J Genet Resour 2019;5(2): 118-129 Homepage: http://sc.journals.umz.ac.ir/ DOI: 10.22080/jgr.2019.2408 RESEARCH ARTICLE

Phylogenetic Assessment of Some of Using DNA Barcoding

Fatemeh Aghighiravan, Majid Shokrpour*, Vahideh Nazeri and Mohammad-Reza Naghavi

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

A R T I C L E I N F O A B S T R A C T Article history: DNA barcoding is a simple method for the identification of any species using a Received 14 April 2019 short genetic sequence from a standard genome section. The present study Accepted 25 June 2019 aimed at examining the nuclear and chloroplast diversity as well as the Available online 12 July 2019 phylogenetic relationships of eight species of saffron including four spring- flowering and five autumn-flowering species from different parts of Iran, using Keywords: the nuclear barcode and chloroplast genes to specify their evolutionary Saffron pathway and to examine their genetic affinity. First, PCR was performed using Wild species the primers designed based on the chloroplast barcodes of matK, trnL, and PCR rbcL, and nuclear barcode of ITS. Then, the PCR products were purified and Cluster analysis DNA marker sequenced. The results of the phylogenetic tree indicated that the examined saffron species of Iran were separated from each other based on the sequences *Corresponding author: of ITS, matK, and trnL barcodes. In the rbcL barcode, the relationships of a  M, Shokrpour number of species remained unresolved, and this genetic region could not [email protected] 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

Print & Online ISSN: comprehensiveness in most species. Moreover, the matK and trnL barcodes p-ISSN 2423-4257 were identified as complementary barcodes. The use of the four genetic regions e-ISSN 2588-2589 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.  2019 UMZ. All rights reserved. Please cite this paper as: Aghighiravan F, Shokrpour M, Nazeri V, Naghavi MR. 2019. Phylogenetic assessment of some species of the Crocus genus using DNA barcoding. J Genet Resour 5(2): 118-129. doi: 10.22080/jgr.2019.2408

Introduction among species regarding the proposed sequences, as a result of which different regions Nowadays, marker-based and DNA-based of the chloroplast and nuclear DNA have been methods are used to identify . In introduced as a standard sequence in plants comparison with the classic methods based just (Chase and Fay, 2009). In 2009, the Consortium on morphology (Mathew, 1982), DNA-based for the Barcoding of Life used the chloroplast methods are cheap, fast, and highly accurate in genes to suggest the rbcL + matK marker species identification as well as biodiversity combination as a standard plant barcode, which assessment (Hebert et al., 2003). The term DNA has a desirable sequencing quality and high barcode sequence refers to a very short standard discriminatory power for plant species DNA sequence of a gene or a well-known (Mahadani and Ghosh, 2013). The rbcL, matK, intergenic region that can be used to identify and ITS barcodes have been examined by many which species the animal, plant, or mushroom researchers in the identification and belongs to. DNA barcoding can be considered as documentation of plant diversity (Chen et al., a novel and inspiring method in taxonomic 2010). ITS region is one of the most widely used research due to its high potential for the nuclear DNA sequences and is often suitable at detection and identification of species (Valentini lower taxonomic levels to reconstruct the et al., 2009). There is a remarkable variation phylogeny and identify the closely-related

This work is licensed under the Creative Commons Attribution 4.0 International License.

