MOLECULAR AND MORPHOLOGICAL DIVERSITY STUDIES OF FIVE (Schott andEndl.) in Ibadan, Nigeria.

ABSTRACT Intra- and inter-species genetic diversity study was carried out comparing the molecular and morphological characters of five Cola species namely , , Cola millenii, Cola lepidota and Cola giganteacollected from five locations in Ibadan, Nigeria. Sixteen Cola accessions were analysed based on 7 quantitative characters, 12 qualitative characters and 8RAPD primers. From the quantitative characters studied, high significant Comment [TCL1]: In full at first mention difference across the location and species was recorded as leaf length had the strongest variability while the species obtained from Botany Nursery showed strongest locational effect on the vegetative leaf characters. All quantitative characters analysed were good determinants for delimiting Cola species. Cola lepidota recorded the highest variability compared to the other Cola species. Qualitative characters studied revealed thatleaf surface, seed colour, floral size, flower colour and height are strong characters for delimiting Cola species. Formatted: Highlight Molecular analysis showed that 309 fragments were recorded, of which 28.5% were Formatted: Highlight polymorphic while 8.4% were monomorphic. Genetic similarities ranged from 0.44 to 0.93 for the 16 Cola accessions with a mean value of 0.67. Deductions from the combined effect of molecular and morphological characters showed high variability for the characters analysed and a likely monophyletic relationship for the 16 Cola accessions.Conclusion

Key words: Accessions, Cola species, Genetic diversity, , medicinal plant. ,molecular characterization

Introduction

The kola nut is the fruit of the medicinal called Colaspecies, which is indigenous to West Africa. The kola plant belongs to the Cola genus of family that has over one hundred and forty species (Onomoet al.,2006). Onomoet al.,(2006) pointed out that Cola Comment [TCL2]: Replace with recent publications, published 3 years or earlier acuminata (Beauvoir) Schott and Endlicher (known as Obi abata in Yoruba) and Cola nitida(Vent.) Schott and Endl. (known as Obi gbanja in Yoruba) are rich in caffeine (2-3%). Theobromin and kolanin are the most important metabolitesin Cola species based on their social, pharmacological and economic importance. Cola gigantea (A. Chev.) traditionally called “ oporoporo” is an African medicinal plant, which thewhose powdered back is used in the cure oftocure venereal diseases, headache, intestinal and lumber pain (Burkill,2000). On the other Comment [TCL3]: This reference is now 20 years old. Get more recent publication

hand, Cola lepidota K. Schum.is a tree of about 18m high that has soft and fibrous bark.Cola millenii and Cola gigantea are widely distributed in wet and dry forest environments (Kuoame and Secande 2006).On a general note, the common morphological traits of the leaves of Cola species are simple, entire and narrowed or rounded towards the base. The arrangement of the leaves on the stem is alternate in some species and verticillatein others, in whorls of 3 threeor 4four. However,the variability within the Cola genus cuts across morphological, phytochemical and anatomical grounds.Molecular systematics is still regarded as the most accurate form of clearing doubts relating to diversity among species. DNA markers are currently used as molecular tool to show any diversity within the evolutionary line and the significant closeness between them. Morphologically, both quantitative and qualitative characters can also be used to characterize and classify within species and between species.

Morphological characters has havebeen widely used in the evaluation of various crops (Kaemeret al, 1995). Exploitation of the qualitative and quantitative traits can be a good taxonomic tool for knowing the genetic variability present within a population of related species. Results obtained from morphological characterization will strongly facilitates genetic diversity studies and future breeding programs based on geographical adaptability. Several morphological descriptors from the leaves, fruits, branches, plant heights and other reproductive parts of the are essential tools for characterization.

On the other hand, Random Amplified Polymorphic DNA(RAPD) markers are Decamer (10 Formatted: Highlight nucleotide length) DNA fragments from PCR amplification of random segments of genomic DNA with single primer of arbitrary nucleotide sequence and which are able to differentiate between genetically distinct individuals, although not necessarily in a reproducible way. It is used to analyse the genetic diversity of an individual by using random primers. RAPD requires only one primer for amplification. Unlike traditional PCR analysis, RAPD does not require any Comment [TCL4]: Write in full at first mention specific knowledge of the DNA sequence of the target organism.

