Value of Seed Protein Profile in the Taxonomy of cultivars of Capsicum in Nigeria

Over a decade, the taxonomy of the genus Capsicum in Nigeria  has remained largely unrevised, unclassified and unidentified. As such, there is a dearth of information on the proper identification of Capsicum spp and relatives found in the country. The aim of this study was to re-examine the taxonomic status of the Capsicum in Nigeria in order to establish genetic diversity between them for proper identification and classification. Sodium dodecyl  polyacrylamide gel electrophoresis of total seed protein was performed on five varieties of Nigerian Capsicum spp., following standard procedures. Six protein bands were observed across the five cultivars of Capsicum, of which 12-14 Kda was the only polymorphic band. Only C. fructescens var. ijosi and C. fructescens var. sombo were unique for manifesting 20-24 and 15-16 Kda bands respectively. Dendrogram of analysis obtained resolved the taxa into two distinct groups. In the first group were cultivars of C. fructescens var. ijosi and sombo while in the second group were C. chinense, which was distinctly separated from C. fructescens var. bawa and C. annum. Artificial dichotomous key was constructed for the identification of members of the genus Capsicum available in Nigeria based on the protein profiles of their seeds. 
Keywords: Capsicum, seed protein, electrophoresis, identification, diversity.


Introduction
In West Africa, peppers are widely grown and are used in a number of ways. They occupy third position in Nigeria among the cultivated vegetables being utilized in the dry state as spice due to their capsaicin content (an alkaloid which is a digestive stimulant) and as vegetable, when supplied for their vitamin content and aroma (USDA, 2015). The crop is utilized both as condiment and food. The thick sweet fleshy or non-pungent varieties are used in salads or stuffed with meat and cooked (Arnarson, 2015). The chemical in chilli peppers (i.e. Capsicum) responsible for the burning sensation is capsaicin which affects only mammals, but not birds. Capsaicin extract is used to make pepper spray, a useful deterrent against aggressive mammals (Grubben and El Tahir, 2004). Pepper fruit accounts for a large portion of vitamins A and C in many Nigerian diets; the most common species of pepper in Nigeria are Capsicum annum L., C. chinense Adepoju et al./ Nig. J. Biotech. Vol. 36 Num. 2 : 1 -8 (December 2019) Jaqc. and Capsicum frutescens L. (DeWitt and Bosland, 2009).
The genus Capsicum in Nigeria has not been thoroughly revised, identified and classified. There is therefore a dearth of information on the exact number of Capsicum spp and varieties found in the country. In addition, some reported works on Nigerian Capsicum misrepresented some Capsicum species due to lack of proper identification. For instance, Aziagba et. al., (2014), erroneously assigned the local name 'shombo' (Yoruba name for a cultivar of C. fructescens) to C. annuum and 'atarugu' (Hausa name for C. chinense) to C. annuum. When the classification of taxa is confused, so is the nomenclature and literally, any information about such taxa is not specific and therefore, less useful. Capsicum has long been regarded as a taxonomically difficult genus by many workers (Pickersgill et. al., 1979;Eshbaugh, 1970Eshbaugh, , 1975Eshbaugh, , 1980Heiser and Pickersgill, 1975;Edeoga et. al., 2010;Zhigila et. al., 2014). In fact, there is no agreement yet among workers with regard to the number of species of Capsicum present in Nigeria.
Presently, there is no satisfactory revision of the taxonomical status of the Nigerian genera of Capsicum. . Moreso, some species of the genus being considered in this study (i.e. Capsicum) are difficult to distinguish because members of the genus have been reported to possess morphological similarities and some are highly phenotypically plastic (Moscone et. al., 2007;Walsh and Hoot, 2001). So far, only one report (Olatunji and Morakinyo, 2015) could be traced on taxonomic markers based on biochemical contents or SDS-PAGE analysis with respect Capsicum species and varieties in Nigeria. Olatunji and Morakinyo (2015) claimed to have worked on C. fructescens and C. annuum varieties only, leaving out C. chinense, which is a commonly used variety in Nigeria. The results of their study also indicated some levels of confusion, as there were reports of consistencies between the protein profile from the leaves when compared with those of the seeds, in their study. These problems clearly justify the necessity for a proper identification of all members of the Nigerian Capsicum taxon by their seed protein composition, especially because a preliminary survey of the wild and cultivated Nigerian species of the Capsicum genus revealed several cases of confusion regarding uncertainty in names and genetic distances.
The aim of this study was to evaluate the seed proteins of the plant species using Sodium Dodecyl Polyacrylamide Gel Electrophoresis (SDS PAGE) with a view to establish genetic diversity between species and also document those protein markers that could be utilized for diagnosing the varieties and species of Capsicum in Nigeria.

Materials and Methods
Sample Collection and regeneration: Protein extraction About 200mg seeds from each genotype was homogenized with mortar and pestle using 0.01M Tris-Hcl buffer (pH 7.5). The resulting homogenates were centrifuged at 15000rpm for 10 minutes, the supernatants were filtered with 541 Whatmann filter paper. The residues were boiled at 90 O C for five minutes with 1:1 ratio of 1.0M Tris (pH 6.8), 10% SDS; 2% βmercaptoethanol, 10% glycerol and 0.002% bromophenol blue (following the method of Kumar and Tata (2010) with gel composition as indicated in Table 1. Electrophoresis SDS-PAGE of total seed protein was carried out in vertical slab gel in discontinuous buffer system following the method that was modified by Essiet and Illoh (2008) as follows: On cooling to room temperature, three drops of 10% Sodium dodecyl sulfate (SDS), 1% of 2mercaptoethanol and sucrose crystals was added to the sample in order to weigh down the protein molecules. Then, one drop of 0.05% bromophenol which served as a tracer dye was added. Four drops of the resultant mixture obtained from the product was directly added to the gels. The tubes were then placed in column acrylamide gel apparatus with tri-glycine buffer in both the upper and the lower vessels. A current of 1.5mA per gel was applied. The current was thereafter increased to 3mA per gel until the dye front was 1 cm from the bottom of the gel.