Aghighiravan et al., J Genet Resour, 2019;5(2): 118-129 species and sexes (Chase et al., 2005; Kress et can be harvested in the spring. Vavilov stated al., 2005; Yousefzadeh et al., 2012;). The trnL-F that the Middle East including Asia Minor, region is one of the important regions in the Turkistan, and Iran is the original source of chloroplast genome, which has been used in saffron. Recent research has indicated that many studies to examine the phylogenetic Greece is the first place where the domestication relationships among plant species (Yousefzadeh of saffron began, and the C. Cartwrightianus et al., 2014; 2019). Many studies on species has been introduced as the closest cyanobacteria, algae, and plants have been relative of saffron (Frello and Heslop-Harrison, derived from the evaluation of their evolutionary 2000). Furthermore, C. pallasii species have history using the intron trnL (Borsch et al., been considered as the ancestors of saffron in 2003). Recent research on saffron samples various references (Brandizzi and Grilli Caiola, obtained from different countries as well as Iran 1998; Zubor et al., 2004). Before starting and have confirmed their differences at the DNA designing a saffron breeding program, it is level (Grilli Caiola et al., 2004; Siracusa et al., essential to gain thorough information about its 2013). L., commonly known as ancestors. As Iran has a very rich germplasm of saffron, is a member of the family Iris plant species and is one of the most significant () and one of the most important genera regions for plant distribution, especially the of Iridaceae with more than 90 species (Mathew, genus Crocus, and given that the true ancestor of 1982). Since ancient times, this plant has been saffron is one of the mysteries that are still not cultivated in some parts of the world including precisely resolved, the present study aimed at Iran, India, Greece, Morocco, Spain, and Italy. identifying the evolutionary pathways of the Some historical evidence suggests that the native cultivated saffron and specifying its genetic habitat of saffron is Iran, and nine various affinity by examining its chloroplast diversity species of Crocus are native to Iran (Mathew, and phylogenetic relationships and comparing 1982), of which three species are spring- the obtained results with those of the wild flowering and the other six species are autumn- species using the DNA barcoding method. flowering plants (Kafi, 2002). Materials and Methods The stigma of Crocus sativus contains glycoside- derived esters, which are responsible for its Sample collection properties of aroma (safranal), flavor Samples of wild species of saffron were (picrocrocin), and color (crocin) (Siracusa et al., collected from the selected regions mentioned in 2013). The C. pallasii subsp. haussknechtii the flora of Iran in winter and spring (Mazhari, species, with the vernacular name of “pišuk”, is 2000) Leaf samples were frozen in the nitrogen one of the most widely used volunteer plants in liquid and stored in a freezer at -80°C before the western provinces of Iran including performing DNA extraction (Table 1). Kermanshah, Ilam, Lorestan, and Hamadan and

Table 1. Samples collection sites. Species province locality Altitude (m) longitude (m) latitude (m) C. almehensis Golestan Alme 2165 56.38 37.22 C. pallasii subsp. Kordestan Ghorve 1900 49.24 33.55 hausskenechtii C. caspius Gilan Talesh 1200 48.49 37.22 C. speciosus Alborz Karaj 1261 50.56 35.46 C. michelsonii North Khorasan North of Ghuchan 1947 58.2 37.22 C. cancellatus Markazi Shazand 1700 48.55 33.25 C. biflorous Kordestan Sanandaj 1894 46.48 35.46

DNA extraction and quality of genomic DNA were evaluated using agarose gel electrophoresis and NanoDrop. Leaf samples were extracted using the GeneAll The GeneAll kit, the ComboGp-50p model, was kit (South Korea) according to the used for DNA purification. manufacturer's instructions. Then, the quantity

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PCR and gel electrophoresis 35 cycles at 95°C for 45 seconds, 65-68°C (depending on the different primers) for 40 In the present study, the sequence of the ITS seconds, and 72°C for 2 minutes. At the end of region of the nuclear genome and the sequences the reaction, a final expansion step was of the chloroplast matK, rbcL, and trnL were performed at 72°C for 10 minutes. The products used to evaluate the phylogenetic of the of the polymerase chain reaction were loaded examined taxa (Table 2). The polymerase chain onto a 1% agarose gel and electrophoresed at 85 reaction (PCR) was performed using the Bio- volts. The results of the PCR amplification of Rad thermocycler (at a 30 μl volume including these regions were sent to Macro Corporation of 15 μM Master Mix, 1.5 μL Primer, and genomic Korea to perform sequencing. 200-50 ng). The thermal cycles consisted of an initial stage of priming at 95 ° C for 5 minutes,

