Protocol: a simple gel-free method for SNP genotyping using allele-specific primers in rice and other plant species.
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.
Hirotsu
Naoki
N
Division of Plant Sciences, National Institute of Agrobiological Sciences, Kannondai 2-1-2, Tsukuba, Ibaraki 305-8602, Japan. kenshi@nias.affrc.go.jp.
Murakami
Naomi
N
Kashiwagi
Takayuki
T
Ujiie
Kazuhiro
K
Ishimaru
Ken
K
Theor Appl Genet. 2008 Mar;116(5):603-12
18097643
Nucleic Acids Res. 2001 Oct 1;29(19):E93
11574695
Plant Physiol. 2006 Jan;140(1):12-7
16403737
Nat Rev Genet. 2001 Dec;2(12):930-42
11733746
Anal Biochem. 1999 Dec 1;276(1):59-64
10585744
Clin Chem. 2001 Feb;47(2):164-72
11159763
Science. 2002 Apr 5;296(5565):79-92
11935017
Plant Physiol. 2003 May;132(1):84-91
12746514
Plant Physiol. 2004 Jul;135(3):1198-205
15266053
Nucleic Acids Res. 1990 Feb 25;18(4):999-1005
2179874
Biochimie. 1996;78(5):364-9
8905155
Nucleic Acids Res. 1980 Oct 10;8(19):4321-5
7433111
DNA Res. 2002 Oct 31;9(5):163-71
12465716
Plant Physiol. 2000 Dec;124(4):1483-92
11115864
DNA Res. 2002 Dec 31;9(6):199-207
12597276
Nucleic Acids Res. 2004 Jul 12;32(12):e103
15249599
Nat Genet. 1999 Oct;23(2):203-7
10508518
PCR Methods Appl. 1995 Jun;4(6):357-62
7580930
Genome Res. 2000 Aug;10(8):1249-58
10958643
Methods Mol Biol. 2001;170:103-16
11357675
Plant Physiol. 2002 Jun;129(2):440-50
12068090
Genome Res. 1998 Dec;8(12):1245-58
9872980
PCR Methods Appl. 1992 Aug;2(1):14-20
1490171
Genetics. 1998 Jan;148(1):479-94
9475757
Genome Res. 1999 May;9(5):492-8
10330129
Nucleic Acids Res. 1989 Apr 11;17(7):2503-16
2785681
Genetics. 2003 Mar;163(3):1123-34
12663549
DNA Res. 2004 Aug 31;11(4):275-83
15500252
Planta. 2008 Feb;227(3):601-9
17960419
Nucleic Acids Res. 1997 Dec 15;25(24):4876-82
9396791
DNA Res. 2006 Apr 30;13(2):43-51
16766512
Proc Natl Acad Sci U S A. 2006 Jun 20;103(25):9578-83
16766658
Theor Appl Genet. 2004 May;108(7):1212-20
14740086
Plant J. 1993 Aug;4(2):403-10
8106085
Genome Res. 2004 Sep;14(9):1812-9
15342564
Nature. 2005 Aug 11;436(7052):793-800
16100779