Jordan Journal of Agricultural Sciences, Volume 12, No.3 2016

Genetic Diversity of Jordanian Wild as Detected by RAPD

Maha Q. Syouf 1, Muhanad Akash2, Moath Al-Gharaibeh1, Rida A. Shibli 2 and Feras Q. Alali 3

ABSTRACT

DNA among eight selected wild Crocus species was evaluated using Random Amplified Polymorphic DNA (RAPD). These are Crocus aleppicus Baker, C. cancellatus Herbert, C.cartwrightianus Herbert,C. damascenus Herbert, C. hermoneus Ky.ex Maw subsp. palaestinus, C. hyemalis Boiss. & Blanche, C. moabiticus Bomm. & Dinsm. ex Bomm, and C. Pallasii supsp. Haussknechtii. The cultivated species; C. sativus and C. vernus were purchased from Italy. Genetic similarities between the Crocus species were evaluated by NTSYS-pc software using Dice coefficient. The dendogram was constructed using the Un weighted Pair Group Method (UPGMA). As a result, tested Crocus species were grouped into four well–separated monophyletic clusters. The cultivated species were clustered separately from the wild collected species. Wild Crocus species collected from different sites for the same species were clustered together. Cluster analysis indicated a high degree of inter and intra species variation within the wild Crocus species. High genetic association was found among some wild species originated from the same collection site. The wild collected C. moabiticus (a Jordanian endemic species) was found to be very close to the cultivated C. sativus. The current results showed close relationship between C. sativus and C. moabiticus. This analysis demonstrated that RAPD-PCR has proved to be a successful tool to determine genetic diversity among studied Crocus species.

Keywords: Crocus, Crocus sativus, Jordan, genetic variation, dendogram, saffron.

INTRODUCTION 1994; Grilli Caiola, 2001). The dry stigmas of C. sativus is currently used as a spice and food colorant that is the Crocus sativus L. commonly known as saffron is a most expensive spice in the world (Alonso et al., 1990; male sterile species (2n=24; X=8) that is reproduced via Escribano et al., 1996; Zeng et al., 2003, Kyriakoudi et corms (Brighton, 1997). Saffron is not found in natural al., 2015). habitats but has been cultivated in the Mediterranean Saffron’s therapeutic medicinal benefits are well basins since the late Bronze Age (Zohary and Hopf, recognized since ancient civilization (Rois et al., 1996; Duke, 2002 and Ferrence and Bendersky, 2004).

1 National Center for Agricultural Research and Extension Recently, the Crocus species received global attention (NCARE), Baqa, Jordan. due to detection of anti-cancer, immuno-modulating, [email protected] 2The University of Jordan, Faculty of Agriculture, anti-inflammatory and anti-oxidant properties effects Department of Horticulture and Crop Science, Amman, (Bhandari, 2015, and Kyriakoudi et al., 2015). The Jordan 3 College of Pharmacy, Qatar University, 2713, Doha, Qatar therapeutic value of saffron in certain types of cancer, University cerebrovascular and cardiovascular diseases, has been Received on 2/2/2015 and Accepted for Publication on 28/10/2015. well documented (Nair et al., 1991; Abdullaev and

© 2016 DAR Publishers/The University of Jordan. All Rights Reserved. -733- Genetic Diversity of… Maha Q. Syouf et al