Table 2. Characteristics of primers used Gene Primer Primer sequence Amplicon Reference name name size (bp) ITS ITS1 5ʹ-TCCGTAGGTGAACCTGCGG-3ʹ 500-700 White et al., 1990 ITS4 5ʹ-TCCTCCGCTTATTGATATGC-3ʹ White et al.,1990 matK matK-F 5ʹ-CGATCTATTCATTCAATATTTC-3ʹ 900-1000 Cuenoud et al., 2002 matK-R 5ʹ-TCTAGCACACGAAAGTCGAAGT-3ʹ Cuenoud et al., 2002 rbcL rbcL-F 5ʹ-ATGTCACCACAAACAGAGACTAAGC-3ʹ 700-800 Levin et al., 2003 rbcL-R 5ʹ-GTAAAATCAAGTCCACCRCG-3ʹ Kress and Erichson, 2009 trnL trnL-F 5ʹ-GGAAATCGGTAGACGCTACG-3ʹ 900-1000 Gismondi et al. 2013 trnL-R 5ʹ-ATTTGAACTGGTGACACGAG-3ʹ Gismondi et al. 2013

Data analysis strongly supported (100%) and is placed as the sister to large sub-clade of series Crociris (C. The entire sequences of PCR products were bannaticus its sole member), Verni, edited by BioEdit v7.2.6, and a combination of Versicolores, and Crocus. The C. pallasii subsp. all the analyzed barcodes (ITS + matK + rbcL + haussknechtii and cultivated C. sativus species, trnL) was aligned using the ClustalW2 ver2.1 with a stability of 100, occurred in one sister software. The final phylogenetic analysis was group with a large evolutionary distance from performed by applying the maximum parsimony other species. Furthermore, the mentioned result and the maximum likelihood methods to analyze was confirmed by the size of the branch length the phylogenetic relationships based on the data created in the phylogenetic tree. The rest of the aligned using the Mrbayes software. In order v3.2 species were also found in the other branches, in to evaluate the validity of branches, the bootstrap which C. caspius with the same species in the method (Felsentein, 1985) was used with a NCBI were found in one sister group with the thousand replications. Specification of the stability of 100. Moreover, C. almehensis and C. percentage of the nucleotide sequence identity biflorus species from series Biflori were isolated with a National Center for Biotechnology from the sister species of C. cancellatus and C. Information Database (NCBI) was performed gargaricus from series Reticulati with the using the Local Alignment Search Tool (Blast). stability of 88 (Fig. 1). The last clade is Results consisting of the remaining species not mentioned yet that within C. speciosus is in Examination of the molecular data sister group with C. pulchellus in series Speciosi. Data obtained from ITS This clade is again in sister group with C. The analysis of data related to the sequence of almehensis from series Biflori and C. cancellatus nrDNA ITS using the Bayesian method from series Reticulati. The unresolved position constructed one phylogenetic tree with two main of these three series has been reported before clades. In the first clade, C. michelsonii with the (Peterson et al., 2008). same species in the NCBI makes a sister group

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Fig. 1. The phylogenic tree derived from the analysis of ITS region data by the Bayesian method, the number above each branch shows the bootstrap value matK gene sequence analysis michelsonii indicated any significant similarity with the crop species, which confirmed the The phylogenetic tree obtained from this results obtained from analyzing the matK gene. analysis had a large trichotomy consist species belonged to section Crocus series Crocus rbcL gene sequence analysis followed by three series of Reticulati, Biflori and The results of this tree analysis also divided the Speciosi together and the last clade consists of species into two main groups (Fig. 3). Within the the only member of section Crociris (C. below clade, the C. Pallasii subsp.-haussknechtii bannaticus) which is in sister group with the C. and the C. sativus are both placed in a large, malyi and C. vernus with the stability of 100. In strongly supported (99%), but very unresolved the sub-branch of this clade, C. michelsoni clade also including the rest of the species. trnL. embedded in the sister group with C. caspius. The analysis of the data obtained from the trnL The position of these species in the sub-branches gene sequence indicated a similar topology to was identical to the position provided by the ITS that of the species and ancestors examined in data analysis. The C. pallasii subsp.- other studies (Ahadi et al., 2015). In the obtained haussknechtii species had the highest similarity tree, the taxa within the group formed a mass with the C. sativus from NCBI and formed a trichotomy. Within this large calde, species of sister group. (Fig. 2). Zarini (2013) examined the section Nusiscapus series Biflori (C. almehensis genotypes of the cultivated and wild saffron and C. biflorus) are embedded with weakly species using the protein band patterns and found supported (55). The other clade consists of a that C. michelsonii species had the minimum large group of all section Crocus, Nudiscapus similarity with the cultivated genotypes. In the and Crociris (C. bannaticus) (Fig. 4). mentioned study, none of the three wild species including C. speciosus, C. cancellatus, and C.