Staining and destaining
After electrophoresis, the gels were stained with Coomassie Brilliant Blue (R250) for 30-40 minutes with continuous shaking, then shifted to another container with destaining solution of methanol and acetic acid for 45 minutes. The gels were further de-stained until the back ground was clear enough for bands scoring. Marker proteins (RPMW): Bovine Serum Albumin (66 kilodaltons), Egg Ovalbumin (45 kilodaltons), Pepsin Porcine Stomach Mucosa (34.7 kilodaltons). Bovine Trypsinogen (24 kilodaltons) and B-Lactoglobulin (18.4 kilodaltons) were used as references. Protein marker used was in form of "MW-SDS-70 Kit" from Sigma Chemical Company, USA. Molecular weights of protein bands were estimated by their relative mobilities. In order to eliminate differences in electrophoretic conditions as a cause of variation in the protein profiles, each genotype protein sample was separated from three independent electrophoretic runs and two separate extractions (Kumar and Tata, 2010). In analyzing the data obtained, presence of a band was scored "1" while absence of the band was scored "0" to generate binary matrix which was used to perform statistical analysis.

Cluster Analyses
The scores of the protein fragment in relative to the standard marker were used as characters to perform a cluster analysis on the species; each of which was taken as operational taxonomic units (OTU). Dendrograms were obtained by adopting a hierarchical cluster analysis using Ward's method applying squared Euclidean Distance (as the distance or similarity measure), both of which could be combined using PAST (Paleontological Statistics Software Package) by Hammer et. al. (2001).

Construction of Dendrogram and Artificial key
An artificial dichotomous key was constructed for the purpose of diagnosing the species in each genus, using the characters obtained. While the qualitative characters were directly used, as observed, the means of quantitative characters were first subjected to statistical significance across the species in each genus to determine which ones were truly diagnostic. The statistical tool used for dendrogram construction was SPSS version 21.

Results
The results of SDS PAGE analysis of the seed proteins in the plants studied are shown in Plate 1 and Table 2. A total of six protein bands was observed across the five cultivars of Capsicum i.e. B1, B2, B3, B4, B5 and B6. The molecular weight of the protein were 35kDa, 28-32 kDa, 25-27 kDa, 20-24 kda, 15-16 kda and 12-14 kda respectively (Table 2).

Discussion
In consonance with the results obtained from morphological, leaf epidermal and wood anatomical evaluation of the cultivars of Capsicum (Adepoju, 2018), the dendrogram obtained from seed protein data (Fig 1) resolved the taxa into three distinct groups. In the first group were two cultivars of C. fructescens clustered (i.e. varieties ijosi and sombo); in the second, C. fructescens var bawa and C. annuum clustered; while C. chinense stood as a distinct cluster on its own.

Biosystematic Implications of the seed protein molecular weights observed in Capsicum
It is clear that the dendrogram in Fig 1, and those obtained from morphological and leaf epidermal data of these Capsicum cultivars by Adepoju (2018) are similar in all respects, indicating that the classificatory value of morphological, leaf epidermal and seed protein characters in this genus are the same. Also, if one places the dendrogram obtained by Adepoju (2018) for wood anatomical characters of the cultivars side by side with Fig 1, one finds out some similarity between the two, particularly as regards the close clustering of C. fructescens var. bawa and C. annuum.
Again, the results as depicted in Figure  1 closely align with capsaicin content profile of the fruits of these plants (Nwokem et. al., 2010 andZeid et. al., 2011). In the first place, the two cultivars with low fruit capsaicin contents clustered together (i.e. C. fructescens var. bawa and C. annuum) while the two cultivars of C. fructescens with high content of this chemical (i.e. var. ijosi and var. sombo) clustered as a group. Lastly, C. chinense which is acknowledged to possess an intermediate value of capsaicin content between the two extremes stood alone as a cluster between the earlier two groups (Fig 1).

LC LC2
LC1 McLeod et. al. (1982) reported a close distance of clustering between C. chinense and the two varieties of C. fructescens and the present study agreed with the authors report. Also, Bhat and Kudesia (2011) studied the protein profile of 5 species of the family Solanaceae (i.e. Solanum melongena, S. xanthocarpum, Datura alba, Lycopersicon esculentum and Capsicum annum) using SDSpoly acrylamide gel electrophoresis. Their results revealed that the genus Lycopersicon was very close to the genus Solanum and that the species Datura alba and Solanum melongena were closer at molecular level compared to other species. Furthermore, similarity index was higher for Capsicum annum and Solanum xanthocarpum (22.22%) which are cultivated and wild types respectively, as compared to two exclusively wild species of Datura alba and Solanum xanthocarpum (in which their similarity index was only 11.11%). Yousaf et. al., (2006)    In conclusion, out of the six seed protein bands observed in Capsicum, the band with the molecular weight of 12-14kda was polymorphic in Capsicum. Also, the seed protein profile has similar classificatory value in line with only those clusters based on fruit capsaicin content.