Phylogenetic Assessment of Some Species of Crocus Genus Using DNA Barcoding

DNA barcoding is a simple method for the identification of any species using a short genetic sequence from a standard genome section. The present study aimed at examining the nuclear and chloroplast diversity as well as the phylogenetic relationships of eight species of saffron including four spring-flowering and five autumn-flowering species from different parts of Iran, using the nuclear barcode and chloroplast genes to specify their evolutionary pathway and to examine their genetic affinity. First, PCR was performed using the primers designed based on the chloroplast barcodes of matK, trnL, and rbcL, and nuclear barcode of ITS. Then, the PCR products were purified and sequenced. The results of the phylogenetic tree indicated that the examined saffron species of Iran were separated from each other based on the sequences of ITS, matK, and trnL barcodes. In the rbcL barcode, the relationships of a number of species remained unresolved, and this genetic region could not appropriately discriminate the species. The ITS barcode was introduced as the best barcode due to its discriminatory power, a high number of SNPs, and its comprehensiveness in most species. Moreover, the matK and trnL barcodes were identified as complementary barcodes. The use of the four genetic regions in comparison with their individual use has caused these species to be well separated. Overall, the obtained phylogram showed that the cultivated species were more similar to C. pallasii subsp. haussknechtii species.