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Fig. 2. The phylogenic tree derived from the analysis of matK region data by the Bayesian method, the number above each branch shows the bootstrap value.

Fig. 3. The phylogenic tree derived from the analysis of rbcL region data by the Bayesian method, the number above each branch shows the bootstrap value.

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Fig. 4. The phylogenic tree derived from the analysis of trnL region data by the Bayesian method, the number above each branch shows the bootstrap value.

these branches, C. michelsonii occurred in the Combination analysis of matK and rbcL sister group with its similar species in the NCBI, The phylogenetic analyses of the combined and the remaining species occurred in the other chloroplast loci (matK and rbcL) resulted in larger branch which is divided into two clades: polytomy which is divided into four branches one strongly supported (100) including C. (Fig. 5). The first clade contains section Crocus. almehensis which is in sister to C. cancellatus, In this group, C. pallasii subsp. haussknechtii and these two species form the sister group to had indicated the maximum similarity to C. species of series Speciosi (C. speciosus and C. pallasii subsp. pallasii and occurred in a separate pulchellus). The second group consists of the sister sub-group. The cultivated saffron is remaining species. All of the species along with embedded in the sister group of the Crocus the group of C. cartwrightianus, C. oreocreticus, series. The next clade is C. michelsoni (series C. hardriaticus, and C. sativus obtained from Orientales) which is in sister to C. caspius NCBI occurred in another sister group. (series Biflori). In the third group, C. bannaticus Moreover, in another group, C. pallasii subsp. is embedded in sister with C. vernus with a haussknechtii is sister to C. pallasii subsp. weakly supported (67). In the last branch, the Pallasii and cultivated saffron. These three species of three series of sections Nudiscapus species are in the sister group of species belong (Reticulati, Biflori, and Speciosi) are placed to series Biflori and Reticulati. Apart from some together. The explanation of such a classification differences present the genus include in these is elucidated later in the combination of four series, many of the species of these two series barcodes. are so similar in morphological characteristics, Combination analysis of ITS, matK, and trnL such as corm tunics (Wolter, 1990). Combining these three barcodes did not improve the Analysis of the data from the combination of resolution of the phylogenetic very much in ITS, matK, and trnL barcodes (Fig. 6) divided comparison to using ITS only. the studied species into two clades. In one of

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Fig. 5. The phylogenic tree derived from the analysis of matK+rbcL region data by the Bayesian method, the number above each branch shows the bootstrap value.

Fig. 6. The phylogenic tree derived from the analysis of ITS+matK+trnL region data by the Bayesian method, the number above each branch shows the bootstrap value.

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Combination analysis of ITS, matK, rbcL, and species in NCBI. Series Orientales are winter- trnL flowering and morphologically well-defined primarily by corm tunics with a carpet of fine The whole studied species obtained from the fibers. The third moderately supported includes combination of the nuclear and chloroplast three series of Biflori and Reticulati and Speciosi markers form a polytomy structure including together. Another large strongly supported clade four clades (Fig. 7). In comparison to Mathew’s (96%) includes species of series Crocus and (1982) taxonomic treatment of Crocus, our Nudiscapus as a dichotomy. Our analysis phylogenetic analysis identified congruence and revealed that C. pallasii subsp. haussknechtii is conflicts on different taxonomic levels. The more closely to the C. sativus and C. pallasii analysis of the combined data resulted in a tree subsp. pallasii and are separated from the clade that grouped C. bannaticus, the only member of consisting of species of C. caspius, C. subgenus Crociris, embedded within a clade almehensis, C. biflorus (series Biflori) and also including species of section Crocus (C. malyi) C. cancellatus and C. gargaricus (series and is placed as the sister group with species of Reticulati). The other sub-clade consists of series series Versicolores (weakly supported 64%). The Crocus. Section Crocus is not monophyletic in second strongly clade forms a monophyletic the present data analysis (Fig. 7). group in series Orientales including C. michelsonii as a sister group with the same

Fig. 7. The phylogenic tree derived from the analysis of ITS+matK+rbcL+trnL region data by the Bayesian method, the number above each branch shows the bootstrap value.

