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Identification and Validation of Reference Genes for qRT-PCR Analysis of Petal-Color-Related Genes in Rosa praelucens.

ABSTRACT: The flower's color is regarded as one of the most outstanding features of the rose. Rosa praelucens Byhouwer, an endemic and critically endangered decaploid wild rose species, is abundant in phenotypic diversity, especially in flower color variation, from white to different degrees of pink. The mechanism underlying this variation, e.g., the level of petal-color-related genes, is worth probing. Seven candidate reference genes for qRT-PCR analysis, including tubulin α chain (TUBA), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), histone H2B (Histone2A), eukaryotic translation elongation factor 1-α (EEF1A), 60S ribosomal protein (RPL37), eukaryotic translation initiation factor 1-α (EIF1A), and aquaporins (AQP), were detected from the transcriptome datasets of full blooming flowers of white-petaled and pink-petaled individuals, and their expression stabilities were evaluated through qRT-PCR analysis. According to stability rankings analysis, EEF1A showed the highest stability and could be chosen as the most suitable reference gene. Moreover, the reliability of EEF1A was demonstrated via qRT-PCR analysis of six petal-color-related target genes, the expression patterns of which, through EEF1A normalization, were found to be consistent with the findings of transcriptome analysis. The result provides an optimal reference gene for exploring the expression level of petal-color-related genes in R. praelucens, which will accelerate the dissection of petal-color-variation mechanisms in R. praelucens.

SUBMITTER: Jian H

PROVIDER: S-EPMC10970342 | biostudies-literature | 2024 Feb

REPOSITORIES: biostudies-literature

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Identification and Validation of Reference Genes for qRT-PCR Analysis of Petal-Color-Related Genes in Rosa praelucens.

Jian Hongying H Wang Huichun H Qiu Xianqin X Yan Huijun H Ma Lulin L

Genes 20240223 3

The flower's color is regarded as one of the most outstanding features of the rose. Rosa praelucens Byhouwer, an endemic and critically endangered decaploid wild rose species, is abundant in phenotypic diversity, especially in flower color variation, from white to different degrees of pink. The mechanism underlying this variation, e.g., the level of petal-color-related genes, is worth probing. Seven candidate reference genes for qRT-PCR analysis, including tubulin α chain (TUBA), g ...[more]

PMID: 38540336

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Project description:BackgroundBivalves comprise around 30,000 extant species and have received much attention for their importance in ecosystems, aquaculture and evolutionary studies. Despite the increasing application of real-time quantitative reverse transcription PCR (qRT-PCR) in gene expression studies on bivalve species, little research has been conducted on reference gene selection which is critical for reliable and accurate qRT-PCR analysis. For scallops, systematic evaluation of reference genes that can be used among tissues or embryo/larva stages is lacking, and β-actin (ACT) is most frequently used as qRT-PCR reference gene without validation.ResultsIn this study, 12 commonly used candidate reference genes were selected from the transcriptome data of Yesso scallop (Patinopectenyessoensis) for suitable qRT-PCR reference genes identification. The expression of these genes in 36 tissue samples and 15 embryo/larva samples under normal physiological conditions was examined by qRT-PCR, and their expression stabilities were evaluated using three statistic algorithms, geNorm, NormFinder, and comparative ∆Ct method. Similar results were obtained by the three approaches for the most and the least stably expressed genes. Final comprehensive ranking for the 12 genes combing the results from the three programs showed that, for different tissues, DEAD-box RNA helicase (HELI), ubiquitin (UBQ), and 60S ribosomal protein L16 (RPL16) were the optimal reference genes combination, while for different embryo/larva stages, gene set containing Cytochrome B (CB), Cytochrome C (CC), Histone H3.3 (His3.3), and Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were recommended for qRT-PCR normalization. ACT was among the least stable genes for both adult tissues and embryos/larvae.ConclusionsThis work constitutes the first systematic analysis on reference genes selection for qRT-PCR normalization in scallop under normal conditions. The suitable reference genes we recommended will be useful for the identification of genes related to biological processes in Yesso scallop, and also in the reference gene selection for other scallop or bivalve species.

Dataset Information

Identification and Validation of Reference Genes for qRT-PCR Analysis of Petal-Color-Related Genes in Rosa praelucens.

Publications

Identification and Validation of Reference Genes for qRT-PCR Analysis of Petal-Color-Related Genes in <i>Rosa praelucens</i>.

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