Phylogenetic Assessment of Some Species of Crocus Genus Using DNA Barcoding

Document Type: Research Article


Department of Horticulture, Faculty of Agriculture and Natural Resources, University of Tehran, Karaj, Iran



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.


Agayev Y, Shakib A, Soheilivand S, Fathi M. 2007. Breeding of saffron (Crocus sativus): possibilities and problems. Acta Hortic 739(25): 203-207.
Ahadi E, Salami A, Shokrpour M, Naghavi M, Sorni A. 2015. Evaluation of chloroplast DNA diversity and phylogenetic relationship among 28 Artemisia species in Iran. Iran J Hortic Sci 45(4): 401-405.
Ahrazem O, Trapero A, Gomez MD, Rubio-Moraga A, Gomez-Gomez L. 2010. Genomic analysis and gene structure of the plant carotenoid dioxygenase 4 family: a deeper study in Crocus sativus and its allies. Genomics 96: 239-250.
Alavi-Kia SS, Mohammadi SA, Aharizad S, Moghaddam M. 2008. Analysis of genetic diversity and phylogenetic relationships in Crocus genus of Iran using interretrotransposon amplified polymorphism. Biotechnol Biotechnol Equip 22 (3): 795-800.
Alsayied NF, Fernández JA, Schwarzacher T, Heslop-Harrison JS. 2015. Diversity and relationships of Crocus sativus and its relatives analysed by inter-retroelement amplified polymorphism (IRAP). Ann Bot 116: 359-368.
Beiki AH, Kei F, Mozafari J. 2010. Genetic differentiation of Crocus species by random amplified polymorphic DNA. GEBJ 18: 1-10.
Borsch T, Hilu K.W, Quandt, D, Wilde V, Neinhuis C, Barthlott W. 2003. Noncoding plastid trnL-trnF sequences reveal a well resolved phylogeny of basal angiosperms. J Evol Biol 16: 558-576.
Brandizzi F, Grilli Caiola M. 1998. Flow cytometric analysis of nuclear DNA in Crocus sativus and allies (Iridaceae). Plant Syst Evol 211: 149-154.
Chase MW, Fay MF. 2009. Barcoding of plants and fungi. Science 325(5941): 682-683.
Chase MW, Salamin N, Wilkinson M, Dunwell JM, Kesanakurthi RP. 2005. Land plants and DNA barcodes: short-term and long-term goals. Philos Trans R Soc Lond B Biol Sci 360: 1889-1895.
Chen S, Yao H, Han J, Liu C, Song J, Shi L, Zhu Y, Ma X Gao, T, Pang X, Luo K, Li Y, Li X, Jia X, Lin Y, Leon C. 2010. Validation of the ITS2 region as a novel DNA barcode for identifying medicinal plant species. PLOS One 5: 8613.  
Erol O, Kaya HB, Sik L, Tuna M, Can L, Tanyolac MB. 2014. The genus Crocus, series Crocus (Iridaceae) in Turkey and East Aegean islands: a genetic approach. Turk J Biol 38: 48-62.
Frello S, Heslop-Harrison J. 2000. Repetitive DNA sequences in Crocus vernus Hill (Iridaceae): the genomic organization and distribution of dispersed elements in the genus Crocus and its allies. Genome 43: 902-909.
Gismondi A, Fanali F, Labarga JMM, Caiola MG, Canini A. 2013. Crocus sativus L. genomics and different DNA barcode applications. Plant Syst Evol 299: 1859-1863.
Grilli Caiola M, Caputo P, Zanier R. 2004. RAPD Analysis in Crocus sativus L. accessions and related Crocus species. Biol Plant 48: 375-380.
Harpke D, Meng S. Rutten T, Kerndor H, Blattner FR. 2013. Phylogeny of Crocus (Iridaceae) based on one chloroplast and two nuclear loci: ancient hybridization and chromosome number evolution. Mol Phylogenet Evol 66: 617-627.
Hebert PD, Cywinska A, Ball SL, deWaard JR. 2003. Biological identifications through DNA barcodes. Proc Biol Sci 270: 313-321.
Kafi M. 2002. Saffron Production and Processing. FUM Press, Iran. (In Persian).
Kerndorff H, Pasche E, Harpke D. 2015. Crocus (Liliiflorae, Iridaceae): Lifecycle, morphology, phenotypic characteristics, and taxonomical relevant parameters. Stapfia 103: 27-65.
Kress WJ, Wurdack KJ, Zimmer EA, Weigt LA, Janzen DH. 2005. Use of DNA barcodes to identify flowering plants. Proc Natl Acad Sci U S A 102: 8369-8374.
