Genetic relationship between sesame (Sesamum indicum L.) and related wild species based on chromosome counts and isozyme markers

Sesame is an orphan crop with little research attention in Kenya. Genetic relationship between cultivated sesame and related wild species in Kenya is not well known. The objective of this study was to determine genetic relationship between traditional landraces of sesame and related wild species using somatic chromosome counts and isozyme markers. Somatic chromosome counts of four wild species revealed a consistent chromosome number of 2n = 32, which differed from that of the cultivated sesame (2n = 26), indicating genetic variation in chromosome counts. Only esterase exhibited significant variation and accession-specific esterase bands were identified. Three cathodic and eight anodic bands were observed and the variable bands ranged from 2 to 6 per accession. Cathodic bands with varied relative migration were observed in wild species only, whereas anodic bands were observed for all the accessions. Accessions of cultivated sesame were more genetically diverse compared to wild species. Morogoro, 107UG, Indian-1 and Indian-2 recorded the highest number of esterase bands, while 103w had the lowest number of bands. Few common bands were found between cultivated sesame and related wild species indicating a distant genetic relationship. Few gene markers are available in sesame and related wild species, therefore, esterase isozymes can contribute to studies in the breeding and genetics of sesame.


INTRODUCTION
The genus Sesamum belongs to the family Pedaliaceae and consist about 20 species, most of which are indigenous to tropical Africa, with two disjunct sections found in India (Ashri, 1998;Bedigian, 2011;Bedigian, 2003).However, many wild species in Africa remain unknown, their chromosome numbers have not been determined and distribution areas have not been fully described (Agnew and Agnew, 1994;Ashri, 1998; Figure 1.Map of East Africa showing the collection sites for sesame landraces and related wild species studied.Grubben and Denton, 2004).Sesamum indicum L. is the most commonly cultivated due to edible and useful properties of its oil.Related wild species including Sesamum angustifolium L. and Sesamum angolense L. have been cultivated in Africa providing source of leafy vegetable during famine period and as traditional medicine (Ashri, 1998;Maundu et al., 1999).
Sesame is an orphan crop with little research attention in Kenya.For example, no international agricultural research institute is mandated to conserve and improve sesame (Ashri, 1998).Genetic relationship between cultivated sesame and related wild species in Kenya is not well known.In order to enhance the genetic potential of this crop, there is need to understand genetic relationship that exists between traditional landraces of sesame and related wild species in Kenya.This may enable the broadening of the genetic base of traditional landraces of sesame grown by farmers in Kenya as well as conserving the gene pool of related wild species.Somatic chromosome counts and isozyme markers have the potential to contribute to advances in breeding, genetics and conservation of biodiversity in sesame and related wild species (Diaz et al., 1999;Isshiki and Umezaki, 1997;Kobayashi, 1981;Martinello and Schifino-Wittmann, 2003;Nanthakumar et al., 2000;Parani et al., 1997).Chromosome counts and isozyme were used because they are cost-effective for scientists in developing countries like Kenya.
The aim of this study was to determine genetic relationship between traditional landraces of sesame and related wild species in order to broaden the genetic base of traditional landraces of sesame and conserve the gene pool of related wild species in Kenya.

Plant materials
Forty-six accessions of sesame representing different regions in Kenya, Tanzania and Uganda were used in this study (Figure 1 and Table 1).These materials comprised 34 landraces, 3 commercial varieties, and 9 accessions of 4 wild species.Seeds of related wild species were collected from different parts of Kenya including Sega, Kitale, Busia, Marigat, Kabarnet and Kabimoi centre (Figure 1).These sites were selected because they form natural habitat for most wild Sesamum species and some of these sites were not adequately sampled during earlier work by Agnew and Agnew  (1994) and Grubben and Denton (2004).Herbarium specimens representing wild species were prepared and used to identify species as described in taxonomic literature (Agnew and Agnew, 1994;Grubben and Denton, 2004).Photographs of wild species were also taken using a digital camera (Sony DSC-130) to aid in species identification.

