Rice is an important food source. There are several thousand different cultivars which can differentiated among using DNA. Due to cahracteristics of a plant cell, DNA isolation from plant tissues is not trivial. We demonstrated that DNA can be isolated from both rice leaves and seeds using mortar with liquid nitrogen, followed by incubation in CTAB buffer at 60 °C. The fragmentation of gnomic DNA depended primarily on the source of plant material. Purity of isolated DNA was high enough to perform PCR. We have successfully amplified ITS regions from several samples. There are many molecular markers that can be used to differentiate among rice species. In the Gramene data base there is a selection of 50 most commonly used microsatellite markers. We chose 9 of them to check if they are appropriate to detect differences among selected rice varieties (Hayayuki, Yukihikari, Duborskian, Titanio, Uz Rosk, Ponta Rubra, Vialone Nano, Ribe, unknown variety of cooking rice, red, black and tea rice). We tried to minimize nonspecific PCR products by increasing the annealing temperature. The PCR products of different varieties are expected to have different lengths, the difference being less than 30 bp. Separation by AGE was not appropriate to detect such small differences. We proved that we can detect a difference of 3 bp by PAGE instead. Nevertheless, we could see no size difference among varieties using markers RM536 and RM431. The purified PCR product of the marker RM431 has been sequenced. The quality of analysed sequences was not good enough to perform a comparison between all rice samples. The sequences of Hayayuki and Ponta Rubra were identical to the reference sequence of RM431.
|