Sesame (Sesamum indicum L. 2n=26), is a self-pollinated [1] member of the order Tubiflorae, family Pedaliaceae, and is one of the ancient and most important sources of a quality plant oil used by human beings [2]. It is typically an annual oilseed crop and grown in the tropics and warm subtropics [3]. Sesame seed is rich in nutrition value and due to the quality of oils, this sesame is known as the “Queen of oilseeds” [4]. The lignan compound and its derivatives prevent oxidation of the oil and help it to have long shelf life and stability [5]. Sesame seed contains 50-60% oil, 49.1% fat, 5.3% ash and 21.6% carbohydrates, [6] proteins (18-25%), minerals such as calcium, phosphorus, iron, magnesium, manganese, zinc, and vitamin B1 [7, 8]. For many developing countries, it is an extra rich source of protein [9].

Ethiopia is among the most known and major producers of sesame in sub-Saharan Africa, and Ethiopian sesame is among the highest quality in the world; especially seeds produced in the Humera, northern part of the country are very famous due to their colour, taste and good aroma [10]. One of the oilseeds for which Ethiopia is known in the international market (Fig. 1) is sesame which is a good source of foreign currency after coffee from the agricultural commodities [11]. In the last decade, sesame production and marketing have shown a very significant growth. Between 2011 and 2017, the total area of production (42370 ha, 327,741 tons) and the quantity (293,645 ha, 7,873 hectograms per hectare) of sesame produced grown eightfold [12, 13]. The major contributor of this production achievement is the northwest of Ethiopia, particularly Humera and Gondar [14].

To meet the need for seed certification and to obtain an affordable and maximum yield, we should use high-quality seed, i.e., the seed should be physically as well as genetically pure. To produce quality seeds, different multiplication stages are needed. But a number of factors play a vital role in affecting the quality of the seed, including admixture, cross-pollination, and genetic drift as affected by drought, temperature, edaphic factors, and seed-borne diseases. In order to maintain the required genetic purity standards in the seed fields, field inspection, seed, and seedling inspection and grown out, tests are required.

Farmers and seed producers need an assurance that they are being supplied with quality seed material to know the characteristic of a specific cultivar and be assured of its quality. Thus, there is a need to investigate for quick and reliable methods of cultivar identification and for seed purity testing. The production and distribution of quality seeds of improved varieties can alone result in 20-25 percent increased yield in self-pollinated crops and it is significantly higher cross-pollinated crops. According to the central statistical authority report of 2017/18, the sesame production in Ethiopia was 2,559,039.30 Quintal with a yield of 6.91Q/ha [15]. In the Tigray region, the yield of sesame is almost similar to the national yield i.e., 6.10 Q/t and the area under sesame cultivation is 746,142.65 ha [16]. The most suitable area for sesame production in Ethiopia is low and mild altitude between 500-1300 m above the sea level [17]. However, the productivity per unit area of the available nationally released sesame genotypes is lower compared with other producing countries. The low productivity of sesame (0.72 t/ha) in Ethiopia, particularly in Tigray is often attributed to a lack of certified quality seed of improved varieties. To produce quality seed, improved genetic purity is the primary criteria to yield the inherent potential of improved genotypes. It is not questionable that one factor constraining quality seed production in Ethiopia, particularly in Humera, is a lack of characterized sesame varieties [11].

Fig. (1). Ethiopian sesame exported from 2011/12 to 2016/17) [18] adopted from [14].

Seedling and plant are the most widely used methods to identify varietal characteristics based on morphological features. According to the International Union for Protection of New Plant Varieties (UPOV), any new characteristic used in varietal characterization should be clearly defined, accepted and should have a standard method of observation, least or not affected by the environment, accessible to breeders, associated with reasonable costs and efforts. By conducting a growth out test and for varietal identification through morphological characters, the plant and seed characters are well studied and thoroughly documented. The National Test Guidelines need to be developed for the conduct of DUS testing. Such characterization studies are lacking in Ethiopian sesame genotypes.

The most interesting fact is the morphological characteristics that we currently used are not included in all these criteria. Besides, varietal characterization using morphological characterization possesses several unwanted characters such as large space requirement, seasonal dependence, time consumption, tedious and environmental influence. The possible ways to avoid these limitations and to speed up the testing procedures is to use chemical tests or electrophoresis, in addition to morphological markers. The use of DNA profiling techniques will help to establish the varietal identity, to measure the genetic relationship and to assess genetic diversity. Genetic diversity in sesame has been detected by universal markers such as Amplified Fragment Length Polymorphism (AFLP) [18, 19] Sequence-Related Amplified Polymorphisms (SRAP) [20], Random Amplified Polymorphic DNA (RAPD) [21, 22], and inter-simple sequence repeat (ISSR) [23, 24].

