Allele-specific hybridization using streptavidin-coated magnetic beads for species identification, S genotyping, and SNP analysis in plants

Kaoru Tonosaki, Junpei Kudo, Hiroyasu Kitashiba, Takeshi Nishio

Research output: Contribution to journalArticle

8 Citations (Scopus)

Abstract

Dot-blot hybridization has been successfully used for the construction of single nucleotide polymorphism (SNP)-based linkage maps, quantitative trait locus analysis, marker-assisted selection, and the identification of species and cultivars. This method is, however, time-consuming, even for a small number of plant samples. We propose a method in which streptavidin-coated magnetic beads replace the nylon membrane for immobilization of the PCR products and are hybridized with allele-specific oligonucleotide probes and a digoxigenin-labeled oligonucleotide hybridized with the allele-specific oligonucleotide probe. After amplification of plant DNA by PCR with the biotinylated primers, those oligonucleotide probes having species-specific or allele-specific sequences were mixed together with the digoxigenin-labeled oligonucleotide and the streptavidin-coated magnetic beads at a temperature suitable for each probe. Species-specific internal transcribed spacer 1 (ITS1) sequences and allele-specific sequences of the hypervariable region I of S-locus receptor kinase (SRK) specifically detected ITS1 sequences and SRK alleles in Brassica species, respectively. SNPs were also successfully analyzed by using allele-specific oligonucleotide probes and competitive oligonucleotides. In the SNP analysis, PCR products were indirectly captured by magnetic beads. SNP alleles of eight cultivars each of Brassica rapa and Raphanus sativus were analyzed using streptavidin-coated magnetic beads. The genotyping results corresponded well with those of dot-blot-SNP analysis. Although allele-specific hybridization using streptavidin-coated magnetic beads is somewhat costly, it is easier and more rapid than dot-blot hybridization. This method is suitable for the analysis of a small number of plant samples with a large number of DNA markers.

Original languageEnglish
Pages (from-to)419-428
Number of pages10
JournalMolecular Breeding
Volume31
Issue number2
DOIs
Publication statusPublished - 2013 Jan 1

Keywords

  • Brassica
  • DNA markers
  • Dot-blot hybridization
  • ITS1
  • SRK
  • Self-incompatibility

ASJC Scopus subject areas

  • Biotechnology
  • Molecular Biology
  • Agronomy and Crop Science
  • Genetics
  • Plant Science

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