Abstract
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.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Asamizu E, Nakamura Y, Sato S, Tabata S (2000) A large scale analysis of cDNA in Arabidopsis thaliana: generation of 12,028 non-redundant expressed sequence tags from normalized and size-selected cDNA libraries. DNA Res 7:175–180
Ashikari M, Sakakibara H, Lin S, Yamamoto T, Takashi T, Nishimura A, Angeles ER, Qian Q, Kitano H, Matsuoka M (2005) Cytokinin oxidase regulates rice grain production. Science 309:741–745
Doi K, Izawa T, Fuse T, Yamanouchi U, Kubo T, Shimatani Z, Yano M, Yoshimura A (2004) Ehd1, a B-type response regulator in rice, confers short-day promotion of flowering and controls FT-like gene expression independently of Hd1. Genes Dev 18:926–936
Edwards K, Johnstone C, Thompson C (1991) A simple and rapid method for the preparation of plant genomic DNA for PCR analysis. Nucleic Acids Res 19:1349
Fischer SG, Lerman LS (1983) DNA fragments differing by single base-pair substitutions are separated in denaturing gradient gels: correspondence with melting theory. Proc Natl Acad Sci U S A 80:1579–1583
Fujii K, Hayano-Saito Y, Saito K, Sugiura N, Hayashi N, Tsuji T, Izawa T, Iwasaki M (2000) Identification of a RFLP marker tightly linked to the panicle blast resistance gene, Pb1, in rice. Breed Sci 50:183–188
Hayano-Saito Y, Tsuji T, Fujii K, Saito K, Iwasaki M, Saito A (1998) Localization of the rice stripe disease resistance gene, Stv−b i, by graphical genotyping and linkage analyses with molecular markers. Theor Appl Genet 96:1044–1049
Internationl Rice Genome Sequencing Project (2005) The map-based sequence of the rice genome. Nature 436:793–800
Jain M, Thorstenson YR, Faulkner DM, Pourmand N, Jones T, Au M, Oefner PJ, White KP, Davis RW (2003) Genotyping African haplotypes in ATM using a co-spotted single-base extension assay. Hum Mutat 22:214–221
Latorra D, Campbell K, Wolter A, Hurley JM (2003) Enhanced allele-specific PCR discrimination in SNP genotyping using 3′ locked nucleic acid (LNA) primers. Hum Mutat 22:79–85
Matsubara Y, Kure S (2003) Detection of single nucleotide substitution by competitive allele-specific short oligonucleotide hybridization (CASSOH) with immunochromatographic strip. Hum Mutat 22:166–172
Michaels SD, Amasino RM (1998) A robust method for detecting single-nucleotide changes as polymorphic markers by PCR. Plant J 14:381–385
Nasu S, Suzuki J, Ohta R, Hasegawa K, Yui R, Kitazawa N, Monna L, Minobe Y (2002) Search for and analysis of single nucleotide polymorphisms (SNPs) in rice (Oryza sativa, Oryza rufipogon) and establishment of SNP markers. DNA Res 9:163–171
Orita M, Iwahana H, Kanazawa H, Hayashi K, Sekiya T (1989) Detection of polymorphisms of human DNA by gel electrophoresis as single-strand conformation polymorphisms. Proc Natl Acad Sci USA 86:2766–2770
Ross PL, Lee K, Belgrader P (1997) Discrimination of single-nucleotide polymorphisms in human DNA using peptide nucleic acid probes detected by MALDI-TOF mass spectrometry. Anal Chem 69:4197–4202
Saghai Maroof MA, Biyashev RM, Yang GP, Zhang Q, Allard RW (1994) Extraordinarily polymorphic microsatellite DNA in barley: species diversity, chromosomal locations, and population dynamics. Proc Natl Acad Sci USA 91:5466–5470
Saiki RK, Bugawan TL, Horn GT, Mullis KB, Erlich HA (1986) Analysis of enzymatically amplified beta-globin and HLA-DQ alpha DNA with allele-specific oligonucleotide probes. Nature 324:163–166
Sasaki A, Ashikari M, Ueguchi-Tanaka M, Itoh H, Nishimura A, Swapan D, Ishiyama K, Saito T, Kobayashi M, Khush GS, Kitano H, Matsuoka M (2002) Green revolution: a mutant gibberellin-synthesis gene in rice. Nature 416:701–702