Due to the importance of kola nut in beverages and medicinal use, it became necessary to characterize the plant and possibly establish the relationship between different species. We carried out this comparative study on five Cola species, seven morpho-quantitative characters and twelve qualitative characters of sixteen Cola accessionscollected from five locations in Ibadanand were analysed for their intra- and inter-species relationship. Also, the DNAs of the 16

Cola accessions collected at five locationswere analysed using RAPD forward primers. Thismolecular analysis is expected to reveal the gene diversity, genetic distance, and allele polymorphism among the five different Cola species. Comment [TCL5]: Insert citations

Materials and Methods

Germplasm Location

Cola species were collected from five locations in Ibadan, Nigeria namely; Cocoa Research

Institute of Nigeria (CRIN), National Centre for Genetic Resource and Biotechnology

(NACGRAB), Dabiri village in AkinyeleLGA, Botanical Garden of University of Ibadan (UI) Comment [TCL6]: In full and Botany Department nursery of University of Ibadan. Each location has a unique eco- diversity characterized by dense rainforest environment, tall , rich humus to poorly loamy soil, and tropical agro ecological environment.

Sample Collection

A total of 16 samplesand 10 replicates were collected at random representing five species of Comment [TCL7]: Why 16 samples

Cola species with an uneven spread per location.Various Cola species were collected at different locations in Ibadan. Mature leaves of Cola acuminata, Cola nitida, Cola gigantea, Cola lepidota and Cola millenii from tree stands that are over 10 years of age were collected and properly identified by the Herbarium Unit, Botany Department, University of Ibadan.

Morphological data analysis

A total of 19 macro-morphological characters of both qualitative and quantitative descriptors were collected toacross the five locations. With the aid of transparent rule, masking tape, a rope Comment [TCL8]: Which criteria was followed to select the 5 locations and visual aid, quantitative measurements were taken of leaf apex, leaf margin, leaf petiole

length, leaf base, collar length, petiole diameter, leaf shape, leaf length, plant height, leaf width, leaf mid veins, and leaf surface. The quantitative characters were obtained by measurement of the leaves in centimetres and counting characters observed. Qualitative characters of the samples collected were scored based on visual evaluation i.e., by scoring the variations in leaf apex, leaf Comment [TCL9]: Avoid using i.e. You can replace it with “like” shape, leaf margin , leaf base and leaf surface, fruit size, flower shape, and seed colour. The quantitative characters were obtained by measurement of the leaves in centimetres and counting characters observed.

Multivariate numerical techniques for the 19-character traits were employed to evaluate the taxonomic relationship from the morphological and molecular data obtained. The data obtained were subjected to the statistical analysis usingSAS version 9.0 (SAS Inc., Cary, NC, USA). Comment [TCL10]: In full

Every character under this statistical analysis werewas given equal weight. Between two Cola species, the overall similarities will bewas a function of their individual similarities in each of the

12 descriptors they were compared with.

The degree of resemblance among species werewasmeasured by the correlation coefficient of the characters across the 16 samplesOperational Taxonomic Units (OTUs). Cluster analysis of the Comment [TCL11]: This is not clear, rephrase or punctuate OTUs based of the percentage similarity was carried out to enable us construct a precise dendrogram showing the degree of resemblance at various levels and the clustering of OTUs according to their similarities.

Comparative morphological studies were carried out through the characterization of the qualitative and quantitative characters of the16 Colastands across five germplasm location in

Ibadan.

Molecular Analysis

Young healthy fresh leaves obtained from young branches of each tree stand were collected from the five species across the five locations. A total 16 leaf sampleswere collected into a temporary preservation box, which were immediately hermetically sealed and packed in ice for the transport to the laboratory. The samples were subsequently stored in the freezer at -20Cuntil required for Comment [TCL12]: Do you mean 20oC? analysis. The molecular analysis was carried out in the Bioscience Lab, IITA, Ibadan, Nigeria. Comment [TCL13]: In full

DNA extraction from Cola samples was done using DNA Kit extraction protocol.