Frenkel, 1992; Abdullaev, 1993; Escribano et al., 1996; destruction, and weak enforcement of protection laws Rois et al., 1996). The known antioxidative activity of (MOE, 2009; 2014). C. sativus is attributed to the tetraterpenes crocins, which In the current study, the relationships between wild give the stigmas their distinctive color, the picrocrocin, eight species collected from different sites in Jordan and which gives the bitterness and safranal which gives its the cultivated C. sativus and C. vernus using RAPD odor (Zarghami and Heinz, 1971; Visvanath et al., 1990; techniques were evaluated. The presence of eight wild Zeng et al., 2003). species of crocus in a small country with limited Saffron cultivation is used for cooking, staining, resources may indicate a proper gene pool for utilization medicine, cosmetics and other purposes (Grilli Caiola in many disciplines and may indicate other alternatives and Canini, 2010). World' cultivation of C. sativus has for cultivation to secure demands by the pharmaceutical decreased due to high cost of production and and other industries urbanization of rural areas (Visvanth et al., 1990; Molina et al., 2005; Chen et al., 2004). Therefore, looking for MATERIALS AND METHODS similar and alternative resources to solve the supply Material and their collection sites problems could help to secure the large demands by the Collection missions targeting wild crocus species were pharmaceutical and other industries. carried out during November to January 2004-2007 to An understanding of genetic diversity is essential to cover different parts of Jordan (Northern, Central and properly maintain and exploit germplasm resources Southern Jordan). The collected species were fully (Fernádez et al., 2011; Candan, 2015), and to devise a identified based on Al-Eisawi (1998) and Zohari and global strategy for better management and more Feinburn-Dothan (1986), eco-geographical data were effective use of variation in collected germplsam recorded and documented for each site using the Global (Brown1989a; 1989b). Random Amplified Polymorphic Positioning Systems (GPS) (GARMIN 12 XL, 1998) and DNA (RAPD) is one of the most extensively used Barometer/Altimeter, 1985). Ecogeographical data molecular techniques for assessing species genetic including longitude, latitude, elevation, province, site diversity (Degani et al., 1998; Ferguson et al., 1998; names and the date of collection for all wild Crocus Lowe et al., 1996; Syouf et al., 2006 and Alsayied et al., collected samples is shown in Table1. Cultivated C. sativus 2015). Syouf et al. (2008) studied genetic relations and the ornamental C. vernus were purchased from Italy. among populations of C. hyemalis from Jordan using RAPD in which high polymorphism was encountered Genomic DNA Isolation inter and intra wild C. hyemalis populations. Nazzal et The DNA of each plant sample was extracted using al. (2011) used the Amplified Fragment Length Wizard Genomic DNA Purification Kit (Promega, Polymorphism to study the variation between two wild USA). The extracted DNA pellets were rehydrated using species of Crocus species collected from north Jordan in 100 µl of DNA rehydration solution, and stored at −20 which it was recommended to study other locations to °C until use. The isolated DNA was checked for purity get a clear image of the diversity of other Crocus and quantity using spectrophotometer as described by species. C. moabiticus is considered one of rare endemic Sambrook et al. (1989). and threatened species for Jordan due to habitat

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RAPD Analysis comparing the banding pattern observed among the The 125 wild DNA Crocus samples and the two samples. Banding patterns were coded by determining cultivated species were subjected to DNA fingerprinting. In the total number of unique bands observed in all of the order to determine the typeability, reproducibility and samples examined. To generate a binary matrix for each discrimination of each primer, separate amplification of primer, band position was assigned as “1”of the presence each primer was conducted (three trials for each primer). of a band and as “0” when there is no band. The The output of each experiment was compared to the similarities and the differences between samples were previous one. determined by the NTSYS-pc version 2.0 (1997) Five 10-base RAPD primers were used (Table 2). The software. Similarity analysis was run based on Dice standard RAPD amplification protocol by Williams et al. coefficient: 2a/ (2a+b+c) where c: the number of bands (1990) was used with some modifications. Amplifications commonly present in individual a and b; and a: the were carried out in 25 µl. reaction mixture containing 1X number of bands present in a but not in b and b: the

PCR buffer mixed with MgCl2 [50 mM KCL,10 mM Tris– numbers of bands present in b but not in a.

