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.