RAPD Analysis All RAPD primers were standardized to a 25 µg/ml stock solution. DNA amplification was performed. The DNA was subjected to the following cocktail mix and condition for the PCR.

10× PCR buffer (1µl), 50mM Mgcl2 (0.4 µl),5pMol forward primer (0.5µl), 5pMol reverse primer (0.5µl), DMSO (0.8µl), 2.5Mm DNTPs (0.8µl), Taq 5u/ul (0.1µl), 100ng/µl DNA (3.0µl) and water (2.9µl). Each reaction tube was then overlaid with mineral oil, in order to seal the tube and prevent evaporation, and amplified on a DNA master cycler (Bio Oven III). The reaction mixture was subjected to a 40 amplification cycles.

Sample Preparation for Electrophoresis

The amplification products were subjected to electrophoresis by mixing 4µl of the DNA fragments and 2 µl of loading dye together and loading them in the well of a 1.5% Agarose gel.

The sample electrophoresed for 1 hour 30minutes at a constant voltage of 80 volt, 300mA 60

Watt. The plates were dismantled and the gels were stained by placing it in Ethiduim bromide

(5mg/ml) for 5 minutes. The gels were also viewed under the illuminator, photographed and the pictures saved.

Scoring of bands

The presence of a DNA band was scored as Present (1) or Absent (0), and each band was regarded as a locus. The result obtained was used to create the binary data set and entered into a binary data matrix as discrete variables to generate a data matrix. The binary data obtained from scoring the RAPD bands were used to evaluate the relationship of the five Cola species across five locations. A similarity matrix was generated from the binary data using Jaccard’s similarity coefficient (Jaccard, 1908). Comment [TCL14]: Why such an old citation?

Results Comment [TCL15]: Categorize into sections or sub‐sections

Among all the vegetative characters used for this study, the leaf length stands out as the character with the highest variability both for the location and species. The high percentage of variance Comment [TCL16]: Where is the data showing this variability. If it is Table 1.1, and the leaf length is the strongest vegetative character for this study to be used for delimiting indicate it here in parenthesis Comment [TCL17]: Where can I find this species. percentage?

From the cluster analysis the dendrogram for quantitative characters showed an interesting relationship pattern that is consistent with earlier taxonomic claims. Similar Cola species irrespective of the location shows likely intra-species relationship. The dendrogram shows three Comment [TCL18]: Put a reference e.g. (plate1.1) to direct the reader where clusters in which one of them is an outlier (non uniformity). Group 1 oneis made up of 8 dendrogram you’re talking about can be found eightuniform OTUs with three subgroups in a seemingly monophyletic relationship. Cola acuminata from three locations namely Botanical Garden, NACGRAB and CRIN as well as

Cola nitida CRIN make up Subgroup 1. This shows an intra-specific relationship in Cola acuminata for the 12 quantitative characters used for study. Within this subgroup is an inter species relationship with Cola nitida CRIN. Two uniform OTUs of Cola nitida from University of Ibadan(UI)Botany DeptNursery and Akinyele LGA in an Intra-species relationship, makes up Comment [TCL19]: Write in full the Subgroup 2. In cluster 2, Cola giganteaAkinyele LGA and Cola lepidota UI Botany

DeptNursery are non-uniform (outlier) while Cola millenii from all the locations shows an Intra specific relationship for sevenmorpho-quantitative traits. Colagigantea NACGRAB shows an inter species relationship with Cola millenii. Cluster 3 is an outgroupout-group from the other

Groups. This shows that Cola gigantea UI Botany Dept nursery may be in a separate relationship with the other Taxa.

From the cluster analysis for qualitative character, 3 Groups were observed with Group 3 showing a great group made up of thirteen individual Cola OTUs all representing the five Cola species. This shows the possibility that these Cola species originating from the same genus. In

Group 2, we see an Intra species relationship of Cola gigantea from two locations namely

Akinyele LGA and NACGRAB and Group 1 , Cola millenii NACGRAB acting as non-uniform outlier.

