Dot-blot-SNP analysis for practical plant breeding and cultivar identification in rice.
We report dot-blot hybridization with allele-specific oligonucleotides for single nucleotide polymorphisms (SNPs) analysis to be applicable for practical plant breeding and cultivar identification. Competitive hybridization of a digoxigenin-labeled oligonucleotide having the sequence of a mutant allele (or a wild-type allele) together with an unlabeled oligonucleotide having the sequence of a wild-type allele (or a mutant allele) was highly effective to reduce background signals in dot-blot hybridization. All 100 tested genes (200 alleles) in rice having SNPs or insertions/deletions were detected in an allele-specific manner. Genotypes of 43 rice cultivars were identified by this technique, and eight SNP markers were found to be sufficient for distinguishing all the cultivars from each other. Dot-blot analysis was also applied to genotyping of Wx and Sd1 of F4 plants in a conventional breeding program. Since dot-blot analysis with competitive hybridization provides a highly reliable, simple, and cost-effective technique for SNP analysis of a large number of samples, this technique is expected to realize the practical use of a novel breeding method, in which plants or breeding lines are selected by SNP analyses of many genes in a laboratory.
Shirasawa
K
K
Graduate School of Agricultural Science, Tohoku University, Aoba-ku, Sendai 981-8555, Japan.
Shiokai
S
S
Yamaguchi
M
M
Kishitani
S
S
Nishio
T
T
D000483:Alleles; D001483:Base Sequence; D001947:Breeding; D017931:DNA Primers / Q000235:genetics; D018744:DNA, Plant / Q000235:genetics; D005838:Genotype; D009693:Nucleic Acid Hybridization; D012275:Oryza / Q000235:genetics*; D020641:Polymorphism, Single Nucleotide
D017931:DNA Primers; D018744:DNA, Plant