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하보근,H. Roger Boerma 한국작물학회 2008 Journal of crop science and biotechnology Vol.11 No.2
Melting curve analysis of fluorescently labeled DNA fragments is used extensively for genotyping single nucleotide polymorphism (SNP). Here, we evaluated a SNP genotyping method by melting curve analysis with the two probe chemistries in a 384-well plate format on a Roche LightCycler 480. The HybProbe chemistry is based on the fluorescence resonance energy transfer (FRET) and the SimpleProbe chemistry uses a terminal self-quenching fluorophore. We evaluated FRET HybProbes and SimpleProbes for two SNP sites closely linked to two quantitative trait loci (QTL) for southern root-knot nematode resistance. These probes were used to genotype the two parents and 94 F₂ plants from the cross of PI 96354 × Bossier. The SNP genotypes of all samples determined by the LightCycler software agreed with previously determined SSR genotypes and the SNP genotypes determined on a Luminex 100 flow cytometry instrument. Multiplexed HybProbes for the two SNPs showed a 98.4% success rate and 100% concordance between repeats of two of the same 96 DNA samples. Also, we developed a HybProbe assay for the Rcs3 gene conditioning broad resistance to the frogeye leaf spot (FLS) disease. The LightCycler 480 provides rapid PCR on 384-well plate and allows simultaneous amplification and analysis in approximately 2 h without any additional steps after amplification. This allowed for a reduction of the potential contamination of PCR products, simplicity, and enablement of a streamlined workflow. The melting curve analysis on the LightCycler 480 provided high-throughput and rapid SNP genotyping and appears highly effective for marker-assisted selection in soybean. Melting curve analysis of fluorescently labeled DNA fragments is used extensively for genotyping single nucleotide polymorphism (SNP). Here, we evaluated a SNP genotyping method by melting curve analysis with the two probe chemistries in a 384-well plate format on a Roche LightCycler 480. The HybProbe chemistry is based on the fluorescence resonance energy transfer (FRET) and the SimpleProbe chemistry uses a terminal self-quenching fluorophore. We evaluated FRET HybProbes and SimpleProbes for two SNP sites closely linked to two quantitative trait loci (QTL) for southern root-knot nematode resistance. These probes were used to genotype the two parents and 94 F₂ plants from the cross of PI 96354 × Bossier. The SNP genotypes of all samples determined by the LightCycler software agreed with previously determined SSR genotypes and the SNP genotypes determined on a Luminex 100 flow cytometry instrument. Multiplexed HybProbes for the two SNPs showed a 98.4% success rate and 100% concordance between repeats of two of the same 96 DNA samples. Also, we developed a HybProbe assay for the Rcs3 gene conditioning broad resistance to the frogeye leaf spot (FLS) disease. The LightCycler 480 provides rapid PCR on 384-well plate and allows simultaneous amplification and analysis in approximately 2 h without any additional steps after amplification. This allowed for a reduction of the potential contamination of PCR products, simplicity, and enablement of a streamlined workflow. The melting curve analysis on the LightCycler 480 provided high-throughput and rapid SNP genotyping and appears highly effective for marker-assisted selection in soybean.