HCL (pH 9.0),1.5 mM MgCl2 and 0.1% triton X-100 ] The dendrogram was constructed using the (Promega, catalog # M2661), 100 µM of each dNTP Unweighted Pair Group Average linkage Method (Promega, USA), 1.5 µl primers at five picomoles/µl, 0.5 (UPGMA), Sneath and Sokal (1973) as described by µl (5u/ µl ) Taq DNA Polymerase (Promega, USA), and 10 Akash and Kang (2009). ng template DNA (Table 2). Amplification reactions were RESULTS performed in a thermal cycler (MJ- Research, Model Table (1) showed the survey result of eight wild PTC200 programmed as follows: initial denaturizing step species collected from 32 different sites in Jordan at 95 °C for 2 min, followed by 35 cycle of denaturizing at (Table1). The most abundant species was C. hyemalis 95 °C for 40 seconds each, annealing at 34 °C for 40 which was collected from eight sites followed by C. seconds, and extension at 72 °C for 2 min. DNA samples moabiticus and C. hermoneus (six sites) followed by C. were subjected to final extension cycle of 72 °C for 5 min, alippicus (five sites) and C. pallasii (four sites.) C. finally the tubes were held at 4 °C for direct use, or stored at damascenes along with C. cancellatus (scarcely found in −20 °C until use. two sites). The longitudes for the collected sites ranged Amplification products were electrophoresed in 1.5% from 35 33 03.3 at Shoubak city in Shoubak district to agarose gels (Bio-RAD PAC 300, USA) at 100 Volt for 36 12 17.3 at Muhareb site at Madaba district. The 120 minutes and the banding patterns were visualized latitude for the collected sites ranged from 30 20 36 at using Ethedium Bromide staining at 0.5 µg/mL (Sigma, Adruh site in Maan district to 32 25 10.9 at Bergish site USA). The size of the PCR products was estimated in Ajloun district (Table 1). using 100 bp DNA ladder (Promega, USA). Gels were It was noticed that C. hyemalis grows in association viewed using the gel documentation system (Vilber with Querqus species in the Mediterranean forests of Lourmat, France) Ajloun and Jerash districts. Being found in patchy and threatened habitats with miner population size such as Data Analysis of RAPD Profiles the C. hermoneus of Shafa Badran and Um Elbasateen at Similarities among the isolates were calculated by Amman district where, only two to three plant species in

-735- Genetic Diversity of… Maha Q. Syouf et al an area of 600 m2 were detected. collected species but separated the cultivated from Amplification of the 127 DNA plant samples from different wild collected Crocus species (Fig. 1). Wild different Crocus species was obtained at 5-10 ng/ µl Crocus species originated from diverse habitats in template concentration. The DNA quality at the ratio of Jordan have been found to vary genetically as revealed UV absorbency at260/280 fell within the range of 1.8- by RAPD. Banding patterns variation is shown in Fig. 5 1.9. The primers OPB-12, OPM-01, OPE-02, OP-03 and and 6. High genetic association between some wild OPM-05 showed clear amplification patterns. The species was found within the same C. hyemalis for number of polymorphic markers across the studied example, the first component of the first cluster shows accessions ranged from 3-8. The maximum number of the species collected from Souf and Junaid at Ajloun markers across the accessions with primer OPB-12, district (species number L1 and L11) , the collected OPM-01, OPM-02, OPM-O3, and OPM-05, was 6,8,5,3 species from Bergish and Junaid (species number L10 and 5, respectively. The sizes of the amplified DNA and L15), the species collected from Junaid and Samta fragment ranged from 0.28 to 1.5 Kb. Table 3 shows the (species number L12 and L18) and the collected species RAPD markers produced by the five primers and their from the same locality of Rehaba at Ajloun district distribution across the accessions. (species number L24, L25 and L35). In the second The primers generated a total of 27 polymorphic component of the first cluster, high genetic association markers; these primers produced a total of 1714 was encountered in C. hyemalis species collected from differently sized fragments (bands) across the tested Souf and Samta sites (species number L5 and L20), as accessions. well as those collected from the same locality at Based on Dice (1945) mathematical models, a Bergish (species number L6 and L7). High genetic similarity matrix was constructed to assess the genetic similarity was also encountered within Samta and identity among the Crocus species. Tested Crocus Rehaba wild Crocus species (species number L21, species were grouped into four well–separated L27and L34). monophyletic clusters corresponding to the main The third component of the first cluster comprises C. taxonomic identification by Zohari, and Feinburn- pallasii collected from Fujej at Shoubak district (Fig. 1) Dothan (1986) and Al-Eisawi 1998 (Fig. 1). encountered high genetic similarity (species number First cluster contains C. hyemalis, C. hermoneus and L103 and L104) and C. cancellatus (species number L59 C. palasii. Second cluster contains C. damascenus, C. and L60) collected from Atross, Madaba district. C. aleppicus and C. cancellatus. Third cluster contains C. hermoneus (species number L51) in the third component moabiticus (Fig 2) and C. cartwrightianus (Fig. 3). of the first cluster out grouped the first cluster. In the Interestingly, the fourth cluster grouped the cultivated second cluster, high genetic similarity was encountered species C. sativus (Fig. 4) and C. vernus, separately. The in the C. aleppicus (species number L83-L85) collected inclusion of the high inter and intra polymorphisms were from the same locality of Adruh at Maan district and C. found for the wild Crocus species collected from the 32 aleppicus (species number L91 and L93) collected from different locations from different eco-geographical Shoubak locality at Shoubak district, also high genetic regions from Jordan (Table 1). RAPD analysis did not association was found in C. cancellatus collected from identify locations specific differences within the wild Rakeen locality, Karak district (species number L78 -