In this study, the deductions from the molecular analysis shows that the purity of DNA of optical density of 260/280 ratio ranges from 0.96 to 1.78 for the 16 Cola OTUs/samples.

The selected primers generated distinctive products in the range of 78–1651bp. The maximum and minimum number of bands produced by the primers are OPT-07 (52), and OPH-02(18), respectively. A total number of 309 amplified fragments was scored across sixteen operational taxonomic units of Cola genus for the selected primers, and was used to estimate genetic relationships among themselves. Out of 309 fragments obtained, 88 fragments (28.5%) were polymorphic while 26 fragments (8.4%) were monomorphic.

Deductions from the amplified products of 16 OTUs in pair-wise genetic similarities ranged from 0.44 to 0.93 in all OTUs with a mean value of 0.67. The matrix values indicated that these taxa were distantly related to each other. Based on the dendrogram, a great group was observed.

Subgroup 1A containCola acuminata (CRIN), Cola millenii (NAGRAB) and Cola millenii with similarity of 74% indicating Intra species relationship. In Sub group 1B, Cola millenii (Botanical

Garden), Cola gigantea (Botanical Garden), Cola gigantea (Akinyele LGA), Cola nitida (CRIN) and Cola nitida (NAGRAB) has a similarity coefficient of 79.1%indicating Intra species relationship. In subgroup 1C, similarity is shown by Cola nitida (Botanical Garden), Cola millenii (Akinyele) ,Cola nitida (NACGRAB) that is 73%indicating Intra species relationship.

Meanwhile the similarity between Group 1 and Group 2 is 69%. In lower Group 2, Cola nitida

(Akinyele LGA) is out group with similarity coefficient of 63.8%. Group 3 is made up of Cola acuminata (Botanical Garden), Cola acuminata (Akinyele), and Cola nitida (UI Botany

Deptnursery) with similarity of 70.4% while the last Group 4 is Cola lepidotawith genetic similarity of 55%.

Discussion

According to Takhtajan (1973), all kinds of data including chemical data, micro- and macro- morphological data, molecular data, as long as it is measurable and can bring distinction in organisms, can be used in classification. Comment [TCL20]: Start with the major result, explain the trend and then cite other authors. You can’t start with a citation in this From the results obtained for the quantitative analysis, a strong Inter species and Intra species section variability were observed for the five species of Cola. Deductions from this study showed that these quantitative descriptors are good determinants for characterizing Cola species.

The performance of each species showed that the different Cola species are similar for one or more characters and are related to each other and could therefore be grouped together. This confirms the work of Whittal (2004) who suggested that some species of the same genus can be Comment [TCL21]: Good. Write like this..

morphologically very similar and may be grouped into the same species despite the fact that they represent separate taxonomic entities.

Deductions from the results showed that each location contributed to variation in the vegetative morphological characters. This was attributed to the environmental factors that influenced the nature of the leaf vegetative characters. On the other hand, the species also revealed high significance for the quantitative descriptors chosen. This species effect indicates the inherent hereditary contributions to the phenotypic variations observed in the vegetative characters. The deduction from this study is that the uniqueness from individual Cola species is a function of the location and species effect. This environmental factor could arise from the soil factors and climatic factors exerting strong effects on the phenotypic appearance of each Operational

Taxonomic Units (OTUs) used for this study. The environment is known to potentially influence Comment [TCL22]: You don’t have to define the abbreviation several times. OTU the morphology and expression of compounds in plants (Folkerset al., 2008; Shen et al., 2008; was earlier defined

Braga et al., 2006; Cybulskillet al., 2000).