Sato H, Suzuki Y, Sakai M, Imbe T (2002) Molecular characterization of Wx-mq, a novel mutant gene for low-amylose content in endosperm of rice (Oryza sativa L.). Breed Sci 52:2131–2135
Shirasawa K, Monna L, Kishitani S, Nishio T (2004) Single nucleotide polymorphisms in randomly selected genes among japonica rice (Oryza sativa L.) varieties identified by PCR-RF-SSCP. DNA Res 11:275–283
Shirasawa K, Kishitani S, Nishio T (2005) Dot-blot analysis for identification of japonica rice cultivars and genotyping of recombinant inbred lines. Breed Sci 55:187–192
Song WY, Wang GL, Chen LL, Kim HS, Pi LY, Holsten T, Gardner J, Wang B, Zhai WX, Zhu LH, Fauquet C, Ronald P (1995) A receptor kinase-like protein encoded by the rice disease resistance gene, Xa21. Science 270:1804–1806
Stoerker J, Mayo JD, Tetzlaff CN, Sarracino DA, Schwope I, Richert C (2000) Rapid genotyping by MALDI-monitored nuclease selection from probe libraries. Nat Biotechnol 18:1213–1216
Teranishi M, Iwamatsu Y, Hidema J, Kumagai T (2004) Ultraviolet-B sensitivities in Japanese lowland rice cultivars: cyclobutane pyrimidine dimer photolyase activity and gene mutation. Plant Cell Physiol 45:1848–1856
The Arabidopsis Genome Initiative (2000) Analysis of the genome sequence of the flowering plant Arabidopsis thaliana. Nature 408:796–815
Till BJ, Burtner C, Comai L, Henikoff S (2004) Mismatch cleavage by single-strand specific nucleases. Nucleic Acids Res 32:2632–2641
Umemoto T, Aoki N, Lin H, Nakamura Y, Inouchi N, Sato Y, Yano M, Hirabayashi H, Maruyama S (2004) Natural variation in rice starch synthase IIa affects enzyme and starch properties. Funct Plant Biol 31:671–684
Wang H, Qi M, Cutler AJ (1993) A simple method of preparing plant samples for PCR. Nucleic Acids Res 21:4153–4154
Wang HY, Luo M, Tereshchenko IV, Frikker DM, Cui X, Li JY, Hu G, Chu Y, Azaro MA, Lin Y, Shen L, Yang Q, Kambouris ME, Gao R, Shih W, Li H (2005) A genotyping system capable of simultaneously analyzing >1000 single nucleotide polymorphisms in a haploid genome. Genome Res 15:276–283
Wu J, Maehara T, Shimokawa T, Yamamoto S, Harada C, Takazaki Y, Ono N, Mukai Y, Koike K, Yazaki J, Fujii F, Shomura A, Ando T, Kono I, Waki K, Yamamoto K, Yano M, Matsumoto T, Sasaki T (2002) A comprehensive rice transcript map containing 6591 expressed sequence tag sites. Plant Cell 14:525–535
Yano M, Katayose Y, Ashikari M, Yamanouchi U, Monna L, Fuse T, Baba T, Yamamoto K, Umehara Y, Nagamura Y, Sasaki T (2000) Hd1, a major photoperiod sensitivity quantitative trait locus in rice, is closely related to the Arabidopsis flowering time gene CONSTANS. Plant Cell 12:2473–2484
Zhou GH, Shirakura H, Kamahori M, Okano K, Nagai K, Kambara H (2004) A gel-free SNP genotyping method: bioluminometric assay coupled with modified primer extension reactions (BAMPER) directly from double-stranded PCR products. Hum Mutat 24:155–163
Acknowledgments
This work was supported in part by a grant-in-aid (14206001) from the Ministry of Education, Science, Sports and Culture, Japan and Green-Techno project of the Ministry of Agriculture, Forestry, and Fisheries, Japan. K. Shirasawa is a recipient of the Research Fellowships of the Japan Society for the Promotion of Science for Young Scientists.
Author information
Authors and Affiliations
Corresponding author
Additional information
Communicated by M. Xu
Electronic supplementary material
Rights and permissions
About this article
Cite this article
Shirasawa, K., Shiokai, S., Yamaguchi, M. et al. Dot-blot-SNP analysis for practical plant breeding and cultivar identification in rice. Theor Appl Genet 113, 147–155 (2006). https://doi.org/10.1007/s00122-006-0281-7
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00122-006-0281-7