Discussion and distinguishing the studied species. The low diversity of DNA of chloroplast in Crocus The results of this study show that among the species was previously examined in a study studied genetic regions, the nuclear ITS gene conducted by Seberg et al., (2009). Even in the region performed better than the other genes and present study, the use of six genetic regions separated all the examined species from one could not be useful for separating the species. In another. Furthermore, the matK gene region was a study by Haung et al., (2015) In the previous one of the good genetic regions for separating study, the distinguish between cultivated saffron 125

Aghighiravan et al., J Genet Resour, 2019;5(2): 118-129 and some Chinese herbs was assessed resulting The results revealed that all cultivated saffron that ITS could make a distinction at the intra- genotypes indicated the maximum similarity species level using the rbcL and trnH-psbA with C. cancellatus which was in contrast with barcodes at the inter-species level. In another the findings of the present study. Furthermore, study, it was found that the use of the ITS and C. michelsonii and C. almehensis revealed the matK markers was effective in the identification maximum similarity to the cultivated species in of Crocus species (Villa et al., 2016). In the study conducted by Alavi-Kia et al. (2008). addition, it was shown that the markers of ITS, The position of C. banaticus within section matK, trnL, and rbcL performed better to create Crocus resembles also to the results of Petersen interspecies divergence (Li et al., 2011). Based et al., (2008) and Harpke et al., (2015). C. on our data, although the alignment of banaticus has to be construed as a morphological chloroplast trnL and ITS regions have similar peculiar taxon with autapomorphies lengths, the uniparentally inherited chloroplast characteristic such as small leaf diameter, introse trnL region provided relatively few polymorphic anthers, inner perianth-segments being much sites (140) in comparison to the ITS region shorter than the outside and several style (270). Hence, the phylogenetic relationships splitting, lilac branches, slender and elongate among species were not well resolved when the ovoid capsule (Dahlgen et al., 1985; Kerndorff et chloroplast region was analyzed alone. al., 2015). As stated in Mathew’s study (1982) Phylogenetic relationships among species were C. bannaticus had needle-like crystals like those hence not well resolved when analyzing the of series Verni which verify the placement of chloroplast region alone. This was in agreement them in the sister group. The relationship with the previous result indicating five between Biflori, Reticulati and Speciosi is chloroplast regions in Crocus with a low unresolved (Peterson et al., 2008). In Mathew’s mutation rate (Petersen et al., 2008; Seberg and estimation the separation of these sections is still Petersen, 2009). The Consortium for the justified and was believed to discern some of the Barcoding of Life (CBOL) (2009) introduced most challenging taxonomic problems within the matK + rbcL genes as the best common barcode genus, while Harpke et al. (2014) stated for these option for plants. This topology again is nicely species that oxalate-crystals have a clear supported by the chloroplast analysis of Petersen structure with only needle-like and sand-like et al. (2008) that resulted in such a classification (Peterson et al., 2008). and grouping in different series of Crocus. The close relationship between C. sativus and C. Although, the combination of matK and rbcL did pallasii is supported by morphological similarity not improve the resolution of the phylogenetic (Mathew, 1982). C. sativus is a sterile triploid tree apparently. The posterior probabilities along only known from cultivation. It is usually the backbone of the tree increased the combined thought to be hybrid with C. cartwrightianus analysis in comparison to using ITS only. (Ahrazem et al., 2010; Larsen et al., 2015; In all studied sequences, C. pallasii subsp. Harpke et al., 2018) which is in contrast with our hausskenechtii indicated the maximum similarity results. Given that until now it has been to the cultivated species. The study conducted by presented that C. cartwrightianus is the most Sanei et al., (2007) which examined C. pallasii probable and also the sole ancestor of the according to karyotype data and the studies cultivated saffron (Nemati et al., 2019), but performed by Alsayied et al. (2015) and Harpke efforts should be devoted to finding the probable et al. (2013) using nuclear, ribosomal and parents of saffron. However, despite the results plastidy single-copy gene sequences confirmed of the mentioned studies, it is conjectured that C. the findings of the present study. Similarly, the pallasii is one of the potential parents of saffron result of the present research was in line with the (Gismondi, 2013; Erol, 2014), which is report of Namayandeh et al. (2012) on wild and confirmed by the findings of the present study. cultivated saffron species using the microsatellite In the point of flowering time, the species vary markers. Beiki et al. (2010) examined the from late autumn through to early and there be variability and genetic relationships of 30 autumn-, winter-, and early spring-flowering different Crocus genotypes using RAPD marker. populations of Crocus (Mathew, 1982). C.