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L79 and L 80-81). Most interestingly, in the third genetic relations among these wild accessions as they cluster C. moabiticus and C. cartwrightianus grouped in share common leaf characteristics with dark green color one cluster with very close relationship to the cultivated that was visually noticed (Fig. 1). species of C. sativus and C. vernus. Morphological similarity between C. moabiticus and C. cartwrightianus collected from Atross locality, C. cartwrightianus in flower shape and stigma color is Madaba district encountered high genetic similarity shown in Fig (2, 3 and 4). The well separation of the within the second component of the third cluster and wild accessions from the cultivated in the fourth cluster high genetic similarity was also observed in C. in the present study is in harmony with the results of an moabiticus collected from Khanasri, Mafraq districts earlier study on one wild C. hyemalis species which was (species number L 61-L62 of and L117-120). The fourth based on RAPD (Syouf et al., 2008). Also, these cluster grouped the cultivated species together with very findings confirm Mathew’s hypothesis (1982 and 1999) close relationship to the third cluster of C. moabiticus and Grilli Caiola et al. (2004) on the origin of the C. and C. cartwrightianus. sativus. and the possibility of the close relationships of both C. moabiticus and C. cartwrightianus that DISCUSION indicating the possibility of being as ancestors to the An understanding of the genetic relationships within cultivated species. These results may facilitate re- wild Crocus species is a key for efficient conservation synthesizing saffron with improved characteristics and and sustainable utilization to Crocus breeders. High show the need for research on these wild relative with variation encountered inter and intra wild species conservation and collection action plans. C. indicated that Jordan host a rich reservoir of wild Crocus cartwrightianus was found to be as one of the close wild species (Table 1 and 3). This variation could be the relatives to C. sativus or more likely parent of saffron outcome of continuous gene flow within a relatively (Grilli Caiola and Canini, 2010; Alsayied et al., 2015). small area characterized by diverse habitats (Al- Eisawi, In fact, the phenogram obtained in this study are 1998; Syouf et al., 2006). compatible with Brandizzi and Grilli Caiola (1998) in The most variable species we encountered is C. relation to the direct origin of C. sativus from a gene hyemalis as evident in the first cluster. Fig. 1 suggests pool similar to that of by means of autopolyploidy. The some sort of biogeographically disjunction in the history unique sharing of banding pattern of C. moabiticus along of evolution, diversification and spread of this accession. with C. sativus suggests the close relationship with the This is mainly restricted to northern Mediterranean cultivated species. This study showed that there is an region in Jordan. urgent need to integrate knowledge derived from There were close association of C. hermoneus with C. genetic, demographic and ecological in order to hyemalis in the first cluster. This result suggests C. formulate management strategies to conserve such hermoneus could be as basal sister group to the rest of C. important species. These results showed that clustering hyemalis. This could be marked as a far affinity among the of different crocus species were not based on location of cultivated accessions of C. sativus and C. vernus (Fig.1). collection. Moreover, RAPD techniques were simple, The placement of C. damascenus, C. aleppicus and suitable and efficient tool to determined genetic diversity C. cancellatus in the second cluster supports the close among crocus.

