High-Throughput and Accurate Determination of Transgene Copy Number and Zygosity in Transgenic Maize: From DNA Extraction to Data Analysis

It is vital to develop high-throughput methods to determine transgene copy numbers initially and zygosity during subsequent breeding. In this study, the target sequence of the previously reported endogenous reference gene <i>hmg</i> was analyzed using 633 maize inbred lines, and two SNPs...

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Autores principales:	Fang Liu, Jinkui Cheng, Xuhua Liu, Xi-Qing Wang
Formato:	article
Lenguaje:	EN
Publicado:	MDPI AG 2021
Materias:	high throughput transgene copy number zygosity maize endogenous reference gene single-nucleotide polymorphism TaqMan assay Biology (General) QH301-705.5 Chemistry QD1-999
Acceso en línea:	https://doaj.org/article/02d391b7def14c638041b977232097d9
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spelling	oai:doaj.org-article:02d391b7def14c638041b977232097d92021-11-25T17:57:11ZHigh-Throughput and Accurate Determination of Transgene Copy Number and Zygosity in Transgenic Maize: From DNA Extraction to Data Analysis10.3390/ijms2222124871422-00671661-6596https://doaj.org/article/02d391b7def14c638041b977232097d92021-11-01T00:00:00Zhttps://www.mdpi.com/1422-0067/22/22/12487https://doaj.org/toc/1661-6596https://doaj.org/toc/1422-0067It is vital to develop high-throughput methods to determine transgene copy numbers initially and zygosity during subsequent breeding. In this study, the target sequence of the previously reported endogenous reference gene <i>hmg</i> was analyzed using 633 maize inbred lines, and two SNPs were observed. These SNPs significantly increased the PCR efficiency, while the newly developed <i>hmg</i> gene assay (hmg-taq-F2/R2) excluding these SNPs reduced the efficiency into normal ranges. The TaqMan amplification efficiency of <i>bar</i> and <i>hmg</i> with newly developed primers was calculated as 0.993 and 1.000, respectively. The inter-assay coefficient of variation (CV) values for the <i>bar</i> and <i>hmg</i> genes varied from 1.18 to 2.94%. The copy numbers of the transgene <i>bar</i> using new TaqMan assays were identical to those using dPCR. Significantly, the precision of one repetition reached 96.7% of that of three repetitions of single-copy plants analyzed by simple random sampling, and the actual accuracy reached 95.8%, confirmed by T<sub>1</sub> and T<sub>2</sub> progeny. With the high-throughput DNA extraction and automated data analysis procedures developed in this study, nearly 2700 samples could be analyzed within eight hours by two persons. The combined results suggested that the new <i>hmg</i> gene assay developed here could be a universal maize reference gene system, and the new assay has high throughput and high accuracy for large-scale screening of maize varieties around the world.Fang LiuJinkui ChengXuhua LiuXi-Qing WangMDPI AGarticlehigh throughputtransgene copy numberzygositymaize endogenous reference genesingle-nucleotide polymorphismTaqMan assayBiology (General)QH301-705.5ChemistryQD1-999ENInternational Journal of Molecular Sciences, Vol 22, Iss 12487, p 12487 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	high throughput transgene copy number zygosity maize endogenous reference gene single-nucleotide polymorphism TaqMan assay Biology (General) QH301-705.5 Chemistry QD1-999
spellingShingle	high throughput transgene copy number zygosity maize endogenous reference gene single-nucleotide polymorphism TaqMan assay Biology (General) QH301-705.5 Chemistry QD1-999 Fang Liu Jinkui Cheng Xuhua Liu Xi-Qing Wang High-Throughput and Accurate Determination of Transgene Copy Number and Zygosity in Transgenic Maize: From DNA Extraction to Data Analysis
description	It is vital to develop high-throughput methods to determine transgene copy numbers initially and zygosity during subsequent breeding. In this study, the target sequence of the previously reported endogenous reference gene <i>hmg</i> was analyzed using 633 maize inbred lines, and two SNPs were observed. These SNPs significantly increased the PCR efficiency, while the newly developed <i>hmg</i> gene assay (hmg-taq-F2/R2) excluding these SNPs reduced the efficiency into normal ranges. The TaqMan amplification efficiency of <i>bar</i> and <i>hmg</i> with newly developed primers was calculated as 0.993 and 1.000, respectively. The inter-assay coefficient of variation (CV) values for the <i>bar</i> and <i>hmg</i> genes varied from 1.18 to 2.94%. The copy numbers of the transgene <i>bar</i> using new TaqMan assays were identical to those using dPCR. Significantly, the precision of one repetition reached 96.7% of that of three repetitions of single-copy plants analyzed by simple random sampling, and the actual accuracy reached 95.8%, confirmed by T<sub>1</sub> and T<sub>2</sub> progeny. With the high-throughput DNA extraction and automated data analysis procedures developed in this study, nearly 2700 samples could be analyzed within eight hours by two persons. The combined results suggested that the new <i>hmg</i> gene assay developed here could be a universal maize reference gene system, and the new assay has high throughput and high accuracy for large-scale screening of maize varieties around the world.
format	article
author	Fang Liu Jinkui Cheng Xuhua Liu Xi-Qing Wang
author_facet	Fang Liu Jinkui Cheng Xuhua Liu Xi-Qing Wang
author_sort	Fang Liu
title	High-Throughput and Accurate Determination of Transgene Copy Number and Zygosity in Transgenic Maize: From DNA Extraction to Data Analysis
title_short	High-Throughput and Accurate Determination of Transgene Copy Number and Zygosity in Transgenic Maize: From DNA Extraction to Data Analysis
title_full	High-Throughput and Accurate Determination of Transgene Copy Number and Zygosity in Transgenic Maize: From DNA Extraction to Data Analysis
title_fullStr	High-Throughput and Accurate Determination of Transgene Copy Number and Zygosity in Transgenic Maize: From DNA Extraction to Data Analysis
title_full_unstemmed	High-Throughput and Accurate Determination of Transgene Copy Number and Zygosity in Transgenic Maize: From DNA Extraction to Data Analysis
title_sort	high-throughput and accurate determination of transgene copy number and zygosity in transgenic maize: from dna extraction to data analysis
publisher	MDPI AG
publishDate	2021
url	https://doaj.org/article/02d391b7def14c638041b977232097d9
work_keys_str_mv	AT fangliu highthroughputandaccuratedeterminationoftransgenecopynumberandzygosityintransgenicmaizefromdnaextractiontodataanalysis AT jinkuicheng highthroughputandaccuratedeterminationoftransgenecopynumberandzygosityintransgenicmaizefromdnaextractiontodataanalysis AT xuhualiu highthroughputandaccuratedeterminationoftransgenecopynumberandzygosityintransgenicmaizefromdnaextractiontodataanalysis AT xiqingwang highthroughputandaccuratedeterminationoftransgenecopynumberandzygosityintransgenicmaizefromdnaextractiontodataanalysis
_version_	1718411781524684800

High-Throughput and Accurate Determination of Transgene Copy Number and Zygosity in Transgenic Maize: From DNA Extraction to Data Analysis

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