Preparation of DNA is cumbersome especially in the case of large numbers of plant samples. Several simple plant DNA preparation methods have been developed for use in conjunction with polymerase chain reaction (PCR) analysis. However, those methods have not been adopted widely for rice molecular analysis. We present a new, simple, and inexpensive method using tris-phosphate (TPE) ethylenediaminetetraacetic acid (EDTA) buffer (100 mM tris-HCl pH9.5, 1 M KCl, 10 mM EDTA pH 8.0) without phenol-chloroform extraction and DNA precipitation steps. The method consists of five steps: leaf tissue grinding, incubating in TPE buffer at 65°C for 20 to 90 minutes, diluting extracts with water, centrifuging to sediment tissue debris, and transferring the supernatant for direct use in PCR or storage. Agarose gel analysis of the crude extracts indicated that the method produced intact genomic DNA (gDNA) from young and old leaves of both young seedlings and mature plants. Leaf sample size (0.5 to 8.0 cm long) for DNA preparation was less sensitive to PCR than the previous methods. DNA quality was tested through PCR amplification of various GC content regions and product sizes, and we obtained bands from all samples, indicating that the method produced suitable DNA quality for PCR. gDNAs were stable for longer than eight months at 4°C. This protocol enabled one person to handle several hundred samples in a day and was tested through various PCR-gel analyses such as genotyping of rice T-DNA mutant lines, positional cloning of rice mutant, and high throughput marker-assisted breeding using allele-specific SNP/Indel markers.
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Advances in plant molecular techniques have dramatically widened the applicability of gene identification and pyramiding valuable genes. This study was carried out to pyramid five resistance genes for biotic stress into the
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A total of 23 elite rice cultivars from eight countries were evaluated for cold tolerance using two screening methods at Chuncheon Substation, National Institute of Crop Science (NICS), Republic of Korea. The rice cultivars Jinbu, Mustaqillik, and Avangard showed cold tolerance and high spikelet fertility (63–79%) in cold-water irrigation screening. Under greenhouse screening, five cultivars (Giza 177, Avangard, Mustaqillik, Jinbu, and Jungan) showed high cold tolerance and high spikelet fertility (71–81%). Simple sequence repeat (SSR) marker analysis of 21 genotypes revealed two major clusters, the
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