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CONCLUSION RAPD techniques were simple, suitable and efficient This study represents the first comprehensive attempt tool to determined genetic diversity among Crocus to analyze the genetic diversity of all Crocus species in species. Wide variation encountered in wild population Jordan. The study showed that there is an urgent need to of Crocus species indicated the potential of future integrate knowledge derived from genetic, demographic agricultural and chemotherapy utilization. The close and ecological in order to formulate management genetic relation of C. moabiticus to the cultivated C. strategies to conserve such important species. These sativus may provide a new scientific phylogeny to the results showed that clustering of different crocus species of Crocus. were not based on location of collection. Moreover,

Table 1. Species number, scientific names and eco-geographical data for collected Crocus sp.

Species Latitude. Altitude Site name and Date of collection Scientific names Longitude (E) No. (N) (m) province M/D/Y

L1-L5 C. hyemalis 35 49 21.2 32 20 23.6 1070.4 Souf/Jerash 11/24/2004 L6-L10 C. hyemalis 3 5 45 24.1 32 25 10.9 843.1 Bergish/Ajloune 11/25/2004

L11-L 15 C. hyemalis 35 47 20.5 32 21 27.0 1052.8 Junaid/Ajloune 11/25/2004

L16-L 17 C. hermoneus 35 49 21.3 32 20 23.6 1103.9 Aluke/Amman 12/9/2007

L18-L22 C. hyemalis 35 49 06.2 32 23 21.2 1055 Samta/Ajloune 12/21/2004

L23-L27 C. hyemalis 35 49 15.5 32 24 15.6 1015 Rehaba/Ajloune 12/21/2004

L28-L 32 C. moabiticus 36 00 40.4 31 35 38.2 734.1 Dab'a/Madaba 12/20/2004

L33-L 37 C. hyemalis 35 48 53.6 32 24 30.1 943 Rehaba/Ajloune 12/28/2004

L38L 42 C. moabiticus 35 59 40.4 31 37 15.0 650 Jiza/Madaba 12/20/2004

L43-L46 C. moabiticus 36 12 17.3 31 43 22.5 763 Muhareb/Madaba 12/14/2004

L47-L50, L52 C. hyemalis 35 48 26.2 32 24 09.6 913 Usaim/Ajloune 12/28/2004 L51 C. hermoneus L53-L 57 C. hyemalis 35 46 43.2 32 23 58.6 852 Talbia/Ajloune 12/28/2004

L58-L60 C. cancellatus 35 40 26.6 31 35 18.3 682 Atross/Madaba 12/30/2004

L61-L62 C. cartwrightianus 35 40 26.6 31 35 18.3 682 Atross/Madaba 12/30/2004 L63-66L C. cartwrightianus 35 37 46.1 31 34 09.5 640.5 Mukawer/Madaba 12/30/2004 L 67 C. moabiticus

L68-69L C. damascenus 35 51 20.8 32 19 23.7 1026.4 Souf/Jerash 1/5/2007

L70-72L C .hermoneus 35 51 20.8 32 19 23.7 1026.4 Souf/Jerash 1/5/2007

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L 73-L77 C. cancellatus 35 44 17 31 11 40.2 287.4 Rabah-karak 1/9/2005

