Copy Number Estimation of a Plant-Derived Selectable Marker Gene by High Resolution Melting Analysis: A Tool to Simplify Transgenic Plant Breeding

Transferring a transgenic trait from a lab genotype into a cultivated plant variety requires selection of single-locus T0 plants, a backcrossing breeding program with transgenic progeny selection at each cycle and, generally, attaining the homozygous state for the transgene locus. Polyploidy and outcrossing complicate this process, as in the case of alfalfa, an autotetraploid, cross-fertilizing forage legume. Recently, a new, efficient selectable marker gene, MsGSAgr, derived from the alfalfa glutamate 1-semialdehyde aminotransferase (GSA) gene was described. Since MsGSAgr differs from the wildtype GSA by one nucleotide, high resolution melting (HRM) analysis could be used to screen transgenic plants for MsGSAgr, and linked transgene(s), copy number. An HRM assay was developed by simulating different copy numbers with mixes of plasmids containing mutated and wild-type GSA sequences. The assay was validated by analyzing transgenic alfalfa plants containing one or multiple MsGSAgr loci. HRM enabled us to clearly discriminate transgenic from non-transgenic plants, and the single copy from the multicopy state, thus providing a tool to streamline molecular plant breeding.