Selection criteria for SNP loci to maximize robustness of high-resolution melting analysis for plant breeding

Yoshiyuki Yamagata, Atsushi Yoshimura, Toyoaki Anai, Satoshi Watanabe

Research output: Contribution to journalArticle

4 Citations (Scopus)

Abstract

DNA markers are useful for identifying genes and developing new genetic materials for breeding and genetic research. High-resolution melting (HRM) analysis can detect a single nucleotide polymorphism (SNP) in two polymerase chain reaction (PCR) fragments as a melting temperature (Tm) difference without additional experimental steps, such as gel electrophoresis. To design a method for developing reliable HRM markers that discriminate between homozygous alleles containing SNPs, we tested new evaluation indexes related to the thermodynamics of double-stranded DNA to find one that maximizes the difference in Tm values between PCR fragments. We found that differences in the change in Gibbs free energy (ΔG°) correlated with actual differences in Tm values. Optimization of the nearest neighboring nucleotide (NNN) of a SNP by nucleotide substitution in the primer and reducing the size of the PCR fragment both enlarged the actual differences in Tm. The genetic DNA markers we developed by NNN substitution, termed NNNs-HRM markers, could be precisely mapped within soybean chromosomes by linkage analysis. We developed a Perl script pipeline to enable the automatic design of a massive number of NNNs-HRM markers; these scripts are freely available and would be useful for practical breeding programs for other plant species.

Original languageEnglish
Pages (from-to)488-498
Number of pages11
JournalBreeding Science
Volume68
Issue number4
DOIs
Publication statusPublished - 2018

All Science Journal Classification (ASJC) codes

  • Agronomy and Crop Science
  • Genetics
  • Plant Science

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