These results deduced from quantitative analysis is arein line with Pierre et al (2006) who Comment [TCL23]: Which results? It is better to discuss a particular result, not the entire work ..Mention about the revealed a high coefficient of variation in Cola acuminata. Cronquist (1981) also opined that quantitative parameters in particular that are in line with Pierre et al (2006). there is significant morphological variation in the sterculiaceae family both within and across Comment [TCL24]: Grammar species. This workis in line with Oyefunke’s work (2016) on comparative phytochemical and Comment [TCL25]: It is better to discuss a particular result, not the entire work organoleptic studies on five Cola species where she indicated that similarities in chemical constituents showed the reasons for the five taxa being in the same genus Cola while the differences show the reasons for the five species to exist as distinct species. Pierre et al. (2006) in his work evaluated seventeen accessions of Cameroun Colaacuminatagermplasm in order to examine the variation based on 17 morphological traits and established a list of minimum descriptors observed in all morphological characters with high coefficient of variation of Cola

acuminata and in every phase of growth indicating a large variation of accessions for these traits.

Pierre et al. (2006) also alleged that all morpho-quantitative descriptors studied are discriminant for the identification of Cola acuminata accessions. Importantly, some species of the same genus can be morphologically very similar and may be grouped into the same species despite the fact that they represent separate taxonomic entities (Whittallet al., 2004). In a related study,

Chandran and Pandya (2000) noticed a wide variation of morphological traits in the morphological characterization of 35 accessions belonging to 13 species of Arachis.

Qualitative analysis in this study, shows that there is Inter- and Intra- species similarities and variability. Each Cola OTUs is distinctively delimited from the rest. This phenotypic quality in the morphological traits makes each individual accession distinct from the others even though they might fall within the same species or within the genus. However, the proportion of similarity and dissimilarity for the qualitative traits becomes a determinant for delineating species. The leaf surface, seed colour, and plant height (100%, 75% and 66.7% respectively) show strong characters for delimiting species and accessions. This shows that the 5 out of the 12 qualitative descriptors are polymorphous. We can also deduce that the highest occurring traits are cuneate leaf base, acuminate leaf apex, entire leaf margin, and glabrous leaf surface. These phenotypic characters shows least variability compared to the leaf surface, seed colour, plant height, flower colour and floral size, which records the highest variability.

This result is in line with the findings of Manzanoet al (2001) who suggested that six leaf and Comment [TCL26]: Merge these 2 paragraphs petiole qualitative descriptors contributed in the variability of Colocasiaesculenta (L) Scott germplasm. Pierre et al.(2006)alleged that all morpho-qualitative characters are discriminant for the variability for the five Cola species. In the morphological characterization and agronomic evaluation of Arracaciaxanthorrhizacollection, 26 out of 29 qualitative descriptors were

polymorphous (Rosso et al., 2002). Pierre et al.(2006)noted that clusters of Colaacuminata based on morpho-qualitative traits don’t have the same structure.

The five Cola species shows strong inter species variability for the 16 OTUs used for the study.

The high difference in gene diversity among 16 taxa reveals the presence of strong genetic structure between them and thus significant differences exist in the genotypic diversity among themselves. This finding is supported by results of Mohdet al.(2004) using a RAPD marker, reported that genetic variation occurred among the three ginger cultivars from Malaysia. RAPD analysis has been found to be useful in differentiating closely related species (Zhang et al.,

2001).The genetic relation through RAPD markers may provide reliable method for the identification of species than morphological characters (Palaiet al., 2007). There is a common trend of maintaining high genetic diversity within populations in tropical plants as reported by

Hamrick and Loveless (1989). These results are also confirmatory with the finding of Huang et al., (2003) who reported significant (high) genetic variations by RAPD markers in other species at cultivar level. This outcome is supported by Nayaket al. (2006) who established that the main cause of polymorphism could be intra specific variation among different cultivars.

The results from the three-combine effect of the character sets (Quantitative, Qualitative and

Molecular) used for this study shows that there is a consistent variability and similarities for the five Cola species. These variations are traceable to the environmental effects and inherent genetic effects. Despite the dendrogram for each character set reveal new clusters based on the data and analysis carried out, we see that OTUs of the same species frequently align together to show intra- and inter- species relationship p for different species of Cola. This consistency in Comment [TCL27]: ?? their similarity is an indication that they are of the same ancestor (monophyletic) and the same

genus. The vegetative characters have shown to be inconsistent both for the qualitative and quantitative traits. On the other hand, the reproductive characters like the seed colour, fruit length, seed shape etc. were found to be relatively consistent. Cola acuminata, C. millenii and Comment [TCL28]: Limit the use of etc., i.e. e.g. to only parenthesis Cola nitidawere found to be preferable for consistency of character across the five locations and is recommended for breeding purposes. Cola gigantea and Cola lepidota show highest variability.

