Saffron ( Crocus sativus ) is a crop with high economical and medicinal values. This plant is considered as the most expensive agricultural and medicine products in the world and it has special position among industrial and exportial products of Iran .Iran now is the largest producer and exporter of saffron in all over the world. Because of the importance of saffron and its high price, it is supply in market often mixed with other ingredients such as safflower, maize stigma, Calendula and daylily. Separating impurities is done using decomposition reactions such as coloured reactions, high performance liquid chromatography (HPLC), near infrared spectroscopy (NIR) and proton nuclear magnetic resonance (NMR), which are highly costly and time-consuming. DNA molecules have been recently adapted to detect impurities such as safflower, yet they are not widely used since the method does not reveal the exact quantity of impurity. Real Time PCR is nowadays employed to precisely detect and quantify chemical substances added to the main material or compound. To this reason, the present study used DNA molecule quantification to estimate purity of fake saffron compared to the genuine product. To reach this goal, several chloroplastic genes such as rbcl, mat K, trn L- trn F and ITS region were received from NCBI for several species of saffron and safflower. The primers designed based on these regions were analyzed and the results showed that the ITS primers designed could specifically amplify the region in saffron and safflower. After optimizing conditions, performed Real Time PCR reaction with SYBR Green method. The suitable dilutions for the standard curve was optimized and used in next experiments with mixed sample of safflower-saffron and commercial sample. In this study observed that the concentration of tested commercial sample is similar to concentration of DNA of pure saffron. Also the limit of saffron detection was almost equal to 0.0064 %. Therefore, the proposed method is recommended as a precise and trustful procedure to quantify impurities in the available saffron products. In this study genetic diversity among 28 genotypes of saffron which were collected from different regions of Iran was also assessed using the SCoT molecular marker. Using 13 SCoT primers, 188 polymorphic bands were amplified with average of 14.46 bands, and the average PIC was equal to 0.228. Drawing phylogenetic tree based on the data from SCoT and UPGMA algorithm revealed that most of the genotypes in the study were categorized into a same Key worlds: Absolute Quantitation, Real Time PCR, Saffron, ITS, SCoT.
