versão On-line ISSN 0717-3458
Electron. J. Biotechnol. v.10 n.1 Valparaíso jan. 2007
Assessment of genetic diversity in sesame (Sesamum indicum L.) detected by Amplified Fragment Length Polymorphism markers
Ghulam M. Ali*
Financial support: This work was performed under the fellowship for G.M. Ali. Authors are grateful to Japan Society for Promotion of Science and Natural Science and Engineering Research Council Canada for this award and financial support.
Keywords: AFLP, genetic diversity, geographical origin, morphology, sesame.
Sesame (Sesamum indicum L.) is one of the oldest oil crops and is widely cultivated in Asia and
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,
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.
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,
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
For selective amplification, thermocycler was programmed to a touchdown temperature cycle at
PCR products were loaded on 0.8% Bis, 30% Acryl-amide,
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
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
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.
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-
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
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
However, there are some exceptions, accession S63 from
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,
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