Conclusion

It is important to note that each taxonomic entity used for this study is distinctively and phenotypically different from the other even though they are of the same species or the same genus. From this comparative analysis, it clear that the high polymorphic and variability effects noticed in both morphological and molecular analysis areis traceable to the environmental effects and inherent genetic effects. This consistency in their similarity as observed with the cluster analysis and dendogram is an indication that they are of the same ancestor (monophyletic) and the same genus. Morphological and molecular dissimilarity is an indication of species uniqueness and habitat influence. The vegetative characters have shown to be inconsistent both for the qualitative and quantitative traits. On the other hand, the reproductive characters like the seed colour, fruit length, see shape etc. werefound to be relatively consistent. The results showed Comment [TCL29]: It is a good habit to proof read your work before submitting to a that the OTUs whose cultivation regions are very close shows maximum similarity among them journal to avoid outright rejection by the editor. as compared to OTUs which are farther apart. These findings will also provide an important contribution in determining resourceful management strategies for breeders for Cola improvement program.

Figure1: A, B, C , D : Dendrograms showing relationship among the 16 Cola accessions/OTUs

(A) Cluster relationship based on quantitative data (B)Cluster relationship based on quanlitativedata Comment [TCL30]: Spelling

(C) Cluster relationship based on molecular data (D) Cluster relationship based on quantitative data +

qualitative + molecular

Table 1.1 Performance of Cola species on Quantitative Characters

Species Leaf Leaf Leaf Number Collar Leaf Comment [TCL31]: Is it possible to measure length width petiole of Mid length petiole number in cm? (cm) length vein diameter (cm) (cm) (cm)

( cm) (cm)

C. lepidota 8.84c 5.13c 4.44c 1.64c 0.31c 0.04c

C. gigantea 32.47a 18.11a 18.67a 9.67a 1.1a 0.23a

C. acuminata 12.14c 5.02c 3.79c 5.18b 0.54b 0.16b

C. millenii 26.53b 13.30b 11.93b 5.60b 0.64b 0.20ab

C. nitida 13.43c 5.23c 2.84c 5.02b 0.25c 0.16b

Each value is the mean of 10 replicates. Values with the same alphabet are not significantly (p<0.05) different from one another across the column according to Duncan’s Multiple Range

Table 1.2 Correlation Matrix showing the relationship among Quantitative Characters, Location and Species Effects

LL LW LPL NLMV CL LPD LN LOCATION SPECIESComment [TCL32]: Where can one find these abbreviations defined LW 0.92**

LPL 0.92** 0.90**

NLMV 0.86** 0.82** 0.82**

CL 0.86** 0.86** 0.85** 0.81**

LPD 0.84** 0.77** 0.78** 0.87** 0.80**

LN 0.77** 0.78** 0.84** 0.79** 0.71** 0.72**

LOCATION -0.45* -0.42* -0.40* -0.43* -0.37 -0.39 -0.3

SPECIES 0.05ns -0.05ns -0.12ns 0.08ns -0.12ns 0.20ns -0.10ns -0.10ns

SAMPLES -0.08ns -0.04ns -0.05ns -0.01ns 0.03ns 0.04ns 0.01ns 0.01ns 0

Highly significant (p<0.1) = **, Significant (p<0.5) = *, ns = not significant,

Table 2.1 : 12 qualitative characters and their occurrence in 16 Cola accessions Comment [TCL33]: What does 0,1,2,3… mean? Leaf Leaf Leaf Leaf Leaf Plant Flower Fruit Seed Floral Fruit Fruit OTUs Apex Margin Shape Base Surface Height Colour Surface Colour Size Width Length GCA 0 0 0 0 0 1 0 0 0 0 0 3

