Plant Syst Evol (2014) 300:1941–1944 DOI 10.1007/s00606-014-1018-8
SHORT COMMUNICATION
Variation in Saffron (Crocus sativus L.) accessions and Crocus wild species by RAPD analysis F. Izadpanah • S. Kalantari • M. E. Hassani M. R. Naghavi • M. Shokrpour
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Received: 6 June 2013 / Accepted: 15 January 2014 / Published online: 8 March 2014 Ó Springer-Verlag Wien 2014
Abstract Crocus, a genus of Iridaceae is mostly grown in areas with Mediterranean environment as the best region. Saffron (C. sativus) is a perennial plant and is cultivated as an industrial crop in several regions of Iran. In this research, five accessions of cultivated Saffron from five areas in Khorasan, and Esfahan including Gonabad, Ferdows, Torbat-e-Heyidariye, Estahbanat, Gopayegan were used. Other nine species of saffron were grown naturally in Iran; so we collected two wild species (C. caspius and C. speciosus) from north of Iran (Gilan Province). RAPD markers were used to classify these species and to find their relationships. In the results of this study, the cluster analysis showed two distinct groups. Also, the maximum similarity was seen between C. caspius and C. speciosus (0.82) and the minimum was between Estahbanat, Ferdows accessions and C. speciosus (0.33). Finally, this method as a convenience procedure could be used to separate different accessions and species of Crocus as well. Keywords Saffron (C. sativus) Wild species Relationship RAPD markers
F. Izadpanah (&) S. Kalantari M. Shokrpour Department of Horticultural Sciences, Faculty of Agriculture, University of Tehran, 31587 Karaj, Iran e-mail:
[email protected] M. E. Hassani Faculty of Agriculture, Food and Natural Resources, University of Sydney, Darlington, Australia M. R. Naghavi Department of Plant Breeding, Faculty of Agriculture, University of Tehran, 31587 Karaj, Iran
Introduction The original saffron is being obtained from C. sativus L. since ancient time. In addition to cultivated saffron (C. sativus), nine species of that are found in nature of which three of them are endemic to Iran (C. gilanicus, C. michelsonii, C. almehensis) (Rashed-Mohassel 2007). For identification of Crocus species based on vegetative growth, floral and post-floral structure different characteristics exist. C. speciosus is a flowering fall or early winter species in which the corm has membranous or leathery tunics, without conspicuous fibers. The leaves are dark green and width of mature leaves is 4–5 mm. Flowers have other colors, sometimes yellow at throat and the style has three arms. C. caspius species is a flowering fall or early winter species in which corm tunics at leaf stage are fibrous, membranous or leathery, without conspicuous fibers and corm sheath is not quite annular at the base. The leaves are dark green and width of mature leaves is 1–3 mm. Flowers have other colors, sometimes yellow at throat and style is three lobate or somehow lobate. This species flowering after leafing and flower throat is dark yellow. Saffron is a perennial and triploid crop (2n = 3x = 24), corm tunics are rough or fine, with reticulate or parallel fiber and tunics at leaf stage are fibrous or membranous without ring at the base. C. sativus L. has 9–10 grayish leaves with no distinct vein on either side of the leaves. Flowers have other colors, sometimes yellow at throat. It’s flowering fall species which flowers open before leafing. Flower’s color is blue violet or pink, stigma arms broad, 25–35 mm and style has three distinctive lobes, yellow or orange red (Rashed-Mohassel 2007). Saffron is well adapted to arid and semi-arid lands, temperate and sub-tropical climates. Mediterranean environment is recognized worldwide as the best region to
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produce saffron stigma annually, with regard to its quality, which is attributed to many factors (Lage and Cantrell 2009). In recent years technological advances in DNA marker techniques were made available to determine variability present in the genotypes for breeding purposes. RAPD markers have been used for measuring genetic diversity in different plants. We also used this technique to clarify the genetic relationships among saffron accessions of Crocus species. Materials and methods Plant material The five cultivated Saffron (C. sativus) accessions were collected from different fields in north east of Iran (Khorasan Province). The wild species, C. caspius and C. speciosus, were gathered from two natural regions of Iran during October 2009 (Gilan Province). The sampling was performed with cooperation of local Agricultural Extension Offices and producers.
