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

Bantong Xue,Youxiong Que,Luguang Wu,Jinlong Guo,Liping Xu,Zhiwei Fu

doi:10.3390/ijms15058846

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.

Highlights

Sugarcane (Saccharum spp. hybrids) is the world’s largest crop, accounting for 80% of all sugar produced in the world, cultivated in 101 countries and the cultivated area was about 26.1 million hectares in 2012 according to the FAO estimates, with a worldwide harvest of 1.83 billion tons
Nineteen specific primer pairs were designed for six candidate reference genes (Table 1), each of which was tested for single fragment gene amplification in the preliminary experiments of Polymerase Chain Reaction (PCR), using the genomic DNA, isolated from sugarcane cultivar ROC22 and Badila, as templates
Gel purification, sequencing and alignment of the amplified fragments, a total of ten primer pairs were selected based on the above selection criterion for subsequent quantitative real-time PCR (qPCR)

Summary

Introduction

Sugarcane (Saccharum spp. hybrids) is the world’s largest crop, accounting for 80% of all sugar produced in the world, cultivated in 101 countries and the cultivated area was about 26.1 million hectares in 2012 according to the FAO estimates, with a worldwide harvest of 1.83 billion tons. Genetic transformation has a potential to introduce desirable traits into target crops and to supplement traditional plant breeding techniques [3], resulting in a revolution in sugarcane breeding and sucrose production [4]. Sugarcane has several advantages that make it an ideal candidate for improvement via genetic engineering. It is rarely flowering and producing fewer viable seeds from commercial sugarcane cultivars especially in the commercial cultivation, which greatly reduces the potential of genetic drifting from genetically modified (GM) sugarcane. Unlike other GM food crops, GM sugarcane could be accepted by the public and regulatory authorities, as refined white sugar is the most chemically pure food derived from plants and has been found to be free from DNA and proteins expressed from the introduced transgene [5]. Higher copies in transgenic sugarcane can even cause co-suppression, mainly from transcriptional gene silencing (TGS) or post-transcriptional gene silencing (PTGS) [6,7]

Methods

Results

Discussion

Conclusion