It is necessary to use reliable methods to identify and group sesame genotypes. The most preferred and reliable method is the use of DNA fingerprinting to evaluate the genetic purity and to assess the genetic variability in different crops [25-30] The research on cultivar identification in sesame is limited and only seed morphological characteristics cannot distinguish the closely related cultivars. Therefore, more emphasis is needed onvarietal identification of sesame. Hence, the current study was designed with the following objectives:1) identification and grouping of varieties through morphological characters and 2) Identification and grouping of varieties through molecular markers.

Among the ten sesame genotypes studied, the number of primary branches per plant was categorized as ‘more’ (5.93) and more (24.13) number of nodes per plant were recorded for M-80 while the highest (26.13) number of nodes per plant was scored from Humera-1. Plant height was found as ‘tall’ in all the sesame genotypes studied. Likewise, the number of nodes per plant in all the cultivars was classified as more nodes per plant i.e. greater than 15. The number of nodes per plant ranged from 26.13 to 21.47 with the genotype Humera-1 and breeder seed (S), ranking the highest and the lowest, respectively. This could be due to its adaptation to the environmental condition of the experimental area. Similar results were recorded in another study [38].

Fig. (5). Number of locules per pod and pod dehiscent.

Fig. (6). Banding patterns of ISSR primer 834 for 10 sesame cultivars.

The minimum and maximum plant height belonged to Adi (128.53 cm) and E (150.93 cm), respectively. Plant height is a useful agronomic character that can help select and group sesame cultivars in ‘short’, ‘medium’ and ‘tall’ categories. The high number of three varieties, namely M-80, Arjene and Serkamo were found with a high number of branches; this may indicate their ability to bear more pod, which may in turn contribute to yield. As a result of this it can be said that selection of plants having more branches is preferred. Variation among sesame genotypes in the number of branches has been reported in another study [39, 40].

Sesame genotypes with a high number of leaves per plant have good yield potential as the number of leaves increases their ability to synthesize nutrients. Several genotypes, such as E, M-80, Argene and breeder seed (S) were found with a high number of leaves and branches. These results revealed that the varieties had good potential to be included in future sesame breeding programs aimed at improving the yield potential of sesame in Humera, northern Ethiopia. Agro-orphological characters such as plant height, number of leaves, and number of pods/plants are not affected by genetic influence [41]. The variation was least for qualitative characters like leaf length, flower colour, number of locules per pod, pod pubescence, and type of pod beak. There was only one flower colour i.e. white with purple shading. The inter-nodal length of the main stem influences the plant height and the number of branches [2]. Analysis of variance showed a significant difference with respect to internodal length with a mean of 12.15 and all the genotypes were grouped as long (long:> 4.0 cm). Similar findings in these characters were earlier reported [42] in castor varieties and hybrids. The variation is genetically controlled and further influenced by environmental conditions and agronomic practices.

The stem pigmentation is among the most important parameters in genotype identification. Based on the stem pigmentation, the varieties were classified as medium (seven varieties) and strongly (three varieties) pigmented. Similar results on stem pigmentation were previously reported [43, 44]. Stem pigmentation can be affected by environmental factors such as temperature and light intensity and it is also genetically controlled [45]. The leaf petiole pigmentation at the peak flowering stage varied among the ten sesamum genotypes. Similar results were also reported by Jain et al. [43], in mung bean varieties. Rajendra et al. [37], grouped sunflower genotypes as present or absent based on their leaf petiole pigmentation. The variation in leaf petiole pigmentation among the genotypes may be due to varied intensity of anthocyanin pigmentation, which is governed genetically and also influenced by the nutritional status of the soil under which the sesame plant is grown.

Fig. (7). UPFMA clustering dendrogram of sesame genotypes based on genetic distance using ISSR markers.

For arid areas like Humera, early maturing genotypes are more preferred than late matured ones. The genotypes Tate, E, S, M-80, and K-74 had the maximum values for days to flowering (50%) while Humera-1 was found with less value i.e, it takes less time in flowering. Based on flower petal, colour varieties were grouped as light pink (four varieties), medium pink (two varieties) and dark pink (two varieties). The genotypes were further divided into three groups based on the flower hairiness. Among the genotypes, one genotype was low, five genotypes were medium and two genotypes were dense pubescence types. Hairiness is an important morphological character of sesame and found in different parts of the plant [6].

