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Int. J. Mol. Sci. 2014, 15(5), 8846-8862; doi:10.3390/ijms15058846

Selection of Suitable Endogenous Reference Genes for Relative Copy Number Detection in Sugarcane

1
Key Laboratory of Sugarcane Biology and Genetic Breeding, Fujian Agriculture and Forestry University, Ministry of Agriculture, Fuzhou 350002, China
2
School of Agriculture and Food Sciences, University of Queensland, Brisbane, QLD 4072, Australia
These authors contributed equally to this work.
*
Author to whom correspondence should be addressed.
Received: 19 February 2014 / Revised: 20 April 2014 / Accepted: 7 May 2014 / Published: 19 May 2014
(This article belongs to the Section Biochemistry, Molecular and Cellular Biology)
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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. View Full-Text
Keywords: sugarcane; endogenous reference gene; absolute quantification; copy number sugarcane; endogenous reference gene; absolute quantification; copy number
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MDPI and ACS Style

Xue, B.; Guo, J.; Que, Y.; Fu, Z.; Wu, L.; Xu, L. Selection of Suitable Endogenous Reference Genes for Relative Copy Number Detection in Sugarcane. Int. J. Mol. Sci. 2014, 15, 8846-8862.

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