title: Validation of a carnation-specific gene, ANS, used as an endogenous reference gene in qualitative and real-time quantitative PCR for carnations.
abstract: The validation of the anthocyanin synthase (ANS) gene as a carnation endogenous reference gene applicable both in classical and real-time PCR methods is a prerequisite for the development of PCR assays for genetically modified (GM) carnation detection. This is important due to the fact that GM carnation lines, developed by Florigene Pty Ltd, have been approved for commercialization. In this study, both methods were tested on 14 different carnation cultivars, and identical amplification products were obtained with all of them. No amplification products were observed with samples from 14 other plant species, which demonstrated that the system was specific to carnation. The results of Southern blot analysis confirmed that the ANS gene had a low copy number in the 10 tested carnation varieties. In qualitative and real-time PCR assays, the LOD values of 0.05 and 0.005 ng carnation DNA, respectively, were validated. Moreover, the real-time PCR system was validated with high PCR efficiency and linearity. Thus, the ANS gene had species specificity, low heterogeneity, and low copy number among the tested cultivars. These results provide evidence that the gene can be used as an endogenous reference gene of carnation, as well as in qualitative and quantitative PCR systems.
authors.affiliation: Shanghai Academy of Agricultural Sciences, Biotechnology Research Institute, 2901 Beidi Rd, Shanghai, People's Republic of China, 201106.
authors.forename: Hong
authors.initials: H
authors.lastname: Zhu
authors.forename: Lingxi
authors.initials: L
authors.lastname: Jiang
authors.forename: Shiru
authors.initials: S
authors.lastname: Tao
authors.forename: Heyan
authors.initials: H
authors.lastname: Lin
authors.forename: Jinbin
authors.initials: J
authors.lastname: Wang
authors.forename: Furong
authors.initials: F
authors.lastname: Tan
authors.forename: Kai
authors.initials: K
authors.lastname: Zhao
authors.forename: Xiao
authors.initials: X
authors.lastname: Wu
authors.forename: Peng
authors.initials: P
authors.lastname: Li
authors.forename: Aihu
authors.initials: A
authors.lastname: Pan
authors.forename: Junwei
authors.initials: J
authors.lastname: Jia
authors.forename: Xueming
authors.initials: X
authors.lastname: Tang
mesh_terms: D000872:Anthocyanins / Q000235:genetics / Q000378:metabolism; D018744:DNA, Plant / Q000032:analysis; D029749:Dianthus / Q000235:genetics*; D018506:Gene Expression Regulation, Plant; D017343:Genes, Plant / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013045:Species Specificity
chemical_list: D000872:Anthocyanins; D018744:DNA, Plant