title: Identification and validation of reference genes for quantitative real-time PCR normalization and its applications in lycium.
abstract: Lycium barbarum and L. ruthenicum are extensively used as traditional Chinese medicinal plants. Next generation sequencing technology provides a powerful tool for analyzing transcriptomic profiles of gene expression in non-model species. Such gene expression can then be confirmed with quantitative real-time polymerase chain reaction (qRT-PCR). Therefore, use of systematically identified suitable reference genes is a prerequisite for obtaining reliable gene expression data. Here, we calculated the expression stability of 18 candidate reference genes across samples from different tissues and grown under salt stress using geNorm and NormFinder procedures. The geNorm-determined rank of reference genes was similar to those defined by NormFinder with some differences. Both procedures confirmed that the single most stable reference gene was ACNTIN1 for L. barbarum fruits, H2B1 for L. barbarum roots, and EF1α for L. ruthenicum fruits. PGK3, H2B2, and PGK3 were identified as the best stable reference genes for salt-treated L. ruthenicum leaves, roots, and stems, respectively. H2B1 and GAPDH1+PGK1 for L. ruthenicum and SAMDC2+H2B1 for L. barbarum were the best single and/or combined reference genes across all samples. Finally, expression of salt-responsive gene NAC, fruit ripening candidate gene LrPG, and anthocyanin genes were investigated to confirm the validity of the selected reference genes. Suitable reference genes identified in this study provide a foundation for accurately assessing gene expression and further better understanding of novel gene function to elucidate molecular mechanisms behind particular biological/physiological processes in Lycium.
authors.affiliation: Key Laboratory of Plant Resources Conservation and Sustainable Utilization, South China Botanical Garden, Chinese Academy of Sciences, Guangzhou, P.R. China.
authors.forename: Shaohua
authors.initials: S
authors.lastname: Zeng
authors.affiliation: Key Laboratory of Pant Germplasm Enhancement and Specialty Agriculture, Wuhan Botanical Garden, Chinese Academy of Sciences, Wuhan, Hubei, P.R. China.
authors.forename: Yongliang
authors.initials: Y
authors.lastname: Liu
authors.affiliation: Key Laboratory of Plant Resources Conservation and Sustainable Utilization, South China Botanical Garden, Chinese Academy of Sciences, Guangzhou, P.R. China.
authors.forename: Min
authors.initials: M
authors.lastname: Wu
authors.affiliation: Key Laboratory of Plant Resources Conservation and Sustainable Utilization, South China Botanical Garden, Chinese Academy of Sciences, Guangzhou, P.R. China.
authors.forename: Xiaomin
authors.initials: X
authors.lastname: Liu
authors.affiliation: Key Laboratory of Pant Germplasm Enhancement and Specialty Agriculture, Wuhan Botanical Garden, Chinese Academy of Sciences, Wuhan, Hubei, P.R. China.
authors.forename: Xiaofei
authors.initials: X
authors.lastname: Shen
authors.affiliation: National Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing, P.R. China.
authors.forename: Chunzhao
authors.initials: C
authors.lastname: Liu
authors.affiliation: Key Laboratory of Plant Resources Conservation and Sustainable Utilization, South China Botanical Garden, Chinese Academy of Sciences, Guangzhou, P.R. China; Key Laboratory of Pant Germplasm Enhancement and Specialty Agriculture, Wuhan Botanical Garden, Chinese Academy of Sciences, Wuhan, Hubei, P.R. China.
authors.forename: Ying
authors.initials: Y
authors.lastname: Wang
mesh_terms: D005638:Fruit / Q000235:genetics; D020869:Gene Expression Profiling / Q000592:standards*; D017343:Genes, Plant / Q000235:genetics*; D032305:Lycium / Q000187:drug effects / Q000235:genetics* / Q000502:physiology; D009928:Organ Specificity; D060888:Real-Time Polymerase Chain Reaction / Q000592:standards*; D012015:Reference Standards; D015203:Reproducibility of Results; D012492:Salts / Q000494:pharmacology; D013312:Stress, Physiological / Q000187:drug effects / Q000235:genetics
chemical_list: D012492:Salts