Selection of suitable endogenous reference genes for relative copy number detection in sugarcane

Xue, Bantong, Guo, Jinlong, Que, Youxiong, Fu, Zhiwei, Wu, Luguang and Xu, Liping (2014) Selection of suitable endogenous reference genes for relative copy number detection in sugarcane. International Journal of Molecular Sciences, 15 5: 8846-8862. doi:10.3390/ijms15058846

Author Xue, Bantong
Guo, Jinlong
Que, Youxiong
Fu, Zhiwei
Wu, Luguang
Xu, Liping
Title Selection of suitable endogenous reference genes for relative copy number detection in sugarcane
Journal name International Journal of Molecular Sciences   Check publisher's open access policy
ISSN 1422-0067
Publication date 2014-05-19
Year available 2014
Sub-type Article (original research)
DOI 10.3390/ijms15058846
Open Access Status DOI
Volume 15
Issue 5
Start page 8846
End page 8862
Total pages 17
Place of publication Basel, Switzerland
Publisher Molecular Diversity Preservation International
Language eng
Formatted abstract
Transgene copy number has a great impact on the expression level and stability of exogenous gene in transgenic plants. Proper selection of endogenous reference genes is necessary for detection of genetic components in genetically modification (GM) crops by quantitative real-time PCR (qPCR) or by qualitative PCR approach, especially in sugarcane with polyploid and aneuploid genomic structure. qPCR technique has been widely accepted as an accurate, time-saving method on determination of copy numbers in transgenic plants and on detection of genetically modified plants to meet the regulatory and legislative requirement. In this study, to find a suitable endogenous reference gene and its real-time PCR assay for sugarcane (Saccharum spp. hybrids) DNA content quantification, we evaluated a set of potential "single copy" genes including P4H, APRT, ENOL, CYC, TST and PRR, through qualitative PCR and absolute quantitative PCR. Based on copy number comparisons among different sugarcane genotypes, including five S. officinarum, one S. spontaneum and two S. spp. hybrids, these endogenous genes fell into three groups: ENOL-3-high copy number group, TST-1 and PRR-1-medium copy number group, P4H-1, APRT-2 and CYC-2-low copy number group. Among these tested genes, P4H, APRT and CYC were the most stable, while ENOL and TST were the least stable across different sugarcane genotypes. Therefore, three primer pairs of P4H-3, APRT-2 and CYC-2 were then selected as the suitable reference gene primer pairs for sugarcane. The test of multi-target reference genes revealed that the APRT gene was a specific amplicon, suggesting this gene is the most suitable to be used as an endogenous reference target for sugarcane DNA content quantification. These results should be helpful for establishing accurate and reliable qualitative and quantitative PCR analysis of GM sugarcane.
Keyword Sugarcane
Endogenous reference gene
Absolute quantification
Copy number
Q-Index Code C1
Q-Index Status Confirmed Code
Institutional Status UQ

Document type: Journal Article
Sub-type: Article (original research)
Collections: School of Agriculture and Food Sciences
Official 2015 Collection
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