Abstract
Proper selection of endogenous reference genes and their real-time PCR assays is quite important in genetically modified organisms (GMOs) detection. To find a suitable endogenous reference gene and its real-time PCR assay for common wheat (Triticum aestivum L.) DNA content or copy number quantification, four previously reported wheat endogenous reference genes and their real-time PCR assays were comprehensively evaluated for the target gene sequence variation and their real-time PCR performance among 37 common wheat lines. Three SNPs were observed in the PKABA1 and ALMT1 genes, and these SNPs significantly decreased the efficiency of real-time PCR amplification. GeNorm analysis of the real-time PCR performance of each gene among common wheat lines showed that the Waxy-D1 assay had the lowest M values with the best stability among all tested lines. All results indicated that the Waxy-D1 gene and its real-time PCR assay were most suitable to be used as an endogenous reference gene for common wheat DNA content quantification. The validated Waxy-D1 gene assay will be useful in establishing accurate and creditable qualitative and quantitative PCR analysis of GM wheat.
Highlights
Genetically modified organisms (GMOs) have been developed and marketed by many countries during the past two decades, controversies over safety issues have always been hot topics in public discussions
The expected DNA fragment of Acc1 gene could be amplified from all lines except Aegilops tauschii, Aegilops speltoides, and Triticum urartu; expected DNA fragments of ALMT1 and Waxy-D1 genes were obtained from all lines except Aegilops speltoides, Triticum urartu, and Durum wheat; and expected PKABA1 gene fragment was amplified from all lines except Aegilops tauschii and Aegilops speltoides (Table S1 in File S1)
After blastN and alignment analysis of the obtained DNA sequences from all line, single nucleotide polymorphisms (SNPs) were discovered in the real-time PCR target regions of Acc1 gene, ALMT1 gene, and PKABA1 gene (Figure 1)
Summary
Genetically modified organisms (GMOs) have been developed and marketed by many countries during the past two decades, controversies over safety issues have always been hot topics in public discussions. The efficient and accurate quantification of host genome DNA copy numbers using endogenous reference genes is very important during the process [9]. GM contents are expressed as mass/mass ratio or copy number ratio of GM over non-GM of the assayed organism. Endogenous reference genes and their real-time PCR assays are referred to as “golden standards” in GMO analysis [8]. One desirable endogenous reference gene and its real-time PCR assay should have three characteristics: species specificity, single or low copy number in the genome, and low heterogeneity among different lines [10]. Low heterogeneity is determined by minimum number of single nucleotide polymorphisms (SNPs) in the target DNA sequence and high PCR amplification performance among different lines [11,12]
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