332 PCR-based genetic markers on rice chromosomes

We have developed 332 PCR-based genetic markers including 161 sequence tagged site (STS) markers and 171 cleaved amplified polymorphic sequence (CAPS) markers. The table (chr 1 to chr 12) summarizes all information for these markers, such as chromosomal location, primer sequences, size of amplified fragment (Nipponbare), restriction enzyme, etc. These markers have been developed to detect polymorphism between Nipponbare (japonica) and Kasalath (indica). Although detection of polymorphism of these markers will depend on the combination of varieties or lines, they may be used for the analysis of other combinations.

STS markers

The 161 STS markers have been developed using clone-specific sequences (3'end) designed from the EST sequence derived from several cDNA libraries (Yamamoto et al. 1997). The chromosomal location of each marker has been identified by EST mapping (Wu et al. Plant Cell 2002) using a YAC-based physical map of rice. For all polymorphic markers, we have confirmed the chromosomal location by linkage analysis using 46 randomly selected BILs (Lin et al. 1998).
The reaction mixture for PCR amplification consisted of 25 ng total DNA, 200 mM of each dNTP (Boehringer Mannheim), 20 pmol primers (5'and 3'primers), 2 units of Taq DNA polymerase (Perkin-Elmer), PCR buffer (10 mM Tris pH 8.3 at 25°C, 50 mM KCl, and 0.001% (w/v) gelatin), and 40 mM MgCl2 in 20.0 L volume. Amplification was performed in GeneAmp PCR System 9600 (Perkin-Elmer) with 35 cycles of 94°C (1 min), 60°C (1 min), and 72°C (2 min), and a final cycle of 72°C for 7 min. The amplified DNA products were electrophoresed on 3.0% agarose gels in 0.5 x TBE buffer at 120 V for 2 h, and stained with ethidium bromide. The size standard marker for electrophoretic analysis was mixture of HindIII digests of DNA and HaeIII digests of X174DNA.

CAPS markers

We have developed 171 CAPS markers including 6 derived CAPS (dCAPS) markers (Konieczny and Ausbel 1993, Neff et al. 1998) using the information derived from a high-density RFLP linkage map (A HIGH-DENSITY RICE GENETIC MAP Harushima et al. 1998, The Latest High-Density Rice Genetic Map, Including 3267 Markers Rice Genome Research Program 2000). Using 5' and 3' sequence data for the clones used for RFLP linkage analysis, we designed unique primer pairs for the specific amplification of genome. Then, restriction digestion was carried out to detect polymorphism. In order to confirm the chromosomal location of CAPS markers, linkage analysis was performed using 14 randomly selected F2 plants (Harushima et al. 1998).
PCR amplification was performed using the GeneAmp PCR System 9600 and the following amplification condition: 30 cycles of 94°C (1 min), 60°C (2 min), and 72°C (3 min), and a final cycle of 72°C for 7 min. The amplified products of each primer pair were digested with 28 restriction enzymes (PstI, HindIII, BamHI, EcoRI, ApaI, XhoI, KpnI, HaeIII, DraI, XbaI, SalI, EcoT14I, MspI, HinfI, EcoRV, BglII, SacI, HhaI, EcoT22I, HapII, ScaI, AfaI, MluI, PshBI, MboI, MvaI, SacII, and HincII). For some markers, the amplified products were further digested with 14 more restriction enzymes (AccII, AluI, AvaII, BcnI, Cfr13I, AccI, AvaI, BanII, Cfr10I, EaeI, HaeII, MflI, Bsp1286I, and TthHB8I). The digested products were electrophoresed on 2.0% agarose gels in 0.5 x TBE buffer at 120 V for 2 h, and stained with ethidium bromide. For dCAPS marker development, we performed the genomic DNA sequencing to identify nucleotide polymorphisms between Nipponbare and Kasalath, and created a unique restriction site into the PCR product in one of varieties by combination of the nucleotide polymorphism with mismatch primer sequences. PCR amplifications with dCAPS primers were carried out at the same condition as that with CAPS primers and the resultant products were digested with the enzyme. The digested products was electrophoresed on 4.0% agarose gels in 0.5 x TBE buffer at 120 V for 2 h, and stained with ethidium bromide.

Harushima Y, Yano M, Shomura A, Sato M, Shimano T, Kuboki Y, Yamamoto T, Lin SY, Antonio BA, Parco A, Kajiya H, Huang N, Yamamoto K, Nagamura Y, Kurata N, Khush GS and Sasaki T (1998) A high-density rice genetic linkage map with 2275 markers using a single F2 population. Genetices 148: 479-494.

Konieczny A and Ausbel FM (1993) A procedure for mapping Arabidopsis mutations using co-dominant ecotype-specific PCR-based markers. Plant J. 4: 403-410.

Lin SY, Sasaki T and Yano M (1998) Mapping quantitative trait loci controlling seed dormancy and heading date in rice, Oryza sativa L., using backcross inbred lines. Theor Appl Genet 96: 997-1003.

Neff MM, Neff JD, Chory J and Pepper AE (1998) dCAPS, a simple technique for the genetic analysis of single nucleotide polymorphisms: experimental applications in Arabidopsis taliana genetics. Plant J. 14: 387-392.

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 and Sasaki T (2002) A comprehensive rice transcript map containing 6591 EST site. Plant Cell

Yamamoto K and Sasaki T (1997) Large-scale EST sequencing in rice. Plant Mol Biol 35: 135-144.