Genotyping common SNP and a microsatellite sequence closely linked to waxy gene in rice by DNA based markers

Abstract

The potential of different DNA based molecular markers was examined for the detection of single nucleotide polymorphism (SNP) in the waxy gene and a microsatellite (SSR) sequence closely linked to it in a collection of rice varieties. DNA was extracted from leaf samples of 68 different rice cultivars by the CTAB method and specific primers were designed for the amplification of waxy gene and SSR marker loci. After amplification of the desired fragments by polymerase chain reaction (PCR) genotyping of samples was carried out by the melting curve analysis technique. To confirm the results of the melting curve genotyping, two methods of PCR-SSCP and PBR were used. The presence of two genotypes (TT and GG) in the waxy gene observed from these two methods was perfectly consistent with the results of the melting curve analysis. The frequency of each TT and GG genotype was estimated 25 and 75 percent, respectively. Nine different genotypes including BH, HH, FF, DD, CC, EE, BB, AA and EG were found in the SSR marker site. This indicates the existence of a high genetic variability in the studied varieties. Although, there were no associations between the G-T polymorphism with the traits, the association of different patterns in SSR marker with the GW, TGW and FGN traits was significant. The results showed that the melting curve analysis technique is a fast and inexpensive method that can be used for polymorphism detection. Morever, this SSR marker site can be used as a suitable marker for detecting some economical traits in rice breeding.