Genotyping assay for F2 and F3generations
Fluorescence quantitative PCR and digital PCR were used to identify homozygous (RR), GR heterozygous (RS) or homozygous sensitive (SS) hybrids across different plants of the F2 and F3 populations, and the sequences of the primers and probes used in the present study are shown in Table 1.
Thirty days after transplanting, fresh leaf samples from GM, wild, F2 and F3 were used for total DNA extraction. Approximately 200 mg of leaf tissue was ground to a fine powder in liquid nitrogen, and DNA was extracted using a DNeasy® Plant Mini Kit (Qiagen, Hilden, Germany). The purified DNA was then used as a template for qPCR to amplify the soybean lectin gene and EPSPS gene, and each reaction was repeated three times. To obtain reliable results, Ct values and the ∆Ct between the Ct for the transgene and the Ct for the endogenous control were used to determine which plants contained the transgene. Samples were considered positive for amplicon production when the lectin gene Ct values and EPSPS gene Ct values were both < 35, and the amplification plot clearly demonstrated an exponential increase in the reporter signal in duplicate PCRs. A negative result was assigned when lectin gene Ct values < 35 and no amplification of the EPSPS gene occurred. Samples with lectin gene Ct values < 35 and EPSPS gene Ct values > 35 were considered indeterminant and required repeat testing.
Samples testing positive by real-time PCR were analyzed forlectin and EPSPS copy number by digital PCR. The copy number ratio, which is expressed as a ratio between target and referencelectin genes for each DNA sample, was calculated and directly used as an indicator for identifying heterozygous and homozygous individuals. A copy number ratio close to 1 would suggest that the sample is a homozygous individual, and a copy number ratio close to 0.5 would suggest a heterozygous individual.