title: Protocol: a simple gel-free method for SNP genotyping using allele-specific primers in rice and other plant species.
abstract: BACKGROUND
  Genotype analysis using multiple single nucleotide polymorphisms (SNPs) is a useful but labor-intensive or high-cost procedure in plant research. Here we describe an alternative genotyping method that is suited to multi-sample or multi-locus SNP genotyping and does not require electrophoresis or specialized equipment.
  
  
  RESULTS
  We have developed a simple method for multi-sample or multi-locus SNP genotyping using allele-specific primers (ASP). More specifically, we (1) improved the design of allele-specific primers, (2) established a method to detect PCR products optically without electrophoresis, and (3) standardized PCR conditions for parallel genomic assay using various allele-specific primers. As an illustration of multi-sample SNP genotyping using ASP, we mapped the locus for lodging resistance in a typhoon (lrt5). Additionally, we successfully tested multi-locus ASP-PCR analysis using 96 SNPs located throughout the genomes of rice (Oryza sativa) cultivars 'Koshihikari' and 'Kasalath', and demonstrated its applicability to other diverse cultivars/subspecies, including wild rice (O. rufipogon).
  
  
  CONCLUSIONS
  Our ASP methodology allows characterization of SNPs genotypes without electrophoresis, expensive probes or specialized equipment, and is highly versatile due to the flexibility in the design of primers. The method could be established easily in any molecular biology laboratory, and is applicable to diverse organisms.
authors.affiliation: Division of Plant Sciences, National Institute of Agrobiological Sciences, Kannondai 2-1-2, Tsukuba, Ibaraki 305-8602, Japan. kenshi@nias.affrc.go.jp.
authors.forename: Naoki
authors.initials: N
authors.lastname: Hirotsu
authors.forename: Naomi
authors.initials: N
authors.lastname: Murakami
authors.forename: Takayuki
authors.initials: T
authors.lastname: Kashiwagi
authors.forename: Kazuhiro
authors.initials: K
authors.lastname: Ujiie
authors.forename: Ken
authors.initials: K
authors.lastname: Ishimaru
references.citation: Theor Appl Genet. 2008 Mar;116(5):603-12
references.pmid: 18097643
references.citation: Nucleic Acids Res. 2001 Oct 1;29(19):E93
references.pmid: 11574695
references.citation: Plant Physiol. 2006 Jan;140(1):12-7
references.pmid: 16403737
references.citation: Nat Rev Genet. 2001 Dec;2(12):930-42
references.pmid: 11733746
references.citation: Anal Biochem. 1999 Dec 1;276(1):59-64
references.pmid: 10585744
references.citation: Clin Chem. 2001 Feb;47(2):164-72
references.pmid: 11159763
references.citation: Science. 2002 Apr 5;296(5565):79-92
references.pmid: 11935017
references.citation: Plant Physiol. 2003 May;132(1):84-91
references.pmid: 12746514
references.citation: Plant Physiol. 2004 Jul;135(3):1198-205
references.pmid: 15266053
references.citation: Nucleic Acids Res. 1990 Feb 25;18(4):999-1005
references.pmid: 2179874
references.citation: Biochimie. 1996;78(5):364-9
references.pmid: 8905155
references.citation: Nucleic Acids Res. 1980 Oct 10;8(19):4321-5
references.pmid: 7433111
references.citation: DNA Res. 2002 Oct 31;9(5):163-71
references.pmid: 12465716
references.citation: Plant Physiol. 2000 Dec;124(4):1483-92
references.pmid: 11115864
references.citation: DNA Res. 2002 Dec 31;9(6):199-207
references.pmid: 12597276
references.citation: Nucleic Acids Res. 2004 Jul 12;32(12):e103
references.pmid: 15249599
references.citation: Nat Genet. 1999 Oct;23(2):203-7
references.pmid: 10508518
references.citation: PCR Methods Appl. 1995 Jun;4(6):357-62
references.pmid: 7580930
references.citation: Genome Res. 2000 Aug;10(8):1249-58
references.pmid: 10958643
references.citation: Methods Mol Biol. 2001;170:103-16
references.pmid: 11357675
references.citation: Plant Physiol. 2002 Jun;129(2):440-50
references.pmid: 12068090
references.citation: Genome Res. 1998 Dec;8(12):1245-58
references.pmid: 9872980
references.citation: PCR Methods Appl. 1992 Aug;2(1):14-20
references.pmid: 1490171
references.citation: Genetics. 1998 Jan;148(1):479-94
references.pmid: 9475757
references.citation: Genome Res. 1999 May;9(5):492-8
references.pmid: 10330129
references.citation: Nucleic Acids Res. 1989 Apr 11;17(7):2503-16
references.pmid: 2785681
references.citation: Genetics. 2003 Mar;163(3):1123-34
references.pmid: 12663549
references.citation: DNA Res. 2004 Aug 31;11(4):275-83
references.pmid: 15500252
references.citation: Planta. 2008 Feb;227(3):601-9
references.pmid: 17960419
references.citation: Nucleic Acids Res. 1997 Dec 15;25(24):4876-82
references.pmid: 9396791
references.citation: DNA Res. 2006 Apr 30;13(2):43-51
references.pmid: 16766512
references.citation: Proc Natl Acad Sci U S A. 2006 Jun 20;103(25):9578-83
references.pmid: 16766658
references.citation: Theor Appl Genet. 2004 May;108(7):1212-20
references.pmid: 14740086
references.citation: Plant J. 1993 Aug;4(2):403-10
references.pmid: 8106085
references.citation: Genome Res. 2004 Sep;14(9):1812-9
references.pmid: 15342564
references.citation: Nature. 2005 Aug 11;436(7052):793-800
references.pmid: 16100779