A Taqman-based assay to determine T-DNA zygosity in GM forage

Reid M, Crowther T, Winichayakul S, Roberts NJ, Bryan G and Richardson K

AgResearch Ltd., Grasslands Research Centre, Private Bag 11008, Tennent Drive, Palmerston North 4442, New Zealand.

The identification of plants with a homozygous transgene is essential to the breeding of genetically modified forage grasses. Using rice as a model, we have developed a zygosity assay based on real-time qPCR and TaqMan hydrolysis probes, which allows us to readily distinguish between plants which are either hemizygous or homozygous for the transgene. Plants transformed with a single T-DNA (containing a synthetic DGAT/Oleosin construct designed to accumulate fatty acids in foliar tissue) were identified by Southern blot hybridisation and used to generate T1 families for these experiments. The segregation ratios of T1 progeny, using standard PCR, were as expected for an inbred species and the qPCR assay reliably differentiated hemizygous and homozygous individuals in each family. These results positively correlate with fatty acid methyl ester (FAMES) analyses, where a significant increase in the C18:1/C18:3 and C18:2/C18:3 fatty acid ratios was observed in homozygous progeny. Quantitative immunoblots performed using a sesame-oleosin antibody also positively correlated with the homozygous progeny identified via the qPCR assay results. This T-DNA zygosity assay is easily modified for use with other plant species and has been adapted for use in our GM ryegrass seed production program, where populations containing a homozygous DGAT/Oleosin construct are currently being developed for field trialling in the United States over the next five years. Development of the assay in rice will be presented.