Molecular Characterization of Solanum Species Using EST-SSRs and Analysis of Their Zinc and Iron Contents

Aims: The objectives of study were to establish genetic diversity among the populations of three Solanum sp: Solanum torvum, Solanum aethiopicum and Solanum anguivi and identify species with the highest concentration of iron and zinc which can be recommended for improved maternal and child nutrition. It also aimed to identify polymorphic markers useful in Solanum species diversity studies or screening in Ghana. Place and Duration of Study: CSIR-Crops Research Institute, Molecular Biology Lab, Kumasi. The study was carried between March 2011 and June, 2012. Original Research Article Oppong et al.; AJEA, 6(1): 30-44, 2015; Article no.AJEA.2015.062 31 Methodology: Investigations were carried out on 30 accessions of three Solanum species assembled from five geographical regions of Ghana. EST-SSR and AAS were employed for the estimation of genetic diversity and assessment of mineral concentrations respectively. Data generated were analysed using POPGENE version 1_32, Genstat software version 9 and Microsoft excel software (Windows 2007). Results: All the markers showed allelic polymorphism in all the accessions studied. Overall genetic diversity was considerably high (I =1.0032, He = 0.4942, Nei’s He = 0.4859) and fixation index statistics (Fst) shows that 10% of the variation exists among the population. Clustering of the accessions did not exactly coincide to the populations of the different Solanum sp under study. Outcrossing rate (t = 0.8154) and Gene flow (Nm = 2.2071) of populations of the accessions were extremely high. Assessment of their mineral contents suggests that S. torvum and S. aethiopicum species are rich in Fe and Zn respectively. Conclusion: EST-SSR markers and mineral analysis revealed genetic diversity among the different accessions. However the population studies suggest gradual homogeneity of these accessions due the high gene flow and outcrossing rate over time.


INTRODUCTION
Solanaceae is a plant family comprising about 2300 species, nearly one-half of which belong to the genus Solanum [1]. The genomics in the genus Solanum are evolving at a moderate pace compared to other plant species [2]. Leptostemonum, subgenus of Solanum, comprise almost one third of the genus. They are distributed worldwide and include a number of economically important species of cultivated eggplants which consist of two sub-groups namely melongena and oliganthes [1]. The former includes S. melongena L. (Brinjal egg-plant, aubergine) and S. macrocarpon (Gboma eggplant), while the latter comprise S. aethiopicum L. [3,4]. S. aethiopicum and S. macrocarpon are known to be domesticated in Africa from their wild relatives namely S. anguivi and S. dasyphyllum respectively [5]. These eggplants are considered as very important vegetables in Ghana and West Africa [6,7,8,]. Another important wild relative is the Solanum torvum (Turkey berry) which can also be found in Mexico, Peru, Venezuela, and in the West Indies including Bermuda [9]. Solanum wild species or landraces are rich source of novel genes which can be introgressed into cultivated species [10]. Interestingly, S. torvum has been identified to carry resistance genes to serious diseases of eggplant, particularly bacterial and fungal wilts, and infections caused by nematodes [1,11,12,13]. S. torvum is used as rootstocks for controlling wilt disease in eggplant [14,15].
Fruits of Solanum anguivi can be utilized either in their fresh or dried state. It is believed to have medicinal properties particularly against high blood pressure and diabetes as well as being an appetizer. Nutritional analysis of S. torvum reveals that it has special properties of stimulating the production of blood cells and could be helpful in treating anaemia due to its high iron content. It contains about 30 mg of iron, 454mg of calcium and sizeable amounts of protein with other nutrients per 100 g edible portion [9]. Nutritionally the high iron content in S. torvum can significantly reduce the incidence of iron (Fe) deficiency [16,17,18] in most parts of the world especially when Fe rich genotypes are identified. Identification of mineral rich genotypes may require mineral characterisation and their diversity.
Photometry and Atomic Absorption Spectroscopy (AAS) has over the years been the most popular techniques used for qualitative and quantitative assessment of trace elements in foods, nanomaterials, biomaterials, forensics and industrial waste [19,20]. However, in recent times, emphasis is given to inductively coupled plasma-atomic emission spectroscopy (ICP-AES) due its high reproducibility and quantitative linear range compared to conventional AAS, and reduces molecular interferences due to a higher temperature (7000-8000 K) in the excitation source (plasma) [20]. Nevertheless, ICP-AES is more expensive than conventional AAS, and in complex samples, emission patterns can be difficult to interpret [21,22]. Moreover, ASS is the method of choice for most routine trace mineral element analyses and very effective for the determination of elements such as magnesium, zinc, and copper [19].
Morphological diversity study has often been employed for genetic diversity studies in crop plants [23]. However variations in environmental conditions such as soil types and fertility levels, light, temperature and moisture regimes affect morphological characterization, particularly when experiments are repeated in time and/or space [24,25,26]. In addition, the genetic make-up of seed, field management practices has been reported to influence the morphology of a crop [27].
The weaknesses of morphological diversity or characterisation paved way for Molecular characterisation which offers the best estimate of genetic diversity since they are independent of the confounding effects of the environmental [28,29]. Among molecular markers, Simple Sequence Repeat (SSR) has proven to be effective due to its high polymorphic and codominance nature, reproducibility, affordability, low technical complexity [29] to mention but a few. It has also been widely employed for many Solanum sp diversity studies [30,31,32]. SSRs derived from ESTs or cytoplasmic DNA (cDNAs), are mostly genic SSRs and have the potential for application in gene function characterisation [33], association analysis for gene tagging [34,35,36], and quantitative trait loci (QTL) analysis [37,38,39]. EST-SSR designed for Solanum sp is currently employed for diversity study [30,40].
In Ghana, despite the role of eggplants on the livelihood of people [7], information on their mineral content and diversity has not been well documented especially, the S. anguivi, S. aethiopicum and S. torvum accessions. This study was therefore designed to establish genetic diversity among three populations of Solanum species: Solanum torvum, Solanum aethiopicum and Solanum anguivi employing EST-SSR primers. It also seeks to assess their mineral contents using the Atomic Absorption Spectrophotometry (AAS) and identify accessions possessing high amount of iron and zinc for enhanced child and maternal nutrition in Ghana and beyond [16,17,18]. The study may assist to identify polymorphic primers useful in Solanum species diversity studies or screening in Ghana.

