Cases for co-dominant DNA marker development between two Japanese two-rowed hulled barleys using Newly designed temperature-switch PCR primers, and consideration of optimized primer concentration for improvement of success rate
2020
Haraguchi, Y. (Fukuoka Agriculture and Forestry Research Center, Chikushino, Fukuoka (Japan)) | Todoroki, T. | Kai, H.
Temperature-switch PCR (TSP) marker is an efficient codominant DNA marker to identify single nucleotide polymorphisms (SNPs) in a single PCR experiment. SNPs can be detected using two locus-specific primers, which requires amplification of a fragment from template DNA containing the SNP, and one allele-specific (as) primer which has a nucleotide that corresponds to a specific SNP site at the 3’ terminal. Although TSP marker is an easy method for SNP genotyping, there are few reports about TSP marker development. To accumulate cases of TSP marker development, we designed 77 TSP primer sets for genotyping of 77 SNPs in two-rowed barley. As a result, success rate of codominant markers was 32.5% (25/77) under standard conditions of reaction solution composition, and reaction temperature as previously reported. The success rate increased to 41.6% (32/77) when the as primer concentration was adjusted appropriately. As another element to obtain higher detection efficiency of SNPs it was considered that the difference in values of melting temperatures between the locus-specific primer and the as primer should be 6-8℃. This information can be used for improvement of success rate for TSP marker development.
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