title: Validation of Reference Genes for RT-qPCR Studies of Gene Expression in Preharvest and Postharvest Longan Fruits under Different Experimental Conditions.
abstract: Reverse transcription quantitative PCR (RT-qPCR) as the accurate and sensitive method is use for gene expression analysis, but the veracity and reliability result depends on whether select appropriate reference gene or not. To date, several reliable reference gene validations have been reported in fruits trees, but none have been done on preharvest and postharvest longan fruits. In this study, 12 candidate reference genes, namely, CYP, RPL, GAPDH, TUA, TUB, Fe-SOD, Mn-SOD, Cu/Zn-SOD, 18SrRNA, Actin, Histone H3, and EF-1a, were selected. Expression stability of these genes in 150 longan samples was evaluated and analyzed using geNorm and NormFinder algorithms. Preharvest samples consisted of seven experimental sets, including different developmental stages, organs, hormone stimuli (NAA, 2,4-D, and ethephon) and abiotic stresses (bagging and girdling with defoliation). Postharvest samples consisted of different temperature treatments (4 and 22°C) and varieties. Our findings indicate that appropriate reference gene(s) should be picked for each experimental condition. Our data further showed that the commonly used reference gene Actin does not exhibit stable expression across experimental conditions in longan. Expression levels of the DlACO gene, which is a key gene involved in regulating fruit abscission under girdling with defoliation treatment, was evaluated to validate our findings. In conclusion, our data provide a useful framework for choice of suitable reference genes across different experimental conditions for RT-qPCR analysis of preharvest and postharvest longan fruits.
authors.affiliation: Department of Biochemistry, Basic Education College of Lingnan Normal University Zhanjiang, China.
authors.forename: Jianyang
authors.initials: J
authors.lastname: Wu
authors.affiliation: Key Laboratory of Tropical Fruit Biology, South Subtropical Crops Research Institute, Chinese Academy of Tropical Agricultural Sciences, Ministry of Agriculture Zhanjiang, China.
authors.forename: Hongna
authors.initials: H
authors.lastname: Zhang
authors.affiliation: Key Laboratory of Tropical Fruit Biology, South Subtropical Crops Research Institute, Chinese Academy of Tropical Agricultural Sciences, Ministry of Agriculture Zhanjiang, China.
authors.forename: Liqin
authors.initials: L
authors.lastname: Liu
authors.affiliation: Key Laboratory of Tropical Fruit Biology, South Subtropical Crops Research Institute, Chinese Academy of Tropical Agricultural Sciences, Ministry of Agriculture Zhanjiang, China.
authors.forename: Weicai
authors.initials: W
authors.lastname: Li
authors.affiliation: Key Laboratory of Tropical Fruit Biology, South Subtropical Crops Research Institute, Chinese Academy of Tropical Agricultural Sciences, Ministry of Agriculture Zhanjiang, China.
authors.forename: Yongzan
authors.initials: Y
authors.lastname: Wei
authors.affiliation: Key Laboratory of Tropical Fruit Biology, South Subtropical Crops Research Institute, Chinese Academy of Tropical Agricultural Sciences, Ministry of Agriculture Zhanjiang, China.
authors.forename: Shengyou
authors.initials: S
authors.lastname: Shi
keywords: ACO gene; Longan (Dimocarpus longan Lour.); RT-qPCR; gene expression; normalization; preharvest and postharvest fruits; reference genes