|
Images of gel electrophoresis
|
RGP caps
|
10.18908/lsdba.nbdc00318-05-002
|
Detailed information and images of gel electrophoresis of each marker.
|
Gel electrophoresis
|
STS markers :
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 μL 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 λDNA and HaeIII digests of φX174DNA.
CAPS markers :
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.
|
Data detail
open_in_full
|
|
Information of the markers in each chromosome
|
RGP caps
|
10.18908/lsdba.nbdc00318-05-001
|
Information of the STS and CAPS markers in each chromosome.
|
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 Plant Mol Biol 35: 135-144).
CAPS markers : We have developed 171 CAPS markers including 6 derived CAPS (dCAPS) markers (Konieczny and Ausbel 1993 Plant J. 4: 403-410, Neff et al. 1998 Plant J. 14: 387-392) 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).
|
STS markers : The chromosomal location of each marker has been identified by EST mapping (Wu et al. 2002 Plant Cell 14: 525--535) 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 Theor Appl Genet 96: 997-1003). Amplification was performed in GeneAmp PCR System 9600 (Perkin-Elmer).
CAPS markers : 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 Genetices 148: 479-494). Amplification was performed in GeneAmp PCR System 9600 (Perkin-Elmer).
|
Data detail
open_in_full
|
|
Details of rice EST mapping results
|
RGP estmap2001
|
10.18908/lsdba.nbdc00318-04-002
|
The data contains the detailed results of PCR-based YAC screening with the clone-specific EST primers.
|
We used clone-specific primer pairs designed from 6713 unique EST sequences (3' end) derived from 19 cDNA libraries.
|
SEGMAP (version 3.48) was used for the analysis of YAC screening data and the establishment of the EST map.
DNA gel blot hybridization of YAC or rice genomic DNA with genetic markers or EST clones was conducted using the enhanced chemiluminescence system (Amersham, Buckinghamshire, UK).
Notes
#; floating markers on the genetic map
c; chimeric YAC clones
*; PCR band size longer than predicted
Ann T; 60 ℃ unless otherwise indicated
NI; YACs with unknown insert
|
Data detail
open_in_full
|
