COMPARISON OF FOUR GENOMIC DNA ISOLATION METHODS FROM SINGLE DRY SEED OF WHEAT, BARLEY AND RYE

Abstract

Modern breeding programs are based on the differences that distinguish one plant from another one, differences encoded in the plant’s genetic material, the DNA. Genotypic selection, particularly at the DNA level, can be exploited in Marker Assisted Selection (MAS) to identify desirable recombinants among segregating populations. Successful DNA amplification is vital for the detection of specific DNA targets, and this depends on the ability of DNA isolation methods to produce good quality DNA. DNA isolation from plant tissues remains difficult because of the presence of a rigid cell wall surrounding the plant cells. DNA isolation methods are affected by several factors like the amount of tissue needed and its availability, the number of steps involved and the chemicals used. In this study four different DNA isolation methods, based on CTAB and SDS (three methods), applied on single dry seed of wheat, barley and rye, were tested and compared. The quality of DNA was assessed by spectrophotometric measurements, gel electrophoresis and PCR reactions. The results of the experiments showed that DNA isolated by all four isolation methods is not excessively fragmented, A260/A280 ratio was between 1.6-1.9 and the concentration ranged between 20-194 ng/μl. The quality of the DNA was good and allowed the amplification of specific fragments by PCR. CTAB method had better A260/A280 ratio followed by SDS1, SDS2 and SDS3. Electrophoretic pattern showed better results with SDS2 method, followed by SDS3, SDS1 and CTAB. Furthermore, the CTAB and SDS1 methods need more time than SDS2 and SDS3, even though the number of steps of each method are almost equal (±2 steps). An estimative cost per sample showed that the cheapest method is the SDS3, the other three having similar costs.