In order to meet consumers' demand for food, a large quantity of rice (Oryza sativa L.) is produced in many parts of the world. Aromatic rice types, i.e. rice varieties having a stronger aroma than ordinary rice, have become increasingly popular in Southeast Asian countries, Europe and the U.S.A.

An aromatic variety, Khao Dawk Mali 105 (KDML105), is mainly produced in Northeast Thailand. Being recognized for its better quality, market demand for this variety has increased both in Thailand and overseas. Although increase of production is urgently needed, the cultivation is limited due to infertile and drought-striken sandy soils. Moreover, erratic rainfall at the beginning of the rainy season and labor shortage for transplanting are other constraints on the production of KDML 105. As a matter of fact, no objective method for the identification of such an aromatic variety has been developed. As a result, varieties different from KDML 105 are often marketed under the name of KDML 105.

In Japan, since rice is distributed as brown rice which can germinate, information about varieties can be obtained. In contrast, in Southeast Asia, rice is mainly marketed as milled rice unable to germinate. To identify varieties of milled rice in a mixture with varieties of similar appearance, conventional methods such as determination of the length-width ratio are not suitable.

Molecular marker techniques based on the polymerase chain reaction (PCR), have provided more objective methods for the identification of grain varieties. These techniques (i.e., microsatellite analysis, randomly amplified polymorphic DNA (RAPD) and amplified fragment length polymorphism (AFLP)) could be very useful for plant-based identification; microsatellite analysis is particularly useful for simple and rapid analysis. To apply this technique to milled rice, however, high quality DNA must be extracted from a single grain of milled rice. It is difficult to extract DNA, because the main components (starch and proteins) of milled rice show the same solubility as that of DNA. It should be emphasized that enzyme-inhibiting polysaccharides often contaminate the "purified" DNA.

Extracting procedures for simple and rapid identification of grain varieties require breakage of the cell wall, disruption of the cell membrane, protection of DNA from endogenous nucleases and finally removal of polysaccharides.

In our laboratory, we developed a DNA extraction method based on a freeze-and-thaw cycle for extracting purified, high molecular weight DNA from grains. The developed technique does not require expensive equipment and materials, and is not a time-consuming procedure.

Microsatellite analytical method for cultivar identification was used for extracting DNA. We analyzed rice samples obtained from Bangkok, Suwannaphum (Northeast Thailand) and Kuala Lumpur markets as well as selected varieties different from KDML 105 (Bangkok and Kuala Lumpur market rice samples). Fig. 1 shows the results of microsatellite analysis of market samples on agarose gel. Some of the rice samples originating from Kuala Lumpur and Bangkok markets did not match the KDML 105 variety.

A simple and rapid method for cultivar identification of milled rice is illustrated in Fig. 2. By applying the improved method, we were able to obtain DNA for microsatellite analysis within 2 h and the whole process of identification required 6 h. Time saving and the possibility to avoid the use of expensive instruments or toxic chemicals in the present method are an attractive alternative to existing methods of identification. The results obtained showed that DNA extracted from tested samples was of high quality and hence could be used in PCR-based techniques for cultivar identification even in laboratories with a moderate level of technology. Another feature of this developed technique was the saving in time and cost as well as the safety of the procedure.