DNA Quantification and Gel Electrophoresis
DNA quality was checked on 0.8% agarose in 1x TAE (Tris-acetic EDTA) buffer by gel electrophoresis. Electrophoresis of the DNA was carried at 100V for 40mins and then visualized with a UV transilluminator. The concentration of the DNA was projected by the intensity and comparison to 1kb lambda DNA mass ladder (1kb, Invitrogen). The quantification of the DNA was evaluated by reading absorbance at 260nm and 280nm with the spectrophotometer. The DNA was then diluted to 10 ng/µl for PCR

PCR Amplification
PCR amplification of DNA was carried out using a 96 well plate AB Applied Biosystem Thermocycler. The PCR conditions were optimized for cycling number, concentrations of the primer, MgCl 2 and DNA template. The reaction mixture (10 ul) contained 6.075 µl of Nuclease free sterile water (DNA grade water),1 µl of 10x PCR Buffer,0.9 µl of MgCl 2 (25 mM), 0.4 µl dNTPs (10 mM), primer 0.25 µl (50 µg/ml) of each forward and reverse, 0.125 µl of Supertherm Taq Polymerase (1unit) and 1 µl of 10 ng DNA template. The PCR cycling conditions followed an initial denaturing step of 94°C for 5mins, a touchdown PCR protocol of 11 cycles of 94°C for 30 secs, 60°C for 30 secs decreasing by 0.5 per cycle and 72°C for 60 secs followed by 30 cycles of 94°C for 30 secs, 55°C for 30 secs and a final elongation step of 72°C for 60 secs. In each PCR, a negative control was included to detect contaminations. Twelve set of EST-SSR primers ( Table 2) were used in the experiment. PCR products were electropholysed on 6% polyacrylamide gel (Water, 10x TBE, 4% acrylamide (19:1), 10% APS and TEMED). A 100bp ladder (Gene Ruler TM , Fermentas) was used as a size marker. Amplified bands were visualized and scored.