L78-82L C. cancellatus 35 44 16.6 31 12 48.2 247.2 Rakeen-karak 1/9/2005

L83-85L C. aleppicus. 35 35 38.3 30 20 36 400.5 Adruh/Ma’an 1/11/2005

L86-89 L C. damascenus 35 34 50.0 30 30 24.5 451 Shoubak/Shoubak 1/11/2005

L 90 C. cartwrightianus 35 37 39.8 30 32 46 859 Shoubak/Shoubak 12/9/2007

L91-95L C. aleppicus 35 33 21.8 30 32 00.6 418.2 Shoubak/Shoubak 1/11/2005

L96-L100 C. pallasii 35 33 03.3 30 26 16.4 622.8 Shoubak/Shoubak 1/11/2005

L101 C. pallasii 35 36 38.8 30 31 42 359 Shoubak/Shoubak 1/12/2005

L102-L105 C. pallasii 35 37 18.8 30 32 11.2 353.7 Fujej/Shoubak 1/12/2005

L 106 C. moabiticus 36 12 17.3 31 43 22.5 763 Muhareb/Madaba 12/14/2007

L 107-111 L C. aleppicus 35 37 03.7 30 39 44.6 577.8 Dana/Tafela 1/12/2005

L 112-116 L C. aleppicus 35 36 40.8 30 40 18.7 408.3 Dana/Tafela 1/12/2005

L 117-120 L C. moabiticus 36 03 44.5 32 24 45.6 576.40 Khanasri/Mafraq 12/9/2004

L 121-L 122 C. hermoneus 35 55 11.6 32 19 12.6 790.2 Thagret Asfour/ 12/9/2004 Jerash L 123 C. hermoneus 35 49 27.6 31 50 42.6 851 Um Elbsateem/ 12/20/2004 Amman L 124 C. aleppicus 35 55 02.3 32 03 51.7 880 Dibeen/Ajloune 2/22/2005

L 125 C. hermoneus 35 55 02.2 32 03 51.7 878 Shfa Bdran/ 2/22/2005 Amman L 126 C. vernus Cultivated Cultivated Cultivated Italy 2/10/2005

L 127 C. sativus Cultivated Cultivated Cultivated Italy 8/11/2005

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Table 2. RAPD-PCR primers used in fingerprinting Primer Sequence OPB-12 5'-GGAGGGTGTT-3' OPB M-01 5'-GTTGGTGGCT-3' OPB M-02 5'-ACAACGCCTC-3' OPB M-03 5'-GGGGGATGAG-3' OPB M-05 5'-GGGAACGTGT-3'

Table 3. RAPD primer, total number of markers, polymorphic marker, and total number of bands produced across all samples.

Total number of Total number of Primer Polymorphic polymorphic % bands across the population markers per primer

OPB-12 6 480 28%

OPM-O1 8 337 19.7%

OPM-O2 5 334 19.4%

OPM-O3 3 259 15.2%

OPM-05 5 304 17.7%

Total 27 1714 100%

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Fig. 1. Dendogram of wild and cultivated Crocus species with species codes (L1-L127) based on Dice coefficient using UPGMA method

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Fig. 2. Crocus moabiticus during flowering from Khanasreh Station in Jordan

Fig. 3. The wild C. cartwrightianus at flowering stage collected from Madaba Jordan

Fig 4. The cultivated Crocus sativus at flowering stage at Mushager Research station

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Fig. 5. RAPD fingerprints profile of Crocus sp samples 1-8 (L96) C. pallasii) 9 (L106) C. moabiticus 10-14 (L107- 116) C. aleppicus15-.19 using primer OPB M-02. M (100pb marker).

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Fig. 6. RAPD fingerprints profile of Crocus sp samples 1-8 (L96) C. pallasii) 9 (L106) C. moabiticus 10-14 (L107- 116) C. aleppicus15-.19 using primer OPB M-03. M (100bp marker)