Somatic chromosome counts of wild species
For somatic chromosome counts, seeds were germinated in Petri dishes with moistened filter paper and 1 to 1.5 mm long roots were fixed in 3:1 ethanol-acetic acid for 24 h, and stored in 70% ethanol below 0°C until required.For slide preparation, roots were hydrolyzed in 1 M HCl at 60°C for 10 min as described by Martinello and Schifino-Wittmann (2003) with modification in staining regime.
The roots were stained in 2% aceto-orcein and semi-permanent slides were examined by light microscopy.Only intact and wellspread cells were analyzed and each root tip was considered as one individual.Chromosome numbers were determined from cells at mitotic metaphase using a Zeiss phase contrast microscope.Photomicrographs were taken using Sony DSC-W130 under ×1000 magnification.

Enzyme extraction, electrophoresis and staining
Enzymes were extracted from young leaves of 2-week-old seedlings and separated electrophoretically on 11% horizontal starch slab gels following the procedure described by Tanksley and Orton (1983) and Wendel (1983).Vallejos (1983).

Statistical data analysis
The esterase bands were scored for their presence (1) or absence (0) in each accession.Data entry was done into a binary matrix as discrete variables.Jaccard's similarity matrix was used to compute pair-wise genetic similarity values (Digby and Kempton, 1994).A dendrogram was generated based on similarity coefficients using un-weighted pair group method with arithmetic mean (UPGMA).
The computer package NTSYS-pc, version 2.1 was used for cluster analysis (Rohlf, 2000).

Chromosome counts of Sesamum species
All the 153 cells of the 63 analyzed individuals from the 9 accessions representing 4 wild Sesamum species had 2n = 32 chromosomes (Figure 2 and Table 2).The cultivated sesame had 2n = 26 chromosomes.Thus, since the base chromosome number in the Sesamum genus is x = 8 and x = 13 (Kobayashi, 1981), these species belong to tetraploid group of Sesamum sp (2n = 4x = 32).Accessions from Marigat (104w) and Kambi ya Samaki (104w-a) had less number of individuals analyzed because of poor germination.

Isozyme variation
Out of 10 enzyme systems assayed, only esterase showed significant variations among the examined accessions.Therefore, we focused on the isozymes of esterase in the present study.Genetic variation between traditional landraces of sesame and related wild species was observed for the number of bands and banding intensity.Esterase bands were designated as C 1 , C 2 , C 3 for cathodic and A 1 , A 2 , A 3 , A 4 , A 5 , A 7 , A 8 and A 9 for anodic according to their relative migration.Three cathodic and eight anodic bands were observed and the variable bands ranged from 2 to 6 per accession (Table 3).Cathodic bands with varied relative migration were observed in wild species only, whereas anodic bands were observed for all the accessions.Four accessions namely, Morogoro, 107UG, Indian-1 and Indian-2 recorded the highest number of esterase bands, while 103w had the lowest number of bands.Bands C 1 and A 1 were specific to accession 101w (Sesamum calycinum) in both α and β-esterases (Figure 4 and Table 3).Band A 8 occurred in all the accessions but stained more intensely in the wild species than traditional landraces of sesame (Figure 3 and Table 3).Band A 9 was recorded only in traditional landraces of sesame (Figure 3 and Table 3).Accessions of traditional landraces of sesame and related wild species formed separate clusters at 0.44 similarity level (Figure 5).Genetic distances among the 4 wild species were greater than distance among landraces and cultivars.

Chromosome counts
Somatic chromosome counts of 4 wild Sesamum species namely S. calycinum, S. angolense, Sesamum latifolium and Sesamum spp.revealed a consistent chromosome number of 2n = 32, which differed from that of cultivated sesame (2n = 26), indicating genetic variation in chromosome counts.Variation in chromosome counts between cultivated sesame and related wild species has been reported in previous studies (Ashri, 1998;Grubben and Denton, 2004;Kobayashi, 1981;Maundu et al., 1999;Prabakaran, 1996).Chrosomose count result   suggests a distant genetic relationship between cultivated sesame and related wild species.