Determination of content of mineral elements using atomic absorption spectrophotometry (A.A.S.).
Sample dried seeds of each accession were dehulled (manually) and ground into fine powder. Five hundred milligram (0.5 g) of each sample (accession) was weighed into a Teflon beaker. Amounts of 5ml of HNO 3 (70% HNO 3 ) and 1ml of H 2 O 2 (30% H 2 O 2 ) were added. The Teflon beakers were covered and placed in a rotor for digestion. Each sample was digested in three replicates (Zuink and Planck, 1990). After the digestion, the inside walls of the Teflon beakers were washed with distilled water and made up to a final volume of 20 ml. The samples were then transferred from the Teflon beakers into test tubes. The digested samples were assayed for the presence of iron (Fe) and zinc (Zn) using Atomic absorption spectrophotometry in an acetylene-air flame employing the manufacturers recommended working conditions. Reference standards for the elements of interest, blanks and repeats of the samples were digested the same way as the actual samples. These served as internal positive controls.

Calculation of Concentrations of Specified Mineral Elements
For each of the two elements; Iron (Fe) and zinc (Zn), concentration in the samples was calculated using the formula:

Statistical Analysis
All the calculations were performed using POPGENE 32 version 1.32 [42,44]. A hierarchical analysis of molecular variance (AMOVA) of the Solanum species populations was calculated to partition genetic diversity within and among populations. For AMOVA, the genotype banding patterns were converted into a '1' (present) and '0' (absence) matrix and subjected to analysis, based on Jaccard coefficient using single linked similarity matrix method. Hierarchal AMOVA was analyzed using Genstat software version 9. Correlation between Zinc and iron were generated to determine the degree of association between Zn and Fe of the accessions using Microsoft Excel software (Windows 2007).

Diversity studies
Sixty four alleles were generated for all loci with a mean of 5.333 per locus (Table 2). It was observed that all the EST-SSR markers showed allelic polymorphism (Fig. 1

Cluster Analysis
UPGMA cluster analysis based on the 12 EST-SSR loci employing Jaccard Coefficient single link similarity matrix method (Fig.  3) demonstrated genetic differentiation by revealing six distinct clusters from a genetic similarity distance of 37.3% up to 85%. However at a genetic distance of 37.3%, all accessions of the various populations could be grouped into two main clusters, i.e. Ntoturoso S. aethiopicum1 and the other 29 accessions which further separated into five distinct sub clusters. The accessions with the closest resemblance were revealed at the sixth cluster at a genetic similarity of 85% (Hwidiem S. torvum, A/A Wenkyi S. torvum, A/Dominase S. anguivi, A/ Odumasi S. torvum and ntoturoso S. torvum). The widest separation occurred between Hwidiem S. torvum and Ntoturoso S. aethiopicum 1. The clustering did not exactly coincide with geographical origin as well as population or species groupings of the accessions. This shows a clear genetic variation and differentiation pattern among the individuals of the various species.

Variation Based on Minerals
The result on mineral analysis for the concentrations of Fe and Zn showed that no accession recorded the highest concentrations of both minerals (Table 5). However, one accession (Assin Odumase S. anguivi) recorded the least concentration for both Fe (7.26 mg/Kg) and Zn (9.16 mg/Kg). Averagely the S. torvum and S. aethiopicum population had the highest concentration of Fe and Zn respectively. The highest concentration of Fe was recorded by Assin Odumase S. torvum (261.48 mg/kg) and the highest zinc concentration was recorded by Sefwi Awaso S. aethiopicum (29.16 mg/kg). Statistically insignificant degree of association was observed between Fe and Zn for each species (Table 5).

