Genetically modified crops have been approved worldwide. The most successful and effective transgenic traits thus far are herbicide tolerance and insect resistance (Jauhar, 2006). Transgenic maize events with insect resistance such as Bt11, Event176, MON810, MON863 and TC1507 are widely used in the US. Herbicide tolerance is conferred by 5-enolpyruvlyshikimate-3-phosphate synthase gene (EPSPS) isolated from the soil bacterium Agrobacterium tumefaciens strain CP4. The gene encodes a glyphosate tolerant form of the enzyme EPSPS. Glyphosate, the active ingredient in the herbicide Roundup, specifically binds to and inactivates the enzyme EPSPS in plant, which is part of the shikimate pathway. The shikimate pathway is involved in the biosynthesis of the aromatic amino acids tyrosine, phenylalanine and tryptophan, as well as other aromatic compounds. When conventional plants are treated with glyphosate they cannot produce the aromatic amino acids essential to their survival.

Maize line NK603 harboring two copies of the CP4 EPSPS was developed by Monsanto to allow the use of glyphosate-containing herbicide as a weed control option for maize crops (Heck et al., 2005). It has been approved for feed, food, and food and/or feed by Argentina, Australia, Canada, China, European Union, Japan, South Korea, Mexico, Philippines, South Africa, Taiwan, and United States. As corn breeders continue to integrate the transgenic trait into newly developed inbred lines, NK603 event will continue to play important roles in weed management worldwide.

The common process of integrating NK603 event into local elite corn inbred lines is through backcrossing coupled with application of glyphosate for selection. At the end of backcrossing, two consecutive selfings are needed to make the transgene homozygous at the transgene locus and select homozygotes. If progenies of the second selfing are not segregating for glyphosate tolerance, the selected plant of previous generation is then a homozygote. Quantitative polymerase chain reaction (QPCR) and other technologies (Gupta et al., 2007) can be used for zygosity testing due to their sensitivities and specificities (Ingham et al., 2001; Song et al., 2002; German et al., 2003). QPCR measures a fluorescent signal after every PCR cycle allowing for analysis of data in the logarithmic phase, not at the end point where the PCR products can no longer be quantified. For this application, an endogenous single copy gene is used for data normalization required to measure the relative copy number of the gene of interest. This QPCR assay allows determination of two-fold difference in copy number between hemizygote and homozygote transgenic plants. However, QPCR requires high quality genomic DNA, expensive equipment and chemicals, and skilled technicians, all of which have hindered QPCR in transgene zygosity testing for corn breeders.

This paper described a simple, low-cost, and high throughput method for maize NK603 transgene zygosity testing. With this method, a single person could process hundreds of plant samples from genomic DNA preparation to plant zygosity status in a single day. It was much easier for breeders to select in the field, and was also time & labor saving. Moreover, this strategy could be applied to develop zygosity testing for any other transgenic events as mentioned above provided that their transgene sequences at 5' and 3' junctions were available.

1 Materials and methods
1.1 Plant material and genomic DNA extraction
Non-transgenic corn, NK603 inbred lines and hybrids were used for this study. A high-throughput DNA extraction procedure was adopted from Xin et al. (2002). The advantage of this method was that extensive maceration was not required. It was robust, simple, fast and high-throughput. Briefly, 10~30 mm2 plant leaf tissues were incubated in 50 μL of freshly made extraction buffer (100 mmol/L NaOH and 2% Tween 20) at 95℃ for 10 min. The extraction mixture was then neutralized by 50 μL neutralization buffer (100 mmol/L Tris-HCl and 2 mmol/L EDTA, pH 2.0). After gentle mixing, the supernatant was ready for PCR (1 μL is enough for one 25 μL PCR reaction). Large amount of samples can be processed with 96-well plates.

1.2 Primers, PCR conditions and products analysis
NK603 specific primers were designed from the unique integration junction sequences between the host plant genome DNA and the transgene. A pair of primers was designed from the 5' junction and the other from the 3' junction. At each junction, one primer was designed from flanking genomic DNA and the other from the transgene. In order to multiplex them, they were designed with similar melting temperatures. PCR products with different sizes were less than 500 bp in length. The four primers were as follows:

 5'GP: 5'-GTCAAAGGATGCGGAACTGTT-3';
 5'TP: 5'-GAGTAAGCTTGTTAACGCGG-3';
 3'TP: 5'-ACCATAACTTCTGCTCGTTGC-3';
 3'GP: 5'-AAAGAACAAGTTGGATGCCGC-3'.
 