Isozyme variation
Esterase exhibited only significant genetic variation among sesame accessions in terms of number of bands and staining intensities.These findings are supported by previous studies where one or few isozyme markers contributed to variation among different sesame genotypes (Isshiki and Umezaki, 1997;Diaz et al., 1999;Parani et al., 1997).These results suggest that few gene markers are available in sesame and related wild species.A comparable pattern of isozyme variation was also reported in other plant species such as Elymus (Yan et al., 2000), Phalaris (Matus and Hucl, 1999) and Trifolium (Lange and Schifino-Wittmann, 2000) in terms of number of bands and staining intensities.Although cultivated sesame is self-pollinated, up to 60% levels of out-crossing has been reported (Yermanos, 1980).Outcrossing plants tend to present between 10 to 20% of the genetic variation between populations (Hamrick and Godt, 1989).Therefore, out-crossing among neighbouring fields may explain isozyme variation observed in this study.According to Hamrick and Godt (1997), cultivated crops have more allozyme diversity and allele frequency differences are much greater as whole than other seed plant species, although the mean genetic diversity partitioned within population of crop species is similar to that of other plant species.These results are in agreement with earlier studies which reported a high genetic diversity among accessions of cultivated sesame in East Africa based on morphological traits (Pham et al., 2010;Were et al., 2001Were et al., , 2006)).
The wild species had a lower number of isozyme bands  (Chamberlain, 1998).This could result in the loss of alleles, either temporarily within one reproductive cycle, or permanently depending on the extent of damage.Esterase bands observed in wild species stained more intensely than traditional landraces of sesame indicating high enzyme activity.This might be attributed to the effect of chromosome numbers on the plant species.The increase in chromosome number has been reported to affect gene expression and it is attributed to gene dosage effect (Olng'otie, 1991).Peroxidase activity per cell in fern plants was reported to increase in direct proportion to increase in the genome (DeMaggio and Lambrukus, 1974).High enzyme activity may also be attributed to the influence of both biotic and abiotic factors on these species.Significant levels of esterase were observed in diseased wild species of sesame in India (Lubaina and Murugan, 2013).Plants have been reported to alter their gene expression patterns in relation to environmental changes such as temperature, water availability or the presence of deleterious levels of ions (Hazen et al., 2004).
The accessions of sesame and related wild species formed separate groups indicating a distant genetic relationship.The distinctness observed in this study could be attributed to different modes of evolution coupled with sexual incompatibility barriers that exist in some Seamum species (Kobayashi, 1981).A high number of chromosome (2n = 32) observed in wild species in this study could also explain the genetic divergent between cultivated sesame and related wild species.Genetic divergence between cultivated and related wild species has been reported in a number of plant species including sesame (Lubaina and Murugan, 2013;Nanthakumar et al., 2000;Prabakaran, 1996), sunflower (Cronn et al., 1997) and Trifolium species (Lange and Schifino-Wittmann, 2000).

Conclusion
Generally, accessions of cultivated sesame were genetically diverse compared to wild species.Morogoro, 107UG, Indian-1 and Indian-2 recorded the highest number of esterase bands, while 103w had the lowest number of bands.Genetic distances among the four wild species were greater than distance among landraces and cultivars, indicating that no cross pollination with wild species occurred during sesame domestication.Few gene markers are available in sesame and related wild species, therefore, esterase isozymes can contribute to studies in the breeding and genetics of sesame.

Figure 5 .
Figure 5. Dendrogram showing relationships between cultivated sesame and related wild species.

Table 1 .
List of sesame accessions and place of collection.

Table 2 .
Somatic chromosome counts of wild Sesamum species.

Table 3 .
α-esterase bands and banding intensity in the 46 sesame accessions based on migration distance of the bands from origin.