GCN 0 0 1 1 0 0 2 0 1 0 1 3 GCM 0 1 2 2 0 2 1 1 0 2 1 0 GCG 0 1 2 2 0 0 0 2 0 3 1 4 NCN 0 0 1 1 0 0 2 0 0 0 0 1 NCA 0 0 3 3 0 3 0 0 0 0 0 2 NCL 1 0 4 1 0 0 3 3 1 4 0 2 CCN 2 0 1 1 2 0 2 0 0 0 1 0 CCA 3 0 5 4 0 0 1 0 0 0 2 0 AC N 2 0 5 1 2 0 2 0 1 0 0 2 ACA 0 0 5 4 0 1 0 0 0 0 0 3 ACM 2 1 0 1 0 2 1 1 2 2 1 4 ACG 4 1 7 3 0 4 0 2 0 3 3 3 NA CM 2 1 0 1 0 2 1 1 2 2 2 2 NACA 0 0 5 1 0 0 0 0 0 0 0 0 NACG 4 1 6 2 1 5 0 2 3 3 0 2

Table 2.2:Quantitative frequency of 12 Qualitative characters

70 60 50 40 30 20 10 0 Grp 0Grp 2Grp 4Grp 6Grp 8 Grp 10 base apex margin shape surface

Plate 1- PCR amplification product for RAPD Primers on gel picture

Plate 1.1: RAPD bands generated by RAPD at locus OPT – 20: 1KB+ Marker, 16 Cola samples

Plate 1.2 :RAPD bands generated by RAPD at locus OPT – 07: 1KB+ Marker, 16 Cola samples

Table 3.1: Yield and purity index of extracted Cola genomic DNA

DNA yield (µg/of fresh Sample ID weight) A260/280* ABBREV. 1 Cola acuminata (CRIN) 78.00 0.96 CCA 2 Cola nitida (DABIRI) 205.30 1.35 ACN 3 Cola acuminata (BOT GARDEN) 345.70 1.62 GCA 4 Cola acuminata (DABIRI) 699.80 1.56 ACA 5 Cola nitida (BOT GARDEN) 421.20 1.65 GCN 6 Cola melenii (DABIRI) 989.00 1.65 ACM 7 Cola melenii (NACGRAB) 1139.80 1.72 NACM 8 Cola melenii (BOT. NURSERY) 1651.10 1.78 NCM 9 Cola melenii (BOT. GARDEN) 810.60 1.70 GCM 10 Cola gigantea (NACGRAB) 1265.30 1.23 NACG 11 Cola gigantea (BOT. GARDEN) 444.30 1.21 GCG 12 Cola gigantea (DABIRI) 272.60 1.20 ACG 13 Cola nitida (CRIN) 616.10 1.38 CCN 14 Cola acuminata (NACGRAB) 1374.50 1.40 NACN 15 Cola nitida (BOT. NURSERY) 330.20 1.57 NCN 16 Cola lepidota (BOT. NURSERY) 518.60 1.63 NCL

Table 3.2:Primers Used, Number of Amplified Polymorphic Bands, PIC and Gene Diversity No. of Polymorphic Primer ID Primer Sequence Sampl Polymorphic Allele Gene information e size DNA bands number diversity Content Comment [TCL34]: The sample is the same all‐through. It is better to delete this column OPT – 06 - 5-CAAGGGCAGA – 3 16 10 10 0.24 0.95 OPT – 07 - 5 - GGCAGGCTGT – 3 16 15 6 0.22 0.99 OPT – 20 5 - GACCAATGCC – 3 16 10 10 0.19 0.92 OPB – 10 5 - CTGCTGGGAC – 3 16 11 10 0.19 0.98 OPH – 02 - 5 - TCGGACGTGA – 3 16 7 8 0.13 0.81 OPH – 05 - 5 - AGTCGTCCCC – 3 16 10 8 0.16 0.94 OPT – 01 - 5 - GGGCCACTCA – 3 16 13 8 0.20 0.97 OPT – 04 - 5 - CACAGAGGGA –3 16 12 7 0.19 0.97

Mean 16 11 8.4 0.19 0.94

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