The total PCR volume was 25 μL: 2.5 μL of 10×PCR buffer, 1 μL of DNA template, 1.5 mmol/L MgCl2, 0.2 mmol/L each dATP, dCTP, dTTP, and dGTP, 0.25 μmol/L each of the four primers and 0.5 unit of Taq DNA polymerase. Amplification was carried out with an initial denaturation at 95℃ for 2 min, followed by 40 cycles of 95℃ for 15 s, 56℃ for 25 s, and 72℃ for 1 min, and a final extension step of 5 min at 72℃. PCR products were analyzed by electrophoresis on 3% agarose gels in 0.5×TAE.

2 Results and discussion
2.1 Designing strategy of zygosity testing
Zygosity testingwas achieved with four PCR primers. One primer was designed based on the 5' flanking genomic DNA sequence (5'GP), one was based on the 3' flanking genomic DNA sequence (3'GP), the third was designed from the transgene near the 5' end (5'TP), and the fourth near the 3' end (3'TP) (Figure 1).
 

The four primers were designed in such a way that their PCR products were less than 500 bp in length and were of different sizes so that they were different from each other on agarose gels. If a plant was a null, there was no transgene in the genome, therefore, only genomic DNA between 5'GP and 3'GP were amplified producing a single PCR product. If a plant was a homozygote, DNA between 5'GP and 5'TP, 3'TP and 3'GP were amplified and two PCR products were produced. Taq DNA polymerase catalyzes DNA synthesis at a rate about 1 kb per min. The transgene of NK603 event was longer than 6 kb, so the distance between 5'GP and 3'GP in a homozygote was too far for the DNA in between to be amplified even these two primers do pair with correct orientation. If a plant was a hemizygote, DNA between 5'GP and 3'GP, 5'GP and 5'TP, 3'TP and 3'GP were all amplified and three PCR products were produced. One band, two and three bands were easily distinguished on an agarose gel, which represents null, homozygote and hemizygote at the transgene locus, respectively.

2.2 Development of NK603 specific zygosity testing
NK603 specific primers were designed based on sequences at both 5' and 3' insertion junctions (Figure 2).
 

The primer 5'GP was located at the 5' flanking genomic DNA, while 5'TP was from the transgene at the 5' end (the rice actin1 promoter). This pair of primers gave rise to a single PCR product of 204 bp (Figure 3). The primer 3'GP was located at the 3' flanking genomic DNA, while 3'TP was from the transgene at the 3' end (the NOS terminator).  This pair of primers gave rise to a single PCR product of 265 bp (Figure 3). When 5'GP and 3'GP pair together and there was no transgene in between, a single PCR product of 356 bp was produced (Figure 3). 5'GP/5'TP and 3'TP/3'GP were NK603 specific and were not cross-amplified of genomic DNA from other transgenic events including Bt11, Event176, GA21, MON810, MON863 and TC1507. When these four primers were used together, one band, two bands and three bands were amplified from null, homozygous and hemizygous plants as expected (Figure 3). NK603 zygosity were also detected by using three primers, either 5'GP, 3'TP and 3'GP or 5'GP, 5'TP and 3'GP. In both cases, genomic DNA, transgene, genomic DNA and transgene were amplified from a null, homozygote and hemizygote, respectively (Figure 4). Same zygosity results were obtained as those using four primers as shown before. We validated the procedure with both three and four primer combinations on different NK603 homologous inbred lines and heterologous hybrid verities and various non-transgenic corn materials. Correct zygosity was achieved as expected.
 

 In summary, here we reported a simple, fast and cost-effective PCR-based transgene zygosity testing method which would help corn breeders effectively do trait integrations. Similar strategy can be applied to other corn transgenic events provided their sequences at transgene insertion junctions are available.

Acknowledgements
This research was supported by Shandong Pivotal Project for Agricultural Elite Variety Development ((2008)6).

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