Research Article Open Access An Accurate and Rapid PCR-Based Zygosity Testing Method for Genetically

This article was first published in the Molecular Plant Breeding (Regular Print Version), and here was authorized to redistribute under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.. Preferred citation for this article as: Liu et al, 2009, An Accurate and Rapid PCR-Based Zygosity Testing Method for Genetically Modified Maize, Molecular Plant Breeding, 7(3): 619-623 Abstract Using genetically modified NK603 maize as an example, an accurate and cost-effective PCR-based procedure was described for genetically modified maize zygosity testing. The method involved a simple PCR reaction with four primers. The primers were designed from the inserted transgene at 5 ' and 3 ' ends (5'TP and 3'TP), the 5 ' flanking genomic DNA (5'GP), and the 3' flanking genomic DNA (3'GP), respectively. If the plant was a null (wild type), genomic DNA between 5'GP and 3'GP were amplified and a single PCR product was produced; If the plant was a homozygote, DNA between 5'GP and 5'TP, 3'TP and 3'GP were amplified and two PCR products were produced; If the plant was a hemizygote, DNA between 5'GP and 5'TP, 3'TP and 3'GP, 5GP and 3'GP were all amplified, producing three PCR products. PCR products with different sizes were separated on an agarose gel, and one, two and three bands were easily scored, which represents null, homozygote and hemizygote, respectively. Compared to expensive quantitative PCR and time-consuming bioassay, this method coupled with high throughput genomic DNA extraction is of great assistance to corn breeders for zygosity selection. Hundreds of leaf discs from individual plants can be punched with eppendor