title: Reference gene selection for quantitative real-time reverse-transcriptase PCR in orchardgrass subjected to various abiotic stresses.
abstract: Quantitative real-time reverse-transcriptase PCR (qRT-PCR) is a powerful tool for the measurement of gene expression; however, the accuracy of this approach depends on the stability of reference genes. The objective of the present study was to identify the stable reference genes in orchardgrass (Dactylis glomerata L.), a principal cool-season forage grass in the world. Ten candidate reference genes were selected in this study including ATP-binding [ABC], actin [ACTIN], cyclophilin [CYP2], glyceraldehyde 3-phosphate dehydrogenase [GAPDH], beta-amylase 4 [BAM4], zeitlupe [ZTL], MAP Kinase 4 [MPK4], ubiquitin-conjugating enzyme [UBC], S-adenosylmethionine decarboxylase [SAMDC], and translationally controlled tumor protein [TCTP]. The candidate genes were assessed in orchardgrass leaves and roots under conditions of drought, high salinity, heat, waterlogging, and abscisic acid (ABA) treatments. We used GeNorm, BestKeeper, NormFinder, and RefFinder for qRT-PCR normalization and validation to determine that the expression of these reference genes was stress-dependent. ACTIN, CYP2, and ABC were found to be the most stably expressed genes for drought stress while ACTIN, TCTP, and ABC were the most stable under salt stress. ACTIN, CYP2, and ABC were all found to be good reference genes for studying heat stress. Likewise, CYP2, MPK4, and ABC were most suitable to study waterlogging, and ACTIN, CYP2, and MPK4 were determined as the three best reference genes for ABA studies. Our study identified and validated the possible reference genes in orchardgrass that may be used for quantification of target gene expression under various abiotic stresses.
authors.affiliation: Department of Grassland Science, Animal Science and Technology College, Sichuan Agricultural University, Ya'an, Sichuan 625014, China.
authors.forename: Linkai
authors.initials: L
authors.lastname: Huang
authors.affiliation: Department of Grassland Science, Animal Science and Technology College, Sichuan Agricultural University, Ya'an, Sichuan 625014, China.
authors.forename: Haidong
authors.initials: H
authors.lastname: Yan
authors.affiliation: Department of Grassland Science, Animal Science and Technology College, Sichuan Agricultural University, Ya'an, Sichuan 625014, China.
authors.forename: Xiaomei
authors.initials: X
authors.lastname: Jiang
authors.affiliation: Department of Grassland Science, Animal Science and Technology College, Sichuan Agricultural University, Ya'an, Sichuan 625014, China.
authors.forename: Yu
authors.initials: Y
authors.lastname: Zhang
authors.affiliation: Department of Grassland Science, Animal Science and Technology College, Sichuan Agricultural University, Ya'an, Sichuan 625014, China. Electronic address: zhangxq@sicau.edu.cn.
authors.forename: Xinquan
authors.initials: X
authors.lastname: Zhang
authors.affiliation: Department of Grassland Science, Sichuan Animal Science Academy, Chengdu 610066, China.
authors.forename: Yang
authors.initials: Y
authors.lastname: Ji
authors.affiliation: Department of Animal Science, Southwest University, Rongchang 402460, China.
authors.forename: Bing
authors.initials: B
authors.lastname: Zeng
authors.affiliation: College of Grassland Science, Nanjing Agricultural University, Nanjing, Jiangsu 210095, China.
authors.forename: Bin
authors.initials: B
authors.lastname: Xu
authors.affiliation: Department of Plant Biology and Pathology, Rutgers, The State University of New Jersey, New Brunswick, NJ 08901, United States.
authors.forename: Guohua
authors.initials: G
authors.lastname: Yin
authors.affiliation: Department of Plant Biology and Pathology, Rutgers, The State University of New Jersey, New Brunswick, NJ 08901, United States.
authors.forename: Samantha
authors.initials: S
authors.lastname: Lee
authors.affiliation: Department of Grassland Science, Animal Science and Technology College, Sichuan Agricultural University, Ya'an, Sichuan 625014, China.
authors.forename: Yanhong
authors.initials: Y
authors.lastname: Yan
authors.affiliation: Department of Grassland Science, Animal Science and Technology College, Sichuan Agricultural University, Ya'an, Sichuan 625014, China.
authors.forename: Xiao
authors.initials: X
authors.lastname: Ma
authors.affiliation: Department of Grassland Science, Animal Science and Technology College, Sichuan Agricultural University, Ya'an, Sichuan 625014, China.
authors.forename: Yan
authors.initials: Y
authors.lastname: Peng
mesh_terms: D001483:Base Sequence; D017931:DNA Primers; D017343:Genes, Plant; D006109:Poaceae / Q000235:genetics* / Q000502:physiology; D060888:Real-Time Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013312:Stress, Physiological
chemical_list: D017931:DNA Primers
keywords: Abiotic stress; Orchardgrass; Reference genes; qRT-PCR