Capsule hairiness was found varied among the genotypes. Two genotypes, namely, Tate and Adi were found with no hairs on stem, leaf, and capsule. Strong or profuse hairs were found in Serkamo and E breeding seed genotypes. However, Setit-1 collected from Humera research institute, exhibited a small stem, less leaves and capsule hairiness. Such characters are an important physical barrier prevention of insect pests and sesame diseases. Haljak et al. [44], studied the hairiness of auricles of flag leaf, flag leaf wax of sheath, ear waxiness, ear density, and peduncle length of nine winter wheat genotypes and suggested that these morphological characters are best for the distinctness of the varieties.

Based on pod number, the varieties were classified into three groups as less (< 40 pods per plant), moderate (40-60 pod per plant) and more (> 60 pods per plant). Among the ten genotypes, seven had moderate (Argene, E, S, Serkamo, K-74, and Humera-1) and three had more (Setit-1, Adi, and M-80) pod numbers per plant. Similar results were observed in chickpea genotypes [46]. The variation in pod number may be due to the pod bearing ability of the genotype and nutrient content of the soil to some extent.

The pod dehiscence varied among the genotypes (Table 3). Genotypes such as Tata, Setit-1 and Humera-1 are grouped as dehiscent and six genotypes, namely, Argene, E, S, M-80, K-74, Adi and Serkamo are grouped as indehiscent types. Likewise, similar results have been reported from castor [42] and jute [47] varieties. The pod dehiscence might be influenced by temperature, especially at the time of flowering. Thousand seed weight is considered as a quality requirement for seed export in the international market. In this study, all the genotypes were grouped as a medium in 1000-seed weight (3.5 g) which implies that all the tested varieties in the current study fulfilled the criteria for export standard i.e. a seed weight of greater than 3g, oil content of 40-50%, and white seed colour are the required criteria.

The average mean for seed yield was determined and found as 2458.04 kg per ha. However, a maximum sesame seed yield of 799.9 kg/ha was reported from Humera, northern Ethiopia [48]. This yield variation could be due to the cultivation conditions, the genetic potential of the genotypes and environment variability. The environment x genotype interaction influences the yield performance of genotypes; as a result, genotypes respond differently to environmental changes in which they are grown [49, 50].

To eliminate the influence of environmental factors on the morphological characters, we analysed the sesame genotypes using ISSR primers and the level of polymorphism among the 10 varieties ranged from 56.25% to 100%. The number of polymorphic loci was 16 for primer 880 (least) and 100% for primers 811, 812 and 860. Bhat et al. [21], evaluated genetic diversity of sesame genotypes and they found a high level of genetic diversity among Indian genotypes. UPGMA analyses were used to construct a dendrogram for the 10 sesame cultivars based on the 376 obtained bands with all the 11 dinucleotide primers. Accordingly, in the dendrogram, the sesame varieties named ADI, Humera1, Serkamo, M-80 and E were grouped together in one cluster and varieties Argena, K-74, and S Breeder appeared to have their own cluster separately Fig. (7). However, the rest of the cultivars get free from the main cluster in the UPGMA dendrogram.

Abdellatef et al. [49], studied 10 sesame genotypes and found the maximum and 92.3% polymorphism while the minimum numbers of fragments were 60% polymorphism. A study [51] reported that when the variation between genotypes is high, the use of a few primers is sufficient. Our results were similar to the findings of other researchers [52-.54]. Zhang et al., [54] who studied 14 agronomic traits and SSR markers to select better performing sesame landraces from China’s germplasm collection.

In our study, the information for genotype identification and grouping of sesame genotypes cultivated in Ethiopia through their morphological characteristics has been confirmed with both agromorphological and molecular markers. This study isreliable, and effective in determining polymorphisms in the molecular genotyping of sesame genotypes, reproducible and it can also produce enough polymorphisms to group and identify the genotypes even though most of the molecular markers found polymorphism. To the best of our knowledge, this is the first study on the characterization of Ethiopian sesame genotypes. The findings presented in this paper may be a useful piece in the future to include more ISSR markers and induvial genotype investigation for detailed characterization of sesame germplasm that would be important for future breeding and sesame improvement program.