mgcl2, 200 m dNTPs, 1 U Tag DNA polymerase (7.5 ll) (Cina Gene, Iran), 0.4 M of each decamer primer (1.5 ll) and 4 ll dH2O. Amplification products were resolved by electrophoresis through a 1.2 % (w/v) agarose (Roche Co., Germany) gel in 19 TBE buffer for 120 min under 120 V. DNA bands were stained with ethidium bromide (5 lg/ml visualized under UV light and photographed by a Gel Doc system (UVP, Bio Doc Co., USA) (Figs. 1, 2). DATA analysis Amplified products were scored as present (1) or absent (0) to form a binary matrix. Data analysis was made by SPSS software v. 19. Dendrogram of genetic similarities among accessions was compiled using UPGMA method. Nei and Li (1979) index, Polymorphic Index coefficient (PIC) and Marker Index were assayed as following: PIC ¼ 1 Rp2i RR2p2i p2j MI ¼ PIC no: of polymorphic bands:
Results DNA extraction and RAPD amplification Genetic diversity and cluster analysis Total genomic DNA was extracted from 120 mg of fresh young leaves using the procedure of nuclear DNA protocols which is described by Dellaporta et al. (1983). Genomic DNA quality and quantity were determined by comparing with a standard k DNA set (Fermentas TM Life Sciences) on 1.2 % agarose gel. Seven random 10-mer primers (OPERON & TIB-MOLBOIL Co., Germany) were selected for RAPD analysis (Table 1). In that time, we found few plants in natural growing regions. In order to, there was a limitation in preparation required material for extension the experiment and using several primers. PCR reactions were carried out in a 15 ll volume containing 10 ng/ll genomic DNA (2 ll), 19 PCR buffer, 1.75 Mm Table 1 Sequence of primers employed and the number of scored total and polymorphic bands on RAPD analysis
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A total of 85 polymorphic bands were obtained by 7 primers and showed 100 % polymorphism. The maximum similarity value (Nei and Li) was seen between C. caspius and C. speciosus (0.82) and the minimum was between Estahbanat, Ferdows accessions and C. speciosus (0.33) (Table 2). The primer TIBMBA-10 and TIBMBB-20 had maximum (0.79, 12.64) and minimum (0.64, 5.75) PIC and Marker Index values, respectively. Cluster analysis based on marker data formed two distinct groups (Fig. 3). In the first group, all of the cultivated saffron accessions were located. This branch was divided into two sub-clusters consisting of accessions belonging to
Name of primer
Sequence (50 -30 )
Total no. of bands
No. of polymorphic bands
Percent of polymorphism
PIC
Marker index
BA08 BA10
CCACAGCCGA GGACGTTGAG
14 16
14 16
100 100
0.76 0.79
10.64 12.64
BA11
CCACCTTCAG
12
12
100
0.69
8.28
BA15
GAAGACCTGG
12
12
100
0.74
8.88
BA17
TGTACCCCTG
10
10
100
0.69
6.9
BB20
CCAGGTGTAG
9
9
100
0.64
5.75
BC10
AACGTCGAGG
100
0.74
8.88
12
12
Total
85
85
Average
12.14
12.4
100
Variation in Saffron (Crocus sativus L.) M
1
2
3 4
1943 5
6
7 8
9
10 11 12 13 14 15 16 17 18
19 20 21 M
3000bp 2500bp 2000bp 1500bp 1000bp 750bp 500bp
250bp
Fig. 1 PCR-RAPD profiles generated from cultivated saffron and wild species using primer TIBMBA17, TIBMBA15, TIBMBA10. Lane 1 wild; lane 2 wild; lane 3 cultivated; lane 4 cultivated; lane 5 cultivated; lane 6 wild; lane 7 wild; lane 8 wild; lane 9 wild; lane 10 M
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2
3
4 5 6 7 8
cultivated; lane 11 cultivated; lane 12 cultivated; lane 13 wild; lane 14 wild; Lane 15 wild; lane 16 wild; lane 17 cultivated; lane 18 cultivated; lane 19 cultivated; lane 20 wild; lane 21 wild; lane M 1 kb ladder (Fermentas)
9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 M 3000bp 2500bp 2000bp 1500bp 1000bp 750bp 500bp
250bp
Fig. 2 PCR-RAPD profiles of cultivated saffron and wild species amplified by decamer primer TIBMBA11, TIBMBB20, TIBMBA08 and TIBMBC10. Lane 1 wild; lane 2 wild; lane 3 cultivated; lane 4 cultivated; lane 5 cultivated; lane 6 wild; lane 7 wild; lane 8 wild; lane 9 wild; lane 10: cultivated; lane 11 cultivated; lane 12 cultivated; Table 2 Similarity coefficient among the members of genus Crocus based on data of RAPD primers
1 1
2
lane 13 wild; lane 14 wild; lane 15 wild; lane 16 wild; lane 17 cultivated; lane 18 cultivated; lane 19 cultivated; lane 20 wild; lane 21 wild; lane 22 wild; lane 23 wild; lane 24 cultivated; lane 25 cultivated; lane 26 cultivated; lane 27 wild; lane 28 wild; lane M 1 kb ladder (Fermentas)
3
4
5
6
7
1
2
20.78
3
0.51
0.55
1 1
4
0.52
0.63
0.7
1
5
0.55
0.62
0.54
0.68
1
6
0.38
0.44
0.36
0.41
0.33
1
7
0.33
0.44
0.4
0.48
0.39
0.82
the two provinces, Isfahan and Khorasan, nearly far from each other and accessions from each province were located close. As it is expected, the wild species was grouped in the second group.
Discussion RAPD analysis in comparison with other DNA marker systems is an economical and quick technique providing
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molecular data. In this research, the preliminary analysis of the variability in wild Crocus species and saffron accessions, originating from the main cultivation and the natural areas, using RAPD markers was performed. Using clustering, the five different accessions of cultivated saffron species were located in the first group and the wild Crocus species were located in the second group. In this cluster, clear classification of Crocus species was seen. This result as expected confirmed Rashed-Mohassel (2007) study on six different species of Crocus in Iran. He found a close
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Fig. 3 UPGMA dendrogram of seven members of genus Crocus based on molecular data obtained from seven random RAPD primers
relation between two wild species and also grouped them in distinct cluster rather than cultivated saffron. Grilli Caiola et al. (2004) investigated on saffron accessions and related Crocus species by RAPD markers so the result of their study showed Crocus sativus L. very likely originated from C. cartwrightianus (Fig. 2). Although the number of accessions and species were used in this work does not represent the total diversity of Iranian cultivated Saffron and wild Crocus species, but remarkable genetic divergence was shown. Further investigations and applying more informative markers such as SSRs would improve our understanding of saffron clone’s origin and species in the world (Fig. 3). Acknowledgments The authors wish to thank Dr. Mardi in ABRII institute for his cooperation in preparing materials.
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References Dellaporta SL, Wood J, Hicks JB (1983) A plant DNA minipreparation version II. Plant Mol Biol Report 1:19–21 Grilli Caiola M, Caputo P, Zanier R (2004) RAPD analysis in Crocus sativus L. accessions and related Crocus species. Biol Plant 48:375–380 Lage M, Cantrell CL (2009) Quantification of saffron (Crocus sativus L.) metabolites crocins, picrocrocin and safranal for quality determination of the spice grown under different environmental Moroccan conditions. Sci Hortic 121:366–373 Nei M, Li WH (1979) Mathematical model for studying genetic variation in terms of restriction endonucleases. Proc Natl Acad Sci USA 76:5269–5273 Rashed-Mohassel MH (2007) Saffron from wild to the field. Acta Hort (ISHS) 739:187–193