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Aghighiravan et al., J Genet Resour, 2019;5(2): 118-129 cancellatus occurs in the same area as the cultivated saffron species are sterile and generate species of series Biflori, Reticulati, and Crocus, the same gene pool that makes it difficult for but C. almehensis become of being spring- breeding strategy and crop development in the flowering, endemic to Iran, in contrast to the future. Therefore, genetic enhancement of autumn-flowering species of this clade, which saffron requires the knowledge of the biology of are is widely distributed, make an unlikely source species or wild relatives. Wild species explanation for the unexpected relationship. The often contain useful gene attributes such as disparate flowering time of the species of this drought, heat, and cold tolerance, which can be clade and the other species under discussion is used in breeding programs for cultivated species, probably not of great significance (Peterson et so that hybrid breeding strategies with the al., 2008; Nemati et al., 2018). The suggested pertinent species are effective in yield and relationship between species of series Biflori and quality improvement of saffron (Agayev, 2007; Reticulati are considerably more complicated. shokrpour et al., 2019). Though the phylogeny For the reasons stated above, this is not has clarified some relationships between super- surprising in view of the morphological specific taxa, more data are clearly needed for similarity between them. Morphologically C. fully resolving the phylogenetic tree. Our results caspius is not a typical member of series Biflori confirm the importance of multi-locus studies for so its position in the sister group with Reticulati the inference of phylogenetic relationships in a species is not too unexpected (Peterson et al., series of Crocus. An ideal barcode should be 2008). This possibility can be taken into able to be replicated using a pair of universal consideration that the species occurring in one primers in all species of a genus or even a group have had a different evolutionary pathway family. The intergenic regions of ITS, matK, and due to their growth in different regions and the trnL could separate all the studied species of impact of different environmental conditions on saffron (Kress et al., 2005, 2007; Lahaye et al., them, which ultimately led to diversity in these 2008). Furthermore, the tree derived from the species (Mirzaei et al., 2001). rbcL gene sequence was unable to discriminate The species of section Crocus are placed in a different saffron species, which indicated the clade together with C. bannaticus and a group of ineffectiveness of this gene in this study (Seberg species regularly included in the series Reticulati and Petersen, (2009). Since the interpretation of of section Nudiscapus, accordingly, this position the evolutionary pathway based on the presented of section Nudiscapus makes them non- tree was not reliable; the triple combination of monophyletic (Peterson et al., 2008). ITS, matK, and trnL barcodes was examined Our phylogenetic analysis of Crocus does not along with the four-component combination. The follow the principal classification of Mathew intergenic and nuclear regions of the ITS had an (1982) into subgenera and sections; nevertheless, identification power of 100%, a high PCR the grouping into series is weakly supported. In amplification capability and could separate all the present study, four barcodes which were the examined species. Consequently, according recommended by various researchers were used to the presented results, this genetic region can to investigate their power to separate the wild be recommended as a barcode in the studied and domesticated species of Crocus. The plants. Moreover, it was revealed that the relationship between three series of Biflori and combination of four markers of ITS, matK, trnL, Reticulati and Speciosi was unresolved and rbcL achieved better to create interspecies according to our phylogenetic data and need divergence. Among the studied species of revision as well as the subgenus conception of saffron, C. pallasii subsp. hausskenechtii showed Mathew’s classification. These findings are in the most similarity to the cultivated species. accordance with chloroplast data reported by Acknowledgments Peterson (2008). Since C. sativus lacks genetic diversity or at least We kindly acknowledge the University of has a small variety, improvement of the Tehran for supporting this research work (Grant qualitative and quantitative traits would be N 73131587/6/13). difficult for its breeders and botanists. All

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