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extends the flowering season of saffron (Crocus sativus wild emmer wheat (Triticum dicoccoides (Korn. ex L.). J Hort Sci and Biotech 80:319-326 Asch. Graenber) Schweinf), Genetic Resources and Nair, S.C., B Pannikar, K.R Pannikar, 1991, Antitumour Crop Evolution 53: 1165-1172 activity of saffron (Crocus sativus), Cancer Lett 57:109- Visvanath, S., G.A., Ravishankar L. V. Venkataraman, 114 1990, Induction of crocin, crocetin, picrocrocin and Nazzal, K.W., R.A Shibli., I.M Makhadmeh., M.Q Syouf, safranal synthesis in callus cultures of saffron Crocus 2011, Amplified Fragment Length Polymorphism sativus L. Biotechnol Appl Biochem 12: 336-340 (AFLP) analysis in Crocus spp. Collected from northern Wiiliams, J.G.K., Kabelik, A. R. Livak., J.A Rafalski, and Jordan. Jordan Journal of Agricultural Science 7(1):1-8 S.V Tingey, 1990, DNA Polymorphism Amplified by Rois, J.L., M.C Recio., R.M Giner., S Manez, 1996, An Arbitrary primers are used as Genetic Markers. Nucl. Update Review of saffron and its active constituents. Acid. Res. 18 (22): 6531-6535. Phytotherapy Res 10:189-193 Zarghami, N.S., D.E. Heinz,1971, Monoterpene Aldehydes Sambrook, J. E., F Fritsch., T. Maniatis, 1989, Molecular and Isophorone- related compounds of saffron, Cloning: a Laboratory Manual, 2nd edition, Cold Spring Phytochemistry 10:2755-2760 Harbor, N.Y: Cold Spring Harbor Laboratory Zeng,Y. F., L.Yan, F Chen, 2003, Increased Crocin Sneath, P.HA., R.R. Sokal,1973, Numerical Taxonomy. production and induction frequency of stigma like The principles and practices of numerical classification. structure from floral orangans of Crocus sativus by San Francisco: Freeman WH p 150 precursor feeding plant cell, Tiss. and Org. Cult 72: Syouf, Q.M., M. Al –Gharaibeh, R. A Shibli., F.Q., Alali H 185-191 Migdadi., 2008, Study of genetic diversity in Crocus Zohari, M., N,. Feinburn-Dothan, 1986, Flora Palestina. hyemalis Boiss. and Blanche. Using RAPD techniques. Israel Academy of Science and Humanities 4: 112-127 Jordan Journal of Agricultural Science 3:231-241 Zohary, D., M., Hopf., 1994, Domestication of in the Syouf, Q.M., B.E Abu-Irmaileh., J Valkoun, S Bdour, Old World, 2nd edition, Clarendon Press, Oxford, U.K. 2006, Introgression from Durum Wheat landraces in p189-90

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ا ﻟ ﺗ ﻧ وع اﻟوراﺛﻲ ﻟﻠزﻋﻔران اﻷردﻧﻲ ا ﻟ ﺑ ري ﺑطرﻳﻘﺔ اﻟﺗﻔرﻳد اﻟﻌﺷواﺋﻲ RAPD