Lahaye R, Bank M. 2008. DNA barcoding the floras of biodiversity hotspots. Proc Natl Acad Sci U S A 105(8): 2923.
Larsen B, Orabi J, Pedersen C, Ørgaard M. 2015. Large intraspecific genetic variation within the Saffron-Crocus group (Crocus L., Series Crocus; Iridaceae). Plant Syst Evol 301: 425-437.
Li DZ, Gao LM, Li HT. 2011. Comparative analysis of a large dataset indicates that internal transcribed spacer (ITS) should be incorporated into the core barcode for seed plants. Proc Natl Acad Sci 108 (49): 19641-19646.
Mahadani P, Ghosh SK. 2013. DNA barcoding: a tool for species identification from herbal juices. DNA Barcodes 1: 35-38.
Mathew B. 1982. The Crocus. A revision of the genus Crocus (Iridacea). Timber Press, Portland, Oregon.
Mirzaei Nadoshan H, Shariat A, Asadi Karam F. 2001. Evaluation of genetic diversity in Haloxylon sp. population using electrophoresis. Iran J Rangelands Forests Plant Breed Genet Res 7: 77-117.
Namayandeh A, Nemati Z, Kamelmanesh MM, Mokhtari M, Mardi M. 2012. Genetic relationships among species of Iranian Crocus (Crocus spp.). C B Journal 3(1): 61-67.
Nemati Z, Blattner F, Kerndorff H, Erol O, Harpke, D. 2018. Phylogeny of the saffron-crocus species group, Crocus series Crocus (Iridaceae). Mol Phylogenet Evol 127: 891-897.
Nemati Z, Harpke D, Gemicioglu A, Kerndorff H, Blattner, FR. 2019. Saffron (Crocus sativus) is an autotriploid that evolved in Attica (Greece) from wild Crocus cartwrightianus. Mol Phylogenet Evol 136: 14-20.
Peterson G, Seberg O, Thorose S, Jorgensen T, Mathew B. 2008. A phylogeny of the Crocus (Iridaceae) based on sequence data from five plastid regions. Taxon 57(2): 487-499.
Sanei M, Rahimyan H, Agayev MY, Soheilvand S. 2007. New cytocype of Crocus pallasii subsp. hauskenechtii from west of Iran. Acta Hortic 739: 107-111.
Seberg O, Petersen G. 2009. How many loci does it take to DNA barcode a crocus? PLOS One 4: 4598.
Shokrpour M. 2019. Saffron (Crocus sativus L.) breeding: opportunities and challenges. in: Al-Khayri J., Jain S., Johnson D. (eds) Advances in Plant Breeding Strategies: Industrial and Food Crops. Springer, Cham.
Siracusa L, Gresta F, Avola G, Albertini E, Raggi L, Marconi G, Lombardo GM, Ruberto G. 2013. Agronomic, chemical and genetic variability of saffron (Crocus sativus L.) of different origin by LC-UV-vis-DAD and AFLP analyses. Genet Resour Crop Evol 60: 711-721.
Valentini A, Pompanon FO, Taberlet P. 2009. DNA barcoding for ecologists. Trends Ecol Evol 24(2): 110-117.
Villa C, Costa J, Meira L, Beartiz M, Oliveira PP, Mafra I. 2016. Exploiting DNA mini-barcodes as molecular markers to authenticate saffron (Crocus sativus L.). Food Control 65: 21-31.
Wolter M. 1990. Calciumoxalat-Kristalle in den KnollenHullen von Crocus L. (Iridaceae) und ihre systematische Bedeutung. Bot Jahrab Syst 112: 99-114.
Yosefzadeh H, Hosseinzadeh Colagar A, Tabari M, Sattarian A, Assadi M. 2012. Utility of ITS region sequence and structure for molecular identification of Tilia species from hyrcanian forests, Iran. Plant Systemat Evol 298: 947-961
Yousefzadeh H, Hosseinzadeh Colagar A, Akbarzadeh F. Tippery NP. 2014. Taxonomic status and genetic differentiation of hyrcanian Castanea based on noncoding chloroplast DNA sequences data. Tree Genet Genomes 10 (6):1611-1629.
Yousefzadeh H, Hosseinzadeh Colagar A, Yousefi E, Badbar M, Kozlowski G. 2019. Phylogenetic relationship and genetic differentiation of Populus caspica and Populus alba using cpDNA and ITS noncoding sequences. J Forestry Res 30(2):451-461.
Zubor A, Suranyi G, Gyori Z, Borbély G, Prokisch J. 2004. Molecular biological approach of the systematics of Crocus sativus L. and its allies. Acta Hortic 85-94.