (RGs) for accurate gene expression analyses, such as RNA blot analysis and reverse transcription-quantitative real time PCR (RT-qPCR) analyses. In a genome-wide analysis, we selected three "Commonly used" housekeeping genes (HKGs), fifteen "Traditional" HKGs, and nine novel genes as candidate RGs based on 80 publicly available transcriptome libraries that include data for receptacle ...[more]

PMID: 33509086

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Project description:Real-time reverse transcription PCR (RT-qPCR) has emerged as an accurate and widely used technique for expression profiling of selected genes. However, obtaining reliable measurements depends on the selection of appropriate reference genes for gene expression normalization. The aim of this work was to assess the expression stability of 15 candidate genes to determine which set of reference genes is best suited for transcript normalization in citrus in different tissues and organs and leaves challenged with five pathogens (Alternaria alternata, Phytophthora parasitica, Xylella fastidiosa and Candidatus Liberibacter asiaticus). We tested traditional genes used for transcript normalization in citrus and orthologs of Arabidopsis thaliana genes described as superior reference genes based on transcriptome data. geNorm and NormFinder algorithms were used to find the best reference genes to normalize all samples and conditions tested. Additionally, each biotic stress was individually analyzed by geNorm. In general, FBOX (encoding a member of the F-box family) and GAPC2 (GAPDH) was the most stable candidate gene set assessed under the different conditions and subsets tested, while CYP (cyclophilin), TUB (tubulin) and CtP (cathepsin) were the least stably expressed genes found. Validation of the best suitable reference genes for normalizing the expression level of the WRKY70 transcription factor in leaves infected with Candidatus Liberibacter asiaticus showed that arbitrary use of reference genes without previous testing could lead to misinterpretation of data. Our results revealed FBOX, SAND (a SAND family protein), GAPC2 and UPL7 (ubiquitin protein ligase 7) to be superior reference genes, and we recommend their use in studies of gene expression in citrus species and relatives. This work constitutes the first systematic analysis for the selection of superior reference genes for transcript normalization in different citrus organs and under biotic stress.

Project description:Real-time reverse transcription PCR (RT-qPCR) is a preferred method for rapid and accurate quantification of gene expression studies. Appropriate application of RT-qPCR requires accurate normalization though the use of reference genes. As no single reference gene is universally suitable for all experiments, thus reference gene(s) validation under different experimental conditions is crucial for RT-qPCR analysis. To date, only a few studies on reference genes have been done in other plants but none in papaya. In the present work, we selected 21 candidate reference genes, and evaluated their expression stability in 246 papaya fruit samples using three algorithms, geNorm, NormFinder and RefFinder. The samples consisted of 13 sets collected under different experimental conditions, including various tissues, different storage temperatures, different cultivars, developmental stages, postharvest ripening, modified atmosphere packaging, 1-methylcyclopropene (1-MCP) treatment, hot water treatment, biotic stress and hormone treatment. Our results demonstrated that expression stability varied greatly between reference genes and that different suitable reference gene(s) or combination of reference genes for normalization should be validated according to the experimental conditions. In general, the internal reference genes EIF (Eukaryotic initiation factor 4A), TBP1 (TATA binding protein 1) and TBP2 (TATA binding protein 2) genes had a good performance under most experimental conditions, whereas the most widely present used reference genes, ACTIN (Actin 2), 18S rRNA (18S ribosomal RNA) and GAPDH (Glyceraldehyde-3-phosphate dehydrogenase) were not suitable in many experimental conditions. In addition, two commonly used programs, geNorm and Normfinder, were proved sufficient for the validation. This work provides the first systematic analysis for the selection of superior reference genes for accurate transcript normalization in papaya under different experimental conditions.

Dataset Information

Genome-wide identification of ubiquitin proteasome subunits as superior reference genes for transcript normalization during receptacle development in strawberry cultivars.

Background

Results

Conclusion

Publications

Genome-wide identification of ubiquitin proteasome subunits as superior reference genes for transcript normalization during receptacle development in strawberry cultivars.

Similar Datasets

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