مھا السيوف1، مھند عكاش2، معاذ غرايبة1، رضى شبلي2، فراس عاللي3

ﻣﻠﺧـص

ﺗم ﺗﻘﻳﻳم اﻟﻣﺎدة اﻟوراﺛﻳﺔ (DNA) ﻟﺛﻣﺎﻧﻳﺔ أﺻوﻝ ﻣ ﺧ ﺗ ﺎ رة ﻟﻧﺑﺎﺗﺎت اﻟزﻋﻔران اﻟﺑرﻳﺔ ﺑطرﻳﻘﺔ اﻟﺗﻔرﻳد اﻟﻌﺷواﺋﻲ RAPD وﻫﻲ: زﻋﻔران اﻟرﻗﺎطﻲ ( Crocus aleppicus Baker )، زﻋﻔران ﺛرﻳﺎ (C. cancellatus erbert ) ، اﻟزﻋﻔران اﻟدﻣﺷﻘﻲ ( C. damascenus Herbert C. hermoneus Ky.ex Maw subsp. palaestinus)، اﻟزﻋﻔران اﻟﻳوﻧﺎﻧﻲ .C (cartwrightianus Herbert)، زﻋﻔران ﺑﻠﺑوس ( C. hermoneus Ky.ex Maw subsp palaestinus Feinbr)، زﻋﻔران اﻟﺗوﻳس (hyemalis Boiss. & Blanche )، زﻋﻔران ﻣؤاب & .C. moabiticus Bomm) (Dinsm. ex Bomm ، زﻋﻔران ﻫرﻳف او اﻟﺷﺣوم ( C. Pallasii supsp. Haussknechtii ). واﻟﻧﺑﺎﺗﺎت اﻟﻣﺳﺗزرﻋﺔ، اﻟزﻋﻔران اﻟﻣﺳﺗزرع .C. sativus L وﻧﺑﺎت اﻟزﻳﻧﻪ C. vernus ( L.) Hill ، ﺣﻳث ﺗم ﺷراوﻫﺎ ﻣن اﻳطﺎﻟﻳﺎ. ﺗم دراﺳﻪ ﻣدى اﻟﺗﺷﺎﺑﻪ اﻟﺟﻳﻧﻲ ﻻﻧواع اﻟزﻋﻔران ﺑﻧظﺎم اﻟﺑرﻣﺟﻳﺎت (NTSYS-pc) واﺳﺗﺧدام ﻣﻌﺎﻣﻝ Dice . وﺗم رﺳم ﺷ ﺟ رﻩ ا ﻟ ﺗ ﻘ ﺎ رب اﻟﺟﻳﻧﻲ ﺑﺎﺳﺗﺧدام طرﻳﻘﻪ ( UPGMA ) . ﺣﻳث أ ظ ﻬ رت اﻟﻧﺗﺎﺋﺞ أرﺑﻌﺔ ﺗﺟﻣﻌﺎت ﻷﺻوﻝ اﻟزﻋﻔران اﻟﺑرﻳﺔ واﻧﻔردت اﻷﺻوﻝ اﻟﻣﺳﺗزرﻋﺔ ﻋﻧﻬﺎ. وﺗﺟﻣﻌت اﻷﺻوﻝ اﻟوراﺛﻳﺔ واﻟﻣﺟﻣﻌﺔ ﻣن ﻧﻔس ا ﻟ ﻧ وع وﻣن ﻋدة أﻣﺎﻛن ﻣﻌﺎ. ﺑﻳن اﻟﺗﺣﻠﻳﻝ اﻟﺗﺟﻣﻳﻌﻲ درﺟﻪ ﻋﺎﻟﻳﻪ ﻣن اﻟﺗﻧوع اﻟوراﺛﻲ ﺑﻳن وﻓﻳﻣﺎ ﺑﻳن أﻧواع اﻟزﻋﻔران ا ﻟ ﺑ ري وﺑﻳﻧت اﻟﻧﺗﺎﺋﺞ وﺟود ﻋﻼﻗﺔ وراﺛﻳﺔ ﻓﻳﻣﺎ ﺑﻳن أﻧواع اﻟزﻋﻔران ا ﻟ ﺑ ري واﻟﻣﺟﻣﻌﺔ ﻣن ﻧﻔس اﻟﻣﻛﺎن ﻛﻣﺎ وأ ظ ﻬ رت اﻟﻧﺗﺎﺋﺞ أن زﻋﻔران ﻣؤاب (ﻧﺑﺎت ﻣﺳﺗوطن أردﻧﻲ) ﻛﺎن اﻷﻛﺛر ﺗﻘﺎرﺑﺎ ً ﻣﻊ اﻟزﻋﻔران اﻟﻣﺳﺗزرع . إن اﺳﺗﺧدام ﺗﻘﻧﻳﺔ اﻟﺗﻔرﻳد اﻟﻌﺷواﺋﻲ أﺛﺑﺗت ﻣدى اﻟﺗﻧوع اﻟوراﺛﻲ ﻷﻧواع اﻟزﻋﻔران ﻣﺣض اﻟدراﺳﺔ. اﻟﻛﻠﻣﺎت اﻟداﻟﺔ: اﻟزﻋﻔران ، اﻟزﻋﻔران اﻟﻣﺳﺗزرع، اﻷردن ،اﻻﺧﺗﻼﻓﺎت اﻟوراﺛﻳﺔ وﺷﺟرﻩ ا ﻟ ﺗ ﻘ ﺎ رب.

1 . اﻟﻣرﻛز اﻟوطﻧﻲ ﻟﻠﺑﺣوث اﻟزراﻋﻳﺔ، اﻟﺑﻘﻌﺔ، اﻻردن. [email protected] 2 . ﻗﺳم اﻟﻣﺣﺎﺻﻳﻝ واﻟﺑﺳﺗﻧﺔ، ﻛﻠﻳﺔ اﻟزراﻋﺔ، اﻟﺟﺎﻣﻌﺔ اﻻردﻧﻳﺔ. 3 . ﻛﻠﻳﺔ اﻟﺻﻳدﻟﺔ، ﺟﺎﻣﻌﺔ ﻗطر، ﻗطر. ﺗﺎرﻳﺦ اﺳﺗﻼم اﻟﺑﺣث 2/2/2015 وﺗﺎرﻳﺦ ﻗﺑوﻟﻪ 2015/10/28.

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