title: Identification of suitable reference genes for gene expression normalization in qRT-PCR analysis in watermelon.
abstract: Watermelon is one of the major Cucurbitaceae crops and the recent availability of genome sequence greatly facilitates the fundamental researches on it. Quantitative real-time reverse transcriptase PCR (qRT-PCR) is the preferred method for gene expression analyses, and using validated reference genes for normalization is crucial to ensure the accuracy of this method. However, a systematic validation of reference genes has not been conducted on watermelon. In this study, transcripts of 15 candidate reference genes were quantified in watermelon using qRT-PCR, and the stability of these genes was compared using geNorm and NormFinder. geNorm identified ClTUA and ClACT, ClEF1α and ClACT, and ClCAC and ClTUA as the best pairs of reference genes in watermelon organs and tissues under normal growth conditions, abiotic stress, and biotic stress, respectively. NormFinder identified ClYLS8, ClUBCP, and ClCAC as the best single reference genes under the above experimental conditions, respectively. ClYLS8 and ClPP2A were identified as the best reference genes across all samples. Two to nine reference genes were required for more reliable normalization depending on the experimental conditions. The widely used watermelon reference gene 18SrRNA was less stable than the other reference genes under the experimental conditions. Catalase family genes were identified in watermelon genome, and used to validate the reliability of the identified reference genes. ClCAT1and ClCAT2 were induced and upregulated in the first 24 h, whereas ClCAT3 was downregulated in the leaves under low temperature stress. However, the expression levels of these genes were significantly overestimated and misinterpreted when 18SrRNA was used as a reference gene. These results provide a good starting point for reference gene selection in qRT-PCR analyses involving watermelon.
authors.affiliation: Key Laboratory of Horticultural Plant Biology, Ministry of Education/College of Horticulture and Forestry, Huazhong Agricultural University, Wuhan, China.
authors.forename: Qiusheng
authors.initials: Q
authors.lastname: Kong
authors.affiliation: Key Laboratory of Horticultural Plant Biology, Ministry of Education/College of Horticulture and Forestry, Huazhong Agricultural University, Wuhan, China.
authors.forename: Jingxian
authors.initials: J
authors.lastname: Yuan
authors.affiliation: Key Laboratory of Horticultural Plant Biology, Ministry of Education/College of Horticulture and Forestry, Huazhong Agricultural University, Wuhan, China.
authors.forename: Lingyun
authors.initials: L
authors.lastname: Gao
authors.affiliation: Key Laboratory of Horticultural Plant Biology, Ministry of Education/College of Horticulture and Forestry, Huazhong Agricultural University, Wuhan, China.
authors.forename: Shuang
authors.initials: S
authors.lastname: Zhao
authors.affiliation: Key Laboratory of Horticultural Plant Biology, Ministry of Education/College of Horticulture and Forestry, Huazhong Agricultural University, Wuhan, China.
authors.forename: Wei
authors.initials: W
authors.lastname: Jiang
authors.affiliation: Key Laboratory of Horticultural Plant Biology, Ministry of Education/College of Horticulture and Forestry, Huazhong Agricultural University, Wuhan, China.
authors.forename: Yuan
authors.initials: Y
authors.lastname: Huang
authors.affiliation: Key Laboratory of Horticultural Plant Biology, Ministry of Education/College of Horticulture and Forestry, Huazhong Agricultural University, Wuhan, China.
authors.forename: Zhilong
authors.initials: Z
authors.lastname: Bie
mesh_terms: D000199:Actins / Q000235:genetics; D002374:Catalase / Q000235:genetics; D029773:Citrullus / Q000235:genetics*; D003080:Cold Temperature; D020869:Gene Expression Profiling / Q000379:methods* / Q000592:standards; D018506:Gene Expression Regulation, Plant; D017343:Genes, Plant / Q000235:genetics*; D007527:Isoenzymes / Q000235:genetics; D020648:Peptide Elongation Factor 1 / Q000235:genetics; D018515:Plant Leaves / Q000235:genetics; D010940:Plant Proteins / Q000235:genetics; D012337:RNA, Ribosomal, 18S / Q000235:genetics; D012015:Reference Standards; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D014404:Tubulin / Q000235:genetics
chemical_list: D000199:Actins; D007527:Isoenzymes; D020648:Peptide Elongation Factor 1; D010940:Plant Proteins; D012337:RNA, Ribosomal, 18S; D014404:Tubulin; D002374:Catalase