Screening and Evaluation of Amplicons of Maize Endogenous Reference Genes Based on Next-Generation Sequencing Technology

Abstract

In this study, 13 amplification regions (amplicons) were designed by analyzing the nucleotide sequences of seven endogenous reference genes reported in previous studies. Totally 208 common maize varieties (lines) were used for high-throughput sequencing analysis of these 13 amplicons, and their sequence conservation, specificity, detection stability and dynamic detection range were evaluated to select appropriate amplicons of the endogenous reference genes for quantitative detection of maize DNA content or copy number based on next-generation sequencing technology. The results showed that the detection rates of the 13 amplicons of the seven endogenous reference genes were 94.7%–100% in the 208 maize samples, while they were not detected in 25 non-maize samples of rice, soybean, cotton and Chinese cabbage, indicating that these amplicons met the standards of high intraspecies consistency and interspecies specificity. Intraspecies conservation analysis showed that E3-UBI-1 did not contain single nucleotide polymorphism (SNP) or insertion deletion (InDel), while the remaining 12 amplicons contained at least one SNP or InDel. Stability analysis showed that the most unstable amplicon in these endogenous reference genes was IVR, and the most stable one was ADH1-2. Their dynamic detection range was 0.2–200 ng. Based on these results, ADH1-2 and E3-UBI-1 are suitable amplicons of the endogenous reference genes for quantitative detection of maize DNA content or transgenic maize components on the next-generation sequencing platform.

Keywords

• zea mays; next-generation sequencing; endogenous reference gene; quantitative detection; stability