An optimized PCR protocol with newly designed primers for reliable molecular selection of high oleic type sunflower

High oleic sunflower is one of the most significant oilseed crops due to the stability of its oil in processing and desirable characteristics for health. Determination of high oleic sunflower by standard methods such as gas chromatography is time consuming and expensive. On the other hand, marker-assisted selection analysis with molecular markers associated with high oleic acid trait is a useful and powerful tool in order to facilitate sunflower breeding programs. In this study, we compared three molecular markers which have been used for selection of the high oleic sunflower varieties. We also describe an optimized PCR protocol with newly designed two primer pairs targeting normal sequence of FAD2 gene as internal control and direct analysis of the inserted DNA sequences which is known to be closely linked to the Pervenets mutation. According to results of our study, showing the insertion site which is linked to the Pervenets mutation by the insertion specific PCR protocols is more reliable than the SSR marker for selection of the high oleic sunflower varieties. Because we have not been able to get successful results with the available PCR protocols described for the insertion site, we report here a novel multiplex PCR protocol with newly designed primers enabling reliable discrimination of the high oleic and low oleic sunflower genotypes in a single PCR tube, which offers some advantages to the breeders by means of saving money and time