Diversity Studies
The 100% allelic polymorphism among the individuals of the species generated by the use of the EEMS primers indicates that all the loci are very informative especially EEMS 37, EEMS 20 and EEMS 48 which produced ten, nine and eight alleles respectively with the former producing the highest diversity (I = 1.978). The informative nature of the EST-SSR markers is also consistent with similar finding by Aniko et.al. [26] who employed the same primers utilised in this study for genetic mapping and phylogenetic studies of eggplant with EEMS 48 producing the highest number of alleles. This suggests that in selecting primers for biodiversity evaluation for eggplant or species closely related, the three primers mentioned above can be given priority. The present results based on the polymorphic pattern of 12 selected EST-SSR loci confirmed that Solanum sp or populations under studied possessed relatively high genetic diversity (He = 0.494, Nei's He = 0.486, I = 1.003). These agrees with results by other workers [26,27,35,46] who employed either SSR, AFLP and/or, RAPD markers as well as morphological descriptors for characterization of eggplant. Except a few loci (EEMS 6, EEMS 16 and EEMS 46), the genetic variation (I) observed among the individual accessions of the various species (Table 1) where either comparably close or higher than genetic variation (I) obtained for the three populations. According to Muller et al (2001) [47], this can be attributed to a high genetic overlap as a result of probable common ancestry. Furthermore, the low values of F IS (0.4188) and F IT (0.4779) as well as high values of t (0.8154) and Nm (2.2071) from the EST-SSR analysis suggest that the Solanum sp under study were more cross pollinated with wide pollen-mediated gene flow although they can undergo self-pollination due to their hermaphroditic nature.    The population dendogram estimated from Nei's unbiased measures of genetic identity and genetic distance also confirm the genetic variation among the populations with the population of S. torvum and S. aethiopicum being widely separated from each other. This is more evident in Hwidiem S. torvum and Ntoturoso S. aethiopicum 1 which were two accessions with widest separation (Fig 3). The close relatedness of S. aethiopicum and S. anguivi from the result clearly demonstrate that the two populations have a common ancestry as S. anguivi is the wild type of egg plant from which S. aethiopicum was domesticated [5]. This is consistent with report by Lester [5] that S. aethiopicum and S. macrocarpon were domesticated in Africa from their wild relatives S. anguivi and S. dasyphyllum respectively. Furthermore, UPGMA dendrogram by Sakata and Lester [48] also indicated S. anguivi and S. aethiopicum as duplicates proving their close relatedness. This further explains the reason for wide diversity between S. aethiopicum and S. torvum. In principle, the wide variation between S. torvum and the two other populations offer a broad genetic base with the potential for wider adaptation in a range of agroecosystems. This also indicates the genetic potential of the populations for breeding purposes [42].

Variation via Cluster Analysis
The dendogram reveals that cluster five and six constitutes mainly S. torvum accessions with the exception of A/ Dominase S. anguivi and Hwidiem S. anguivi in cluster five and six respectively. Cluster five and six were captured at a genetic distance of 0.85 (85%) and 0.77 (77%) respectively. The inclusion of the S. anguivi accessions among the S. torvum may be attributed to the high outcrossing rate and gene flow (t = 81.54%; Nm = 2.2071). It can be inferred from the results of the study that about 85% of the S. torvum genetic characteristics has been introgressed into such S. anguivi accessions making them lose their unique genetic characteristics (Fig. 3). This also implies that the S. torvum accessions in cluster five and six should be assembled and conserved for breeding purpose to avoid contamination. Nevertheless, the remaining clusters constitute a mixture of the three populations which grouped not based on geographical origin or species as indicated by the low values of F IS and F IT resulting in probable heterozygosity of those accessions [42].

Variation among Accessions via Mineral Content
The distinct concentration of the Fe and Zn analyzed among the accessions also display variation among the accessions. Diversity in Fe and Zn concentrations in the accessions of the present study agrees with similar findings by several early workers [49,50]. This suggests that micronutrient enrichment traits also exist in the egg plants as evident in other crops such as rice and common beans (Phaseolus vulgaris L.) [51,52] Concentrations obtained for iron is higher than recorded by Chinese red long-grain varieties (64 mg/kg) [53,16]  The study did not reveal any accession among the three egg plant species possessing high concentration of both iron and zinc neither any accessions with high correlation for Fe and Zn.
Therefore the outstanding concentrations recorded by Assin Odumase S. torvum and Sefwi Awaso S. aethiopicum for iron and zinc respectively suggest their usefulness in a breeding program whose objective is to improve iron and zinc concentration. Thus the two species can be utilized in a hybridisation program to develop an eggplant rich in iron and zinc.

CONCLUSION
In conclusion, the EST-SSR markers and mineral analysis revealed genetic diversity among the individual accessions of the populations studied without duplications. However the population studies suggest the gradual homogeneity of these accessions due the high gene flow and outcrossing rate over time. It is therefore recommended that the accessions should be collected and conserved to avoid further contamination especially with the almost distinct S. torvum species. Molecular diversity of the species under study or closely related ones can easily be carried out with EST-SSR primer EEMS 37. Assin Odumase S. torvum and Sefwi Awaso S. aethiopicum can be recommended as a good source for improved child and maternal health in Ghana because of their high content of iron and zinc respectively.