Submitted to: Book Chapter
Publication Type: Book / chapter
Publication Acceptance Date: 6/30/2002
Publication Date: 1/1/2003
Citation: Mcclung, A.M. 2002. Techniques for development of new cultivars. pp. 177-202. In Smith, C.W. and Dilday, R.H. (eds.) 642 pp. (Book Chapter) Interpretive Summary: Previous research in this project has demonstrated that rice breeding lines can be differentiated into different classes of cooking quality by using microsatellite markers. These markers are associated with different alleles of the waxy gene which encodes for granule bound starch synthase enzyme in rice. A single base pair change in one area of the waxy gene results in the rice having a firm or soft cooked texture. We evaluated a new method for detecting single base pair changes in rice DNA in an effort to develop a high throughput assay that would be suitable for a marker assisted breeding program. Rice breeding material which was previously determined to be segregating for the waxy gene using microsatellite markers was evaluated using the READIT system. The READIT system was 100 percent accurate in detecting the homozygous waxy gene classes as well as heterozygotes. This appears to be a fast and reliable method for detecting single base pair polymorphisms and would be a useful technology for use in a marker assiste breeding program.
Technical Abstract: The waxy gene encoding granule-bound starch synthase (GBSS) is responsible for the synthesis of amylose in developing grain. Recent work has shown that a G-T polymorphism in leader intron 5 prime splice site of GBSS plays a key role in determining the cooking and processing quality of rice. Cultivars with sequence AGGTATA at this location splice GBSS pre-mRNA efficiently and produce relatively large amounts of amylose. These varieties generally have a firm texture when cooked and the grains remain separate. In contrast, GBSS pre-mRNA splicing is temperature sensitive and generally less efficient in cultivars with the sequence AGTTATA. As a result, these cultivars generally have lower amylose content and produce soft and sticky cooked rice. We have utilized the READIT system, a novel assay based on the ability of DNA polymerase to perform the reverse of DNA polymerization, or pyrophosphoralysis, to screen the critical G-T polymorphism in a wide range of US and Asian germplasm. We found the READI system to be 100 percent accurate, allowing fast and reliable analysis. The ability to detect heterozygotes as well as the capacity for automation makes the READIT system a feasible option for high throughput marker assisted selection.