An Accurate and Rapid PCR-Based Zygosity Testing Method for Genetically Modified Maize

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 eppendorf tubes in field and plant zygosity result is readily available. Similar strategy can be applied to develop event-specific zygosity testings for other maize genetically modified events such as Bt11, Event176, GA21, MON810, MON863 and TC1507 provided that their sequences at both insertion junctions are available.