High-throughput SNP Genotyping by Melting Curve Analysis for Resistance to Southern Root-knot Nematode and Frogeye Leaf Spot 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