MOLECULAR AND MORPHOLOGICAL DIVERSITY STUDIES OF …sdiarticle4.com/prh/doc/Rev_ARRB_60214_Tum_A.pdf · acuminata (Beauvoir) Schott and Endlicher (known as Obi abata in Yoruba) and

MOLECULAR AND MORPHOLOGICAL DIVERSITY STUDIES

OF FIVE Cola (Schott andEndl.)species 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 acuminata, Cola nitida, 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 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 plant height are strong characters for delimiting Cola species. Molecular analysis showed that 309 fragments were recorded, of which 28.5% were 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, kola nut, medicinal plant. ,molecular characterization

Introduction

The kola nut is the fruit of the medicinal tree called Colaspecies, which is indigenous to West

Africa. The kola plant belongs to the Cola genus of Sterculiaceae family that has over one

hundred and forty species (Onomoet al.,2006). Onomoet al.,(2006) pointed out that Cola

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 [TCL1]: In full at first mention  

Formatted: HighlightFormatted: Highlight

Comment [TCL2]: Replace with recent publications, published 3 years or earlier

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 plants are essential

tools for characterization.

On the other hand, Random Amplified Polymorphic DNA(RAPD) markers are Decamer (10

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

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

Formatted: Highlight

Comment [TCL4]: Write in full at first mention  

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.

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)

and Botany Department nursery of University of Ibadan. Each location has a unique eco-

diversity characterized by dense rainforest environment, tall trees, 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

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

and visual aid, quantitative measurements were taken of leaf apex, leaf margin, leaf petiole

Comment [TCL5]: Insert citations 

Comment [TCL6]: In full 

Comment [TCL7]: Why 16 samples  

Comment [TCL8]: Which criteria was followed to select the 5 locations  

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

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).

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

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

Comment [TCL9]: Avoid using i.e. You can replace it with “like” 

Comment [TCL10]: In full 

Comment [TCL11]: This is not clear, rephrase or punctuate

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

analysis. The molecular analysis was carried out in the Bioscience Lab, IITA, Ibadan, Nigeria.

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

Comment [TCL12]: Do you mean 20oC? 

Comment [TCL13]: In full 

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).

Results

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

and the leaf length is the strongest vegetative character for this study to be used for delimiting

species.

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

clusters in which one of them is an outlier (non uniformity). Group 1 oneis made up of 8

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

the Subgroup 2. In cluster 2, Cola giganteaAkinyele LGA and Cola lepidota UI Botany

Comment [TCL14]: Why such an old citation? 

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

Comment [TCL16]: Where is the data showing this variability. If it is Table 1.1, indicate it here in parenthesis  

Comment [TCL17]: Where can I find this percentage? 

Comment [TCL18]: Put a reference e.g. (plate1.1) to direct the reader where dendrogram you’re talking about can be found

Comment [TCL19]: Write in full 

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.

From the results obtained for the quantitative analysis, a strong Inter species and Intra species

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 [TCL20]: Start with the major result, explain the trend and then cite other authors. You can’t start with a citation in this section 

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

the morphology and expression of compounds in plants (Folkerset al., 2008; Shen et al., 2008;

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

These results deduced from quantitative analysis is arein line with Pierre et al (2006) who

revealed a high coefficient of variation in Cola acuminata. Cronquist (1981) also opined that

there is significant morphological variation in the sterculiaceae family both within and across

species. This workis in line with Oyefunke’s work (2016) on comparative phytochemical and

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

Comment [TCL22]: You don’t have to define the abbreviation several times. OTU was earlier defined  

Comment [TCL23]: Which results?  It is better to discuss a particular result,  not the entire work ..Mention about the quantitative parameters in particular that are in line with Pierre et al (2006).  

Comment [TCL24]: Grammar  

Comment [TCL25]: It is better to discuss a particular result,  not the entire work 

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

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

Comment [TCL26]: Merge these 2 paragraphs 

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

their similarity is an indication that they are of the same ancestor (monophyletic) and the same

Comment [TCL27]: ??

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

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

that the OTUs whose cultivation regions are very close shows maximum similarity among them

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.

Comment [TCL28]: Limit the use of etc., i.e. e.g. to only parenthesis  

Comment [TCL29]: It is a good habit to proof read your work before submitting to a journal to avoid outright rejection by the editor.  

Figure

(A) C

e1: A, B, C ,

Cluster relation

D : Dendrog

nship based on

grams showin

n quantitative

ng relationsh

e data (B)C

hip among th

Cluster relation

he 16 Cola ac

nship based on

ccessions/OT

n quanlitative

Us

edata Comment [TCCL30]: Spelling  

(C) C

qu

Table 1.

Species

Cluster relation

ualitative + m

.1 Performanc

Leaf

length

(cm)

nship based o

molecular

ce of Cola spe

h

Leaf

width

(cm)

on molecular

ecies on Quan

Leaf

petiole

length

(cm)

data (D) Clus

ntitative Char

Number

of Mid

vein

ster relationsh

racters

Collar

length

(cm)

hip based on q

Leaf

petiole

diameter

quantitative ddata +

Comment [TCnumber in cm?

CL31]: Is it poss? 

sible to measuree 

( 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 SPECIES

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

OTUs Leaf Apex

Leaf Margin

Leaf Shape

Leaf Base

Leaf Surface

Plant Height

Flower Colour

Fruit Surface

Seed Colour

Floral Size

Fruit Width

Fruit Length

GCA 0 0 0 0 0 1 0 0 0 0 0 3

Comment [TCL32]: Where can one find these abbreviations defined  

Comment [TCL33]: What does 0,1,2,3… mean? 

GCN

GCM

GCG

NCN

NCA

NCL

CCN

CCA

AC N

ACA

ACM

ACG

NA CM

NACA

NACG

0

0

0

0

0

1

2

3

2

0

2

4

M 2

0

4

Table 2.2

P

0

1

1

0

0

0

0

0

0

0

1

1

1

0

1

2:Quantitative

late 1- PCR a

1 1

2 2

2 2

1 1

3 3

4 1

1 1

5 4

5 1

5 4

0 1

7 3

0 1

5 1

6 2

e frequency of

amplification

0

0

0

0

0

0

2

0

2

0

0

0

0

0

1

f 12 Qualitati

product for R

0

10

20

30

40

50

60

70

Grp 0Grp 2

base apex

0

2

0

0

3

0

0

0

0

1

2

4

2

0

5

ive characters

RAPD Primer

2Grp 4Grp 6Grp

margin s

2 0

1 1

0 2

2 0

0 0

3 3

2 0

1 0

2 0

0 0

1 1

0 2

1 1

0 0

0 2

s

rs on gel pictu

p 8 Grp 10

shape surfac

1

0

0

0

0

1

0

0

1

0

2

0

2

0

3

ure

e

0

2

3

0

0

4

0

0

0

0

2

3

2

0

3

1 3

1 0

1 4

0 1

0 2

0 2

1 0

2 0

0 2

0 3

1 4

3 3

2 2

0 0

0 2

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

Sample ID

DNA yield (µg/of fresh

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

Primer ID Primer Sequence Sample size

No. of Polymorphic DNA bands

Allele number

Gene diversity

Polymorphic information

Content

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

REFERENCE

Comment [TCL34]: The sample is the same all‐through. It is better to delete this column 

Adebola, P.O and Morakinyo, J.A., 2006. Evaluation of morpho-agronomic variability of wild

and Cultivated Kola (Cola Species Schott and Endl.) in South Western Nigeria 53(4):687–694

Adebola, P.O, 2003. Genetic characterization and Biosystematic studies in the genus Cola Schott and Endlicher. Ph.D. thesis, University of Ibadan Pp: 203

Adebola, P.O., Aliyu, O.M. and Badaru, k., 2002. Genetic variability studies in the germplasmcollecton of Kola (Cola nitida (Vent) Schott and Endlicher) in South Western Nigeria .Plant Genetic Resources Newsletter, 132:57-59

Adebola, P.O., Morakinyo, J.A., 2006. Evaluation of morpho-agronomic variability of wild and cultivated kola (Cola species Schott etEndl.) in South Western Nigeria. Genet Resource Crop Evol53:687–694

Adebona, A.C., 1992 Biotechnology of Kola improvement In: Thottappilly, L.M., D.R. Monti, M. Raj and A.W. Moore, (Eds.), Biotechnology: Enhancing research on tropical crops in Africa. CTA/IITA.Co-Publication.IITA, Ibadan, Nigeria, 376.

Adenuga, O., Mapayi, E., Olasupo, F., Olaniyi, O. and Oyedokun, A., 2012. Nigeria’s Cola Genetic Resources: The need for renewed exploration. Asian Journal of Agricultural Sciences, 4(3), 177-182.

Adesoye, A.I., Fasola, T.R. and Akinro, L.A., 2004. Extraction of high quality DNA from Cola nitidaand Cola acuminata. Research in Plant Biology, 4(4): 21-26.

Alverson, W. S., Karol, K. G., Baum, D. A., Chase, M. W., Swensen, S. M., Court, R. M C., and K. J. Sytsma., 1998. Circumscription of the Malvales and relationships to other Rosidae: Evidence from Rbcl Sequence DNA. American Journal of Botany85: 876–887.

Badaru, K., 1989. Advances in Kola breeding research in Nigeria. In: Progress in Tree Crop Research.2nd Edn., Cocoa Research Institute Of Nigeria(CRIN), Ibadan. Dec. A Commemorative Book to mark the 25thAnniversary of CRIN.

Bayer, M. F. Fay, De Bruijin, A.Y., Savolainen, V., Morton, C.M., Kubitzki, K., Alverson,W.S., and Chase, M.W., 1999. Support for an expanded family concept of Malvaceae within a recircumscribed order Malvales: a combined analysis of plastid atpB and rbcL DNA sequences. Botanical Journal of the Linnean Society 129: 267–303.

Bodard, M., 1962.Contributions to the systematic study of the Cola in West Africa.Annals of the Faculty of Sciences of the University Of Dakar.

Braga, M. R., Adiar, P. M., Marabesi, M. A. and de- Godoy, J. R. L., 2006.Effects of elevated CO2 on the phytoalexin production of two soybean cultivars differing in the resistance to stem canker disease.Environ Exp Bot. 58(1–3):85–92.

Burkill, H.M., 2000. The useful plants of West Africa.2nd Edition, Volume 5, Families S-Z, Addenda.Royal Botanical Gardens, Kew, Richmond, United Kingdom, 686.

Chan, R., Baldwin, B.G., and Ornduff, R., 2002. Cryptic Goldfields: A molecular phylogenetic reinvestigation of Lastheniacalifornicasensulato and close relatives (compositae: heliantheaesensulato). Am J Bot89: 1103–1112.

Chandran, K. and Pandya S.M., 2000.Morphological characterization of Arachis species of section, Arachis.Plant Genet.Resourc.Newslett. 121: 38-41.

Chevalier, A., and Perrot, A. 1911.“Les Vegetauxutiles de l'AfriquetropicaleFrancaise,” Challamell, Paris.

Cronquist, A., 1973 Chemical Plant Taxonomy. In: G. Bendz, and J. Santesson (Eds.), Chemistry. In: Botanical Classification. Proceedings of the 25th Nobel Symposium, August 20-25. Academic Press, New York & London.

Cybulskill, W. J., Peterjohn, W .T. and Sullivan, J .H., 2000. The influence of ultraviolet-B radiation on tissue quality and composition of loblolly pine (Pinustaeda L.)needles. Environ Exp Bot.44(3):231–241.

Donoghue, M. J., Alverson, W. S., 2000. A new age of discovery, Ann. Missouri Bot. Gard., Vol. 87, Pp: 110-126.

Doyle, J. A., 1996. Seed plant phylogeny and the relationships of Gnetales, Int.J. PlantSci., 1996, Vol. 157 Pp: S3-S39

Doyle, J. J., 1992. Gene trees and species trees: Molecular systematics as one character taxonomy. Syst. Bot. 17:144-163.

Duminil, J., Caron, H., Scotti, I., Cazal, S.O, & Petit, R.J., 2006. Blind Population Genetics: Survey of Tropical Rainforest Trees. Mol Ecol15: 3505–3513.

Egbe, N.E. and Oladokun, M.A.O., 1987. Factors limiting high yields of kola (Cola nitida) production in Nigeria.Café Cacao Thé.,32(4): 303-310.

Eijnatten, C.,Van, L.M.,1968. The germination of Kolanuts, Cola nitida (Vent.)Schott and Endl.Nigerian Agric. J. 5:72–82.

Faleiro, F.G., Santos, I.S., Bahia, R.C.S., Santos, R.F., Yamada, M.M., Anhert, D., 2002. Optimization of DNA extraction and amplification of Theobroma cacao L.Agrotrópica14: 31-34.

Felsenstein, J., 1978. Cases in which parsimony or compatibility methods will be positively misleading, Syst. Zool., 27: 401-410

Folkers, A., Hüve, K., Ammann, C., Dindorf, T., Kesselmeier, J., Kleist, E., Kuhn, U., Uerlings, R., and Wildt, J., 2008. Methanol emmissions from deciduous tree species: Dependence on temperature and light intensity. Plant Biol.10(1):65–75

Food and Agriculture Organisiation, FAO, (1982).Fruit bearing forest trees.Technical notes.FAO Forestry. 34:43-45.

Food and Agriculture Organisiation, FAO, 1995. “Edible Nuts,” Non-Wood Forest Products Series, Number 5.

Fundura, Z., R. Vera., E. Yaber and O. Barrios, 1992.Variability and classification of cultivars of Arachis hypogea (L.).INIFAT-MINAGRI, La Havana, Pp 25.

Furste, L.J., and Odusolu, E.O., 1974. Notes on the Selection of C.S.1, C.S.2 and C.S.3 Kola trees for yield and rooting ability.Kola pilot projectmemorandum. 5: 1-38.

Gatesy, S.M., and Dial, K. P., 1996.Locomotor modules and the evolution of avian flight, evolution, 50: 331-340.

Goji, T. C and Ayodele, A., 2012.Taxonomic revision in the Genus Cola Schott and Endl.in Nigeria, Pp 34 - 68

Hamrick, J.L., Loveless, M.D.,1989. The genetic structure of tropical tree populations: associations with reproductive biology. In: Bock J.H., Linhart Y.B., editors. Plant Evolutionary Ecology. pp. 131–146.

Hillis, D. M. and Wiens, J. J., 2000. Molecules versus morphology in systematics: Conflicts, Artifacts, and Misconceptions on phylogenetic analysis of morphological data. Washington DC Smithsonian Institution Press, 1-19.

Huang, H., Layne, D.R., Kubisiak, T.L.,2003. Molecular characterization of cultivated pawpaw ( Asiminatriloba ) using RAPD markers. Journal of the American Society for Horticultural Science. 128:85–93.

Huelsenbeck J. P., 1991. When Are Fossils Better Than Extant Taxa In Phylogenetic Analysis?, Syst. Zool., 40: 458-469)

Huelsenbeck, J. P., 1991. When are fossils better than extant taxa in phylogenetic analysis?, Syst. Zool. 40:458-469.

Huelsenbeck, J. P., 1997. Is the Felsenstein Zone a fly trap? Syst. Biol. 46:69–74

Hutchinson, J. &Dalziel, J. M., 1954. Floral Of West Tropical Africa Vol. 1, Part 3.The crown agents for the colonies, london. Pp: 828

Hutchinson, J., and Dalziel, J. M. 1958. “Flora of West Tropical Africa”.Vol 2 Part 2 , Pp: 345

Irvine, F. R. (1956). “Plants of the Gold Coast,” Oxford University Press.

Islam, M.A., Meister, A., Schubert, V., Kloppstech, K., Esch, E.,2007. Genetic diversity and cytogenetic analysis in Curcuma zedoaria (Christm) Roscoe from Bangladesh.Genetic Resources and Crop Evolution.54:149–156

Jaccard, P., 1908. Nouvellesrecherchessur la distribution florale .VaudoiseSci. Natl.44: 223-270.

Jeffer, J.N.R., 1967. Two cases studies in the application of Principal Component Analysis. Applied Statistic, 16: 225-236.

Jendoubi, R., Neffat, M., Henchi, B., and Yobi, A., 2001. System of reproduction and morphophenological variability in Allium roseum( L.) Plant Genetic Resources Newsletter,127: 29-34.

Jenner, R. A., 2004. Accepting partnership by submission? Morphological phylogenetics in a molecular millennium, Syst. Biol., 53: 333-342

Kaemer, D., Weising, k., Bayemann, B., Borner,T., Epplen, J.T. and Kahl, G., 1995. Oligonucleotide fingerprinting of TomatoDann: PlantBreed.,114: 12 -17.

Keay, R. W. J, Onochie, C. F. A and Stanfield, D. P., 1960. Nigerian Trees.Federal Department of Forest Research, Ibadan, 349

Keay, R. W. J., 1958. “Trees of Nigeria,” Oxford University Press, New York. Pp: 383 Kuome, C., and Sacande, M., 2006.Cola nitida (Vent) Scholt and Endl., Seed Leaflet:111 Lovejoy, P.E., 1980. Kola in the history of West Africa.Cahier D Etudes Africaines 77-78, 97-

134. Maddison, W.P., 1997. Gene Trees. In: Species Trees, Syst. Biol., 46: 523- 536 Manzano, B.A., Nodals, A.A.R., Gutierrez, F.Z., Mayor and Alfonso,C.L., 2001. Morphological

and isoenzyme variability of taro (Colocasiaesculenta L. Schott) germplasm in Cuba.PlantGeneticresourcesNewsletter, 126: 31-40

Mohd, M.A., Ibrahim, H., Khalid, N., 2004. Differentiation of three varieties of ZingiberofficinaleRosc.by RAPD fingerprinting. Malaysian Journal of Science. 23:135–139

Monzano, A.R., Nodals, A.A.R., Gutierrez, M.I.R., Mayor, F.Z., and Alfonso, C.I, 2001. Morphological and isoenzyme variability of Taro (Colocasiaesculenta L. Schott) Germplasm in Cuba.Plant Gen. resour.Newslet.,126: 31-40.

Morakinyo, J.A. and Olorode O., 1984.Cytogenetic and morphological studies on Cola acuminata (P. Beauv).Schott and Endlicher; Cola nitida (Vent) Schott and Endlicher and Cola acuminata X Cola nitida F1 Hybrid. Café Cacao, 28 (4): 234

Morakinyo, J.N., Egbe, E. and Oladokun Y.A.O., 1981. Compatibility studies and yield components of recent Cola nitida selections. Café Cacao., 25(2): 121-126.

Mshana, N.R., Abbiw, D.K., Addae-Mensah, I., Adanouhoum, M.R.A., Ahyi, J.A., Ekpere, E.G., Enow-Orock, Z.O., Gbile, G.K., Naomesi M.A., Odei, H. , Odunlami, A.A., Oteng-Yeboah, K. Sarprong, A.,Sofowora and Tackie,A.N., 2000. Traditional Medicine Phaarmacopocia: Contribution to the revision of Ethnobotanical and Floristic Studies in Ghana. (OAU/STRC) ISTT.Kolanut Nigeria Magazine71: 298305.

Nei, M., Fuerst, P.A. and Charaborty R., 1978. Submit molecular weight and genetic variability of protiens in natural populations. Proceedings of National Academy of Science. 75:3359-3362

Ogutuga, D.B.A., 1975. Chemical composition and potential commercial uses of kolanuts, Cola nitida (Vent) Schott and Endlicher. Ghana J. Agric. Sci., 8: 121-125.

Ojo, A.A., Olaniran, Y.A.O., and Oladokun, M.A.O., 1982. Evaluation of selected Cola nitida clones and their crosses. 1. General and Specific Combining Ability estimates. Café Cacao The.,26(1): 43-47.

Okoloko, G. E. and Jacob, V.I., 1971.Kola breeding in Nigeria. Pp: 22-23. Onomo, P.F., Niemenak,N., and Ndoumou, D.O., 2006. Isoenzyme variability of three Cola

(Cola Acuminata (Pal De Beauv,) Schott and Endlicher, Cola nitida (Vent) Schott and Endlicher and Cola anomala Schott. and Endlicher. germplasm in Cameroon.Pak. J. Biol. Sci.,9(3): 391-397.

Opeke, L. K., 1992. Tropical Tree Crops.Ibadan Spectrum books Ltd. Pp180–200.

Orisakeye, O.T., Olugbade, T.A., 2012. Studies on antimicrobial activities and phytochemical analysis of the Plant SterculiatragacanthaLindl.Middle East J .Sci.Res.11(7): 924-927.

Oyefunke , I.S., 2016. Comparative phytochemical and organoleptic studies on the root and bark of some selected species of Cola (Schott. and Endl.) Pp: 56- 75

Pierre, E. O., Niemenak, N., and Ndoumou, D.O., 2006. Morphological variability of Cola acuminata ((Pal. De Beauv) Schott and Endl.Germplasm in Cameroon.Pakistan Journal of Biological Sciences, 9: 398-403.

Pratt, D.B. and Clark, L.G., 2004. Amaranthusrudis and A. tuberculatus, one species or two J Torrey Bot Soc 128: 282–296.

Purseglove, J. W., 1968. “Tropical Crops: Dicotyledons,” Longmans Green & Co Ltd, Pp:56 Quarcoo, T., 1973.Kola Breeding. CRIN Ann Report, Pp: 55-91. Rick, C.M and Holle, M., 1990. Genetic variation and its evolutionary significance: Ecol Bot,44:

69-78.

Rohlf, F.J., 1993. NTSYS-pc.Numerical Taxonomy and Multivariate Analysis system.Exeter, New York, Pp 345.

Rohlf, F.J., 2000. NTSYS-PC, Numerical taxonomy system for the PC, Exeter Software, Ver. 2.1.Applied Biostatistics Inc, Setauket.

Rokas, A., Williams, B. L., King, N., and Carroll, S. B., 2003. Genome-scale approaches to resolving incongruence in molecular phylogenies, nature, 425: 798-80.

Rosso, C.A., Medina, C.I., and Lobo, M., 2002. Morphological characterization and agronomic evaluation of a Colombian collection of Arrachia( ArracaciaxanthorrihizaBancroft.) Plant Genetic Resources Newsletter, 132: 22-29.

Russel, T.A., 1955. The Kola of Nigeria and the Cameroons.Trop. Agric. (Trinidad), 32: 210-240.

Scotland, R. W., Olmstead, R.G., and Bennett, J.R., 2003. Phylogeny reconstruction: The role of morphology. Syst. Biol. 52:539– 548

Shaw, A.J., 2000. Molecular phylogeography and cryptic speciation in the Mosses, Mielichhoferiaelongata and M. Mielichhoferiana (Bryaceae).Mol Ecol9: 595–608.

Shen, H., Tang, Y., Muraoka. H., Washitani, I., 2008. Characteristics of leaf photosyntheis and simulated individual carbon budget in Primulamutans under contrasting light and temperature conditions. J Plant Res., 121:191–200.

Sie, R.S.J., N’Goran, A.K., Montagnