Sesame (Sesamum indicum L.) is one of the oldest oil crops and is widely cultivated in Asia and Africa. To determine the level of genetic diversity in relation to geographical origins and morphological characteristics, a total of 96 accessions have been collected from different parts of the world and were analyzed using AFLP techniques. Twenty-one primer pairs generated a total of 445 bands and among them 157 (35%) were polymorphic. Using UPGMA clustering analysis method based on the similarity coefficient, accessions were separated into two major groups. The first group mostly consists of Eastern Asian origin and another group consists of South Asian origin. Sub-clusters separated the accessions and form distinct diversity among groups. Considering the relatednessof accessions, geographical origin and their morphological characteristics are reflected to the similarity of AFLP pattern.

Sesame (Sesamum indicum L.) family Pedaliaceae, is one of the most ancient oilseeds crop known to mankind. It was cultivated and domesticated on the Indian subcontinent during Harappan and Anatolian eras (Bedigian et al. 1985; Bedigian et al. 2003) but now it is grown in many parts of the world. However, Asia is rich in diversity of cultivated sesame. It is an important source of edible oil and is widely used as a one of the ingredients in food products especially in bakery foods and animal feed. Sesame oil has medicinal and pharmaceutical value and is being used in many health cure products. Sesame seed contains 50-60% oil and 25% protein with antioxidants lignans such as sesamolin, sesamin and has been used as active ingredients in antiseptics, bactericides, viricides, disinfectants, moth repellants, anti-tubercular agents (Bedigian et al. 1985) and considerable source of calcium, tryptophan, methionine and many minerals (Johnson et al. 1979). These lignan contents have beneficial physiological effects in animal and human health (Ashakumary et al. 1999). Composition of fatty acid in sesame oil is variable between different cultivars (Yermanos et al. 1972; Brar, 1982). Effective antioxidant properties of sesame seed oils are characterized by the presence of lignans, sesamin and sesamolin (Fukuda et al. 1986). These highly important characteristics have lead researchers to develop interest in biochemical analysis and in identifying the accessions having rich beneficial oil contents, in order to make efforts for the improvement of this crop using advanced technologies.

Recently, the use of AFLP in genetic marker technologies has become the main tool due to its capability to disclose a high number of polymorphic markers by single reaction (Vos et al. 1995). It is a useful technique for breeders to accelerate plant improvement for a variety of criteria, by using molecular genetics maps to undertake marker-assisted selection and positional cloning for special characters. Molecular markers are more reliable for genetic studies than morphological characteristics because the environment does not affect them. In sesame, few reports have been published on the analysis of the diversity viz., RAPD (Bhat et al. 1999), isozymes (Isshiki and Umezaki, 1997), morphological and agronomic characters (Bedigian et al. 1986) but a little work has been done on sesame using AFLP molecular techniques for evaluating genetic diversity in relatedness with geographical origin.

AFLP markers have successfully been used for analyzing genetic diversity in some other plant species such as peanut (Herselman, 2003), soybean (Ude et al. 2003), and maize (Lübberstedt et al. 2000). These studies have indicated that the AFLP technique is highly applicable for molecular discrimination at the species level.

The identification of genetic relationship among the cultivars based on biochemical and molecular analysis will be used in further genetic improvement. It will also provide support for selection of crossing combinations from bulk parental genotypes and for broadening the genetic basis of breeding programs. Therefore, it is necessary to study cultivars at the molecular level to distinguish them for their special characters and to differentiate varieties collected from different regions of the world.

In this context, the aims of the present study were to find out the relationships between sesame cultivars including breeding lines and, to analyze their genetic relationships for further genotypes identification. First, to determine varietals differences among varieties collected from different regions of the world, and second to describe the genetic similarity between accessions and confirm them by using morphological parameters.

Plant material

Ninety-six accessions including breeding lines, experimental lines and local varieties collected from different regions of the world were analyzed (Table 1) for AFLP. This material was maintained at the National Institute of Crop Sciences Tsukuba, Japan. Each accession had homogeneous material therefore a single plant was used from each one.

AFLP methodology

All accessions were grown in a greenhouse and a total of 100 mg of fresh leaves were collected for DNA isolation using Plant DNA ZOL kit (Invitrogen life technology USA). AFLP analysis was performed according to Vos et al. (1995) method with little modifications. Initially, genomic DNA (120 ng) was digested using 1 µl of EcoR1/Mse1 (1.25) unit enzymes (Invitrogen AFLP Core reagent kit) at 37ºC for 12 hrs. Digested reactions were ligated following manufacture instructions. Diluted ligations (1:10) mixture were pre-amplified using the E00 (GACTGCGTACCAATTC) and M00 (GATGAGTCCTGAGTAA) primers. PCR reactions were performed in a thermal cycler (GeneAmp PCR system 9700, Applied Biosystems, USA) at 94ºC denature for 30 sec, annealing at 56ºC for 60 sec and extension at 72ºC for 60 sec for 20 cycles. Pre-selective PCR products (1:100 diluted) were stored at -20ºC.

For selective amplification, thermocycler was programmed to a touchdown temperature cycle at 94ºC for 30 sec, 65ºC for 30 sec and 72ºC for 60 sec for 13 cycles. The annealing temperature was decreased 0.7ºC each cycle and then, 23 cycles at 94ºC for 30 sec, 56ºC for 30 sec and 72ºC for 60 sec. Combinations of primers showed in Table 2 were used for the AFLP analysis.

PCR products were loaded on 0.8% Bis, 30% Acryl-amide, 1.5 M Tris-HCl (pH 8.8), 10% APS, TEMED gel. Marker VIII ladder (Roche diagnostic GmbH Germany) was used as molecular weight standard. Gels were stained using a Vistra green (Amersham) solution (60 µl in 200 ml sterilized H₂O) for 40 min. Stained gels were washed with 25% ethanol for 1 hr. Bands were scored visually from gel pictures. An example is shown in Figure 1.

AFLP analysis

A total of 21 primer combinations were selected to carry out the analysis in the ninety-six varieties (Table 1). Total bands were scored visually and polymorphic bands were analyzed as presence (1) or absence (0). Phylogenetic relations were determined by the UPGMA method using the Jaccard's similarity coefficient (SPSS - 10 software).

For an initial screening, seven-hundred-four primers combinations were tested in eight varieties (data not shown). From this study, the twenty-one most effective primers were selected by scoring the amount of polymorphic bands. Results showed (Table 2) that E-ACT/M-GTT primer combination produced maximum polymorphic bands (65% of total detected bands) whereas the primers E-AAC/M-GGT, E-AGA/M-GTC, E-AGC/M-GAG and E-ACT/M-TAT produced superior number of polymorphic bands. E-AGG was found to be the best performer primer, having more ability to produce polymorphic bands with other M primers. Among twenty-one selected combinations, eight combinations were composed by the E-AGG primer.

Results for AFLP data and phenotypic data are presented in Figure 2 and Table 1, respectively. Main clusters were related to geographic origin but the small clusters also present a phenotypic relatedness for four morphological traits viz., branching habit, number of flowers per axil, type of capsule and seed coat colour. Molecular data categorized the sesame accessions in two main groups (Figure 2). Group I and II, which discriminate varieties related with geographical origin. Countries were separated in the two main groups with some exceptions; both groups accumulated most of the accessions from countries of close origin. It is clear in cluster analysis that the accessions from Japan, India, Myanmar, and Pakistan showed a close phylogenetic relationship, based on their origin.

It was noticed that due to genetic difference, major genotype clusters were related to main geographic origin. However, small clusters were also formed based on some known characteristics, pedigree relations or belonging to close area of cultivation within main group. Group 1 was divided into nine (a to i) sub groups. Results displayed that both S79 and S80 were sister breeding lines with high lignin contents as could be confirmed by their close distance. AFLP markers produced identical fingerprints between these lines and, one of their parents (S81) was also neighboured within small distance.

Three accessions viz., S4, S5, and S6 gathered in cluster "c" were originated in USA, especially S4 and S6 carried indehiscent character with short molecular distance from another indehiscent accession S90. Whereas three accessions from China were grouped in cluster "d". Cluster "e" bunched four Japanese accessions collected from western region of Japan. Varieties S22, S24 and S83 from Korea as well as others accessions from central Japan were in the cluster "g". Cluster "h" was composed by 18 accessions; one from Korea, two from China and the rest were from central Japan. It was noticeable that most of the accessions from clusters "g" and "h" were from Korea and western region (Shimane prefecture). Some other varieties in these clusters belong to central region that is neighbouring to western Japan, reflecting that geographical association being close position in clusters.

Group II consisted of two main clusters "a" and "b". Cluster "a" was mainly composed by accessions collected from Myanmar, three from India, one from Bangladesh and, one from Sri Lanka r. Cluster "b" was formed by most of the accessions from India, Pakistan, Bangladesh, Sri Lanka, Thailand and Nepal. Dendrogram (Figure 2) confirms that the accessions collected from same countries were closely associated. Pakistan, Thailand and India dominantly showed their association in small units considering molecular similarities. Overall consideration could be that the entire South Asian region was a place of origin from the studied accessions.

Similarities in morphological characters, such as basal branching, one flower, bicarpels and white seed coat colour were also showed in the accessions accumulated in Group-1 a, b, f and g. Basal branching, three flowers and bicarpels were gathered in cluster "c" and "d". Cluster "e" was characterized by accessions with basal branching, no branching, one flower, tetracarpels and white seed colour.

Some of the accessions in cluster "h" had similarity having basal or no branching and bicarples. Group II was divided into two main clusters "a" and "b"; majority of the accessions in cluster "a" produced basal branching habit, one flower bicarples and reddish brown. In cluster "b" most of the accessions produced white seed colour whereas some accessions had few exceptional morphological characters in each sub group, which may differentiate the clusters.

From above results, it has been observed that different geographical regions could be characterized by the presence of AFLP fragments, and a possible correlation between some morphological characters and geographic origin was also evident.

Genetic diversity of different materials can be studied together by morphological traits, the geographical origin and by using molecular marker techniques like RFLPs, RAPDs or AFLPs. Work on the subject has already been described in many other species, especially in cereals (Cho et al. 1998), horticultural crops (Aranzana et al. 2003), medicinal plants, ornamental plants and, oilseed plants (Hansen et al. 2003). Microsatellites and SSRs are also considered a powerful tool to investigate plant variability (Donini et al. 1998; Huang et al. 2002; Khlestkina et al. 2004). Recently, it has been assumed that in plant breeding, diversity can be reduced using biochemical molecular techniques. Present study was carried out on diversity of ninety-six sesame accessions collected from different parts of the world, mainly from the Asian region.

In our work, close genetic relations between the accessions were determined by geographical origin using AFLP markers. The accessions were clustered in two main groups; mainly corresponding to their geographical origin as well morphological characteristics. All accessions from Japan were clustered in Group I and, none of Japanese accessions were outside this group. It is remarkable to mention that accessions from neighboring countries of Japan (Korea and China) were also pooled in this group and showed low diversity.

It is important to stand out that in the collected materials from Japan, most of the accessions from the same or neighbour regions were closely grouped, i.e. accessions S49, S28, S29, S27, S51, S53 and S91 from central region pooled together (g and h clusters). Accessions S38, S39 and S36, S40 and S82 from western region and some Korean accessions (Figure 2) were also grouped together, probably due to a very close place of origin. It has been concluded that sesame cultivated in these countries had a very narrow genetic base. Present results support the evidences of previous studies from Isshiki and Umezaki (1997) and Bhat et al. (1999). Similarly, majority of the accessions from Myanmar were grouped in cluster "a" of group II, while all other south Asian neighboring countries were pooled together in cluster "b". Figure 2 showed a very consistent relationship between these accessions. Our results are in accordance with the conclusion on distribution of genetic diversity for soybean observed by Cui et al. (2000) and Ude et al. (2003).

However, there are some exceptions, accession S63 from Myanmar showed a drastically distinct position (Figure 2) being clustered in "I a" indicating the highest diversity. Bhat et al. (1999) found comparable results using RAPDs for accessions collected in India. As the accessions representing different regions were grouped in different clusters, further strategies could be following for both breeding management and usage.

Considering morphological data (branching habit, number of flowers per axel, capsule type and seed coat colour), some sesame genotypes were closely grouped in sub-clusters. Group I clusters "c" and "d" included basal branching, three flowers and bi-carpals, while accessions from cluster "e" produced one flower with tetra-carpel trait. Clusters I f, g and II b mostly accumulated accessions with basal branching, one flower, white seed coat colour, but f and g might be separated because of tetra carpel character. Whereas "II a" showed similarities on the basal branching, one flower but with different seed coat colour, altogether point toward their relatedness (Kobayashi, 1981; Bisht et al. 1998). Similar results indicating relationship between molecular data with morphological traits have been reporter by Furini and Wunder (2004) for complex Solanum genus and, by Sharma et al. (2000) in Morus genus.

In coincidence with Kobayashi (1981) results, tetra-carpals characters appears mostly in accessions belonging to Japan and far east countries, whereas those belonging to other Asian countries produced bi-carpals. Results of cluster pattern showed a relationship when comparing molecular and morphological data for most of the phenotypic characters. Federici et al. (2001) observed this kind of relationship in rice. In this case, about 90% of the samples having straw hull and short awns were clustered together and, about 75% with black hull and long awns were accumulated separately by AFLP data. Furini and Wunder (2004) also reported consistency between molecular and morphological data in eggplant. Additionally, this relationship has been studied in different crops, i.e. rice (Federici et al. 2001), common vetch (Sharma et al. 2000), Morus (Potokina et al. 2002; Baranger et al. 2004).

Two lines (S79 and S80) with high lignin contents showed strong relation on the basis of biochemical analysis (Sirato-Yasumoto et al. 2001) as was revealed in the dendrogram. Both lines were breed for high lignin contents; which showed feasibility of AFLP technique as a tool for identification of parental genotypes (Marsan et al. 1998). In addition, it was remarkable that accessions S4, S6 and S90 (with indehiscent trait) were closely grouped. Linkage for indehiscent characters in sesame has also been reported by Uzun et al. (2003).

Summarizing, we demonstrated that for genetic relatedness studies in sesame AFLP was a reliable tool. AFLP patterns will be useful to identify the different sesame accessions and to make relatedness by biochemical analysis. Morphological traits, geographical origins, and observations on genotype-specific amplified bands of AFLP will also be useful for their economic value and explore the different genotypes for further classification.

We would like to thank Dr. Ryoji Takahashi for all his technical and moral support for this work and also like to thanks Evangelos D. Leonardos and Javaid Iqbal, Department of Plant Agriculture, University of Guelph, Guelph, Canada for critical reading of the manuscript.

ARANZANA, M.J.; CARBO, J. and ARUS, P. Using amplified fragment-length polymorphisms (AFLPs) to identify peach cultivars. Journal of the American Society for Horticultural Science, September 2003, vol. 128, no. 5, p. 672-677.

ASHAKUMARY, Lakshmikuttyamma; ROUYER, Isabelle; TAKAHASHI, Yoko; IDE, Takashi; FUKUDA, Nobuhiro; AOYAMA, Toshifumi; HASHIMOTO, Takashi; MIZUGAKI, Michinao and SUGANO, Michihiro. Sesamin, a sesame lignan, is a potent inducer of hepatic fatty acid oxidation in the rat. Metabolism: Clinical and Experimental, October 1999, vol. 48, no. 10, p. 1303-1313. [CrossRef]

BARANGER, A.; AUBERT, G.; ARNAU, G.; LAINÉ, A.L.; DENIOT, G.; POTIER, J.; WEINACHTER, C.; LEJEUNE-HÉNAUT, I.; LALLEMAND, J. and BURSTIN, J. Genetic diversity within Pisum sativum using protein- and PCR-based markers. Theoretical and Applied Genetics, May 2004, vol. 108, no. 7, p. 1309-1321. [CrossRef]

BEDIGIAN, D.; SEIGLER, David S. and HARLAN, Jack R. Sesamin, sesamolin and the origin of sesame. Biochemical Systematics and Ecology, May 1985, vol. 13, no. 2, p. 133-139. [CrossRef]

BEDIGIAN, D.; SMYTH, C.A. and HARLAN, J.R. Patterns of morphological variation in sesame. Economic Botany, 1986, vol. 40, p. 353-365.

BEDIGIAN, Dorothea. Evolution of sesame revisited: domestication, diversity and prospects. Genetic Resources and Crop Evolution, November 2003, vol. 50, no. 7, p. 773-778.

BHAT, K. Venkataramana; BABREKAR, Prashant P. and LAKHANPAUL, Suman. Study of genetic diversity in Indian and exotic sesame (Sesamum indicum L.) germplasm using random amplified polymorphic DNA (RAPD) markers. Euphytica, October 1999, vol. 110, no. 1, p. 21-34. [CrossRef]

BISHT, I.S.; MAHAJAN, R.K.; LOKNATHAN, T.R. and AGRAWAL, R.C. Diversity in Indian sesame collection and stratification of germplasm accessions in different diversity groups. Genetic Resources and Crop Evolution, August 1998, vol. 45, no. 4, p. 325-335. [CrossRef]

BRAR, G.S. Variations and correlations in oil content and fatty acid composition of sesame. Indian Journal of Agriculture Science, 1982, vol. 52, p. 434-439.

CHO, Y.G.; MCCOUCH, S.R.; KUIPER, M.; KANG, M.-R.; POT, J.; GROENEN, J.T.M. and EUN, M.Y. Integrated map of AFLP, SSLP and RFLP markers using a recombinant inbred population of rice (Oryza sativa L.). Theoretical and Applied Genetics, August 1998, vol. 97, no. 3, p. 370-380. [CrossRef]

CUI, Zhanglin; CARTER JR., Thomas E. and BURTON, Joseph W. Genetic diversity patterns in Chinese soybean cultivars based on coefficient of parentage. Crop Science, November-December 2000, vol. 40, no. 6, p. 1780-1793.

DONINI, P.; STEPHENSON, P.; BRYAN, G.J. and KOEBNER, R.M.D. The potential of microsatellites for high throughput genetic diversity assessment in wheat and barley. Genetic Resources and Crop Evolution, October 1998, vol. 45, no. 5, p. 415-421. [CrossRef]

FEDERICI, Maria Teresa; VAUGHAN, Duncan; TOMOOKA, Norihiko; KAGA, Akita; WANG, Xin Wang; DOI, Koji; FRANCIS, Marta; ZORRILLA, Gonzalo and SALDAIN, Nestor. Analysis of Uruguayan weedy rice genetic diversity using AFLP molecular markers. Electronic Journal of Biotechnology [online]. 15 December 2001, vol. 4, no. 3 [cited]. Available from: http://www.ejbiotechnology.info/content/vol4/issue3/full/3/index.html. ISSN 0717-3458.

FUKUDA, Y.; NAGATA, M.; OSAWA, T. and NAMIKI, M. Contribution of lignan analogues to antioxidative activity of refined unroasted sesame seed oil. Journal of the American Oil Chemists' Society, August 1986, vol. 63, no. 8, p. 1027-1031.

FURINI, A. and WUNDER, J. Analysis of eggplant (Solanum melongena)-related germplasm: morphological and AFLP data contribute to phylogenetic interpretations and germplasm utilization. Theoretical and Applied Genetics, January 2004, vol. 108, no. 2, p. 197-208. [CrossRef]

HANSEN, L.B.; SIEGISMUND, H.R. and JORGENSEN, R.B. Progressive introgression between Brassica napus (oilseed rape) and Brassica rapa. Heredity, September 2003, vol. 91, no. 3, p. 276-283. [CrossRef]

HERSELMAN, Liezel. Genetic variation among Southern African cultivated peanut (Arachis hypogaea L.) genotypes as revealed by AFLP analysis. Euphytica, September 2003, vol. 133, no. 3, p. 319-327.  [CrossRef]

HUANG, X.Q.; BÖRNER, A.; RÖDER, M.S. and GANAL, M.W. Assessing genetic diversity of wheat (Triticum aestivum L.) germplasm using microsatellite markers. Theoretical and Applied Genetics, October 2002, vol. 105, no. 5, p. 699-707. [CrossRef]

ISSHIKI, Shiro and UMEZAKI, Teruhisa. Genetic variations of isozymes in cultivated sesame (Sesamum indicum L.). Euphytica, February 1997, vol. 93, no. 3, p. 375-377. [CrossRef]

JOHNSON, L.A.; SULEIMAN, T.M. and LUSAS, E.W. Sesame protein: a review and prospectus. Journal of the American Oil Chemists' Society, March 1979, vol. 56, no. 3, p. 463-468.

KHLESTKINA, E.K.; HUANG, X.Q.; QUENUM, F.J.-B.; CHEBOTAR, S.; RODER, M.S. and BORNER, A. Genetic diversity in cultivated plants-loss or stability? Theoretical and Applied Genetics, May 2004, vol. 108, no. 8, p. 1466-1472. [CrossRef]

KOBAYASHI, T. The wild and cultivated species in genus sesamum. Sesame: Status and Improvement. In: Proceedings of Expert Conclusion (8^th - 12^th December 1980, Rome, Italy). FAO Plant Production and Protection Paper, 1981, vol. 29, p. 157-163.

LÜBBERSTEDT, Thomas; MELCHINGER, Albercht E.; DUBLE, Christina; VUYLSTEKE, Marnik and KUIPER, Martin. Relationships among Early European Maize Inbreds: IV. Genetic diversity revealed with AFLP markers and comparison with RFLP, RAPD, and pedigree data. Crop Science, May-June 2000, vol. 40, no. 3, p. 783-791.

MARSAN, P.A.; CASTIGLIONI, P.; FUSARI, F.; KUIPER, M. and MOTTO, M. Genetic diversity and its relationship to hybrid performance in maize as revealed by RFLP and AFLP markers. Theoretical and Applied Genetics, February 1998, vol. 96, no. 2, p. 219-227. [CrossRef]

POTOKINA, E.; BLATTNER, F.R.; ALEXANDROVA, T. and BACHMANN, K. AFLP diversity in the common vetch (Vicia sativa L.) on the world scale. Theoretical and Applied Genetics, July 2002, vol. 105, no. 1, p. 58-67. [CrossRef]

SHARMA, A.; SHARMA, R. and MACHII, H. Assessment of genetic diversity in a Morus germplasm collection using fluorescence-based AFLP markers. Theoretical and Applied Genetics, November 2000, vol. 101, no. 7, p. 1049-1055. [CrossRef]

SIRATO-YASUMOTO, Satoko; KATSUTA, Masumi; OKUYAMA, Yoshinao; TAKAHASHI, Yoko and IDE, Takashi. Effect of sesame seeds rich in sesamin and sesamolin on fatty acid oxidation in rat liver. Journal of Agricultural and Food Chemistry, 2001, vol. 49, no. 5, p. 2647-2651. [CrossRef]

UDE, George N.; KENWORTHY, William J.; COSTA, Jose M.; CREGAN, Perry B. and ALVERNAZ, Jennie. Genetic diversity of soybean cultivars from China, Japan, North America, and North American ancestral lines determined by amplified fragment length polymorphism. Crop Science, September-October 2003, vol. 43, no. 5, p. 1858-1867.

UZUN, B.; LEE, D.; DONINI, P. and CAGIRGAN, M.I. Identification of a molecular marker linked to the closed capsule mutant trait in sesame using AFLP. Plant Breeding, February 2003, vol. 122, no. 1, p. 95-97. [CrossRef]

VOS, Pieter; HOGERS, Rene; BLEEKER, Marjo; REIJANS, Martin; VAN DE LEE, Theo; HORNES, Miranda; FRITERS, Adrie; POT, Jerina; PALEMAN, Johan; KUIPER, Martin and ZABEAU, Marc. AFLP: A new technique for DNA fingerprinting. Nucleic Acids Research, January 1995, vol. 23, no. 21, p. 4407-4414. [CrossRef]

YERMANOS, D.M.; HEMSTREET, S.; SAEEB, W. and HUSZAR, C.K. Oil content and composition of the seed in the world collection of sesame introductions. Journal of the American Oil Chemists' Society, 1972, vol. 49, p. 20-23.