Evaluation of Appropriate Reference Genes for Gene Expression Normalization during Watermelon Fruit Development

PLOS ONE, Dec 2019

Gene expression analysis in watermelon (Citrullus lanatus) fruit has drawn considerable attention with the availability of genome sequences to understand the regulatory mechanism of fruit development and to improve its quality. Real-time quantitative reverse-transcription PCR (qRT-PCR) is a routine technique for gene expression analysis. However, appropriate reference genes for transcript normalization in watermelon fruits have not been well characterized. The aim of this study was to evaluate the appropriateness of 12 genes for their potential use as reference genes in watermelon fruits. Expression variations of these genes were measured in 48 samples obtained from 12 successive developmental stages of parthenocarpic and fertilized fruits of two watermelon genotypes by using qRT-PCR analysis. Considering the effects of genotype, fruit setting method, and developmental stage, geNorm determined clathrin adaptor complex subunit (ClCAC), β-actin (ClACT), and alpha tubulin 5 (ClTUA5) as the multiple reference genes in watermelon fruit. Furthermore, ClCAC alone or together with SAND family protein (ClSAND) was ranked as the single or two best reference genes by NormFinder. By using the top-ranked reference genes to normalize the transcript abundance of phytoene synthase (ClPSY1), a good correlation between lycopene accumulation and ClPSY1 expression pattern was observed in ripening watermelon fruit. These validated reference genes will facilitate the accurate measurement of gene expression in the studies on watermelon fruit biology.

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Evaluation of Appropriate Reference Genes for Gene Expression Normalization during Watermelon Fruit Development

June Evaluation of Appropriate Reference Genes for Gene Expression Normalization during Watermelon Fruit Development Qiusheng Kong 0 1 Jingxian Yuan 0 1 Lingyun Gao 0 1 Liqiang Zhao 0 1 Fei Cheng 0 1 Yuan Huang 0 1 Zhilong Bie 0 1 0 Key Laboratory of Horticultural Plant Biology, Ministry of Education, College of Horticulture and Forestry Sciences, Huazhong Agricultural University , Wuhan , China 1 Editor: Robert W Dettman, Northwestern University , UNITED STATES Gene expression analysis in watermelon (Citrullus lanatus) fruit has drawn considerable attention with the availability of genome sequences to understand the regulatory mechanism of fruit development and to improve its quality. Real-time quantitative reverse-transcription PCR (qRT-PCR) is a routine technique for gene expression analysis. However, appropriate reference genes for transcript normalization in watermelon fruits have not been well characterized. The aim of this study was to evaluate the appropriateness of 12 genes for their potential use as reference genes in watermelon fruits. Expression variations of these genes were measured in 48 samples obtained from 12 successive developmental stages of parthenocarpic and fertilized fruits of two watermelon genotypes by using qRTPCR analysis. Considering the effects of genotype, fruit setting method, and developmental stage, geNorm determined clathrin adaptor complex subunit (ClCAC), β-actin (ClACT), and alpha tubulin 5 (ClTUA5) as the multiple reference genes in watermelon fruit. Furthermore, ClCAC alone or together with SAND family protein (ClSAND) was ranked as the single or two best reference genes by NormFinder. By using the top-ranked reference genes to normalize the transcript abundance of phytoene synthase (ClPSY1), a good correlation between lycopene accumulation and ClPSY1 expression pattern was observed in ripening watermelon fruit. These validated reference genes will facilitate the accurate measurement of gene expression in the studies on watermelon fruit biology. - Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: This work was supported by the National Natural Science Foundation of China (31471894) (QK), the earmarked fund for Modern Agro-industry Technology Research System (CARS-26-16) (ZB), and the Special Fund for Agro-scientific Research in the Public Interest (201203080-2) (ZB). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Watermelon (Citrullus lanatus) is a popular and economically important horticultural crop in terms of production and consumption. During development and ripening, watermelon fruits undergo various physiological and biochemical changes, resulting in diverse sizes, colors, shapes, sweetness, textures, and aromas [1]. Watermelon is gaining popularity as a model plant for studying non-climacteric fruits. Studies have focused on the unique metabolic and regulatory networks of watermelon fruit to improve the crucial nutritional attributes of this fruit [2]. Competing Interests: The authors have declared that no competing interests exist. Moreover, the vast amounts of genomic resources are beneficial for elucidating the biological processes involved in fruit development. Transcriptome sequencing has been conducted to study the transcriptional regulatory networks in watermelon fruit, revealing many key genes involved in sugar, citrulline, and carotenoid metabolisms [1, 3–5]. Target gene expression patterns during fruit development can provide clues to understand its biological functions. Realtime quantitative reverse-transcription PCR (qRT-PCR) is an efficient method to measure gene transcript abundance and to validate gene expression changes detected in RNA-Seq [1, 6] and microarray [2]. In qRT-PCR analysis, gene expression is quantified by normalizing to one or more internal reference genes presumed to be stable throughout a given experiment. Normalization removes considerable non-biological variations associated with the different steps of sample preparations and expression measurements [7]. Accordingly, qRT-PCR accuracy is strongly influenced by internal reference gene stability. The use of unstable reference genes in relative quantification of gene expression could lead to significant biases and misinterpretations of data [8–10]. Moreover, reference genes are not constantly expressed under a wide range of experimental conditions [11, 12]. Thus, systematic validation of reference genes must be conducted on each experiment prior to their use [13, 14]. Statistical algorithms such as geNorm [15] and NormFinder [16] have been developed to determine gene expression stability, which greatly simplified the choice of appropriate reference genes for normalization in qRT-PCR analysis. Suitable reference genes have been identified in watermelon under normal growth conditions as well as biotic and abiotic stresses, providing (...truncated)


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Qiusheng Kong, Jingxian Yuan, Lingyun Gao, Liqiang Zhao, Fei Cheng, Yuan Huang, Zhilong Bie. Evaluation of Appropriate Reference Genes for Gene Expression Normalization during Watermelon Fruit Development, PLOS ONE, 2015, 6, DOI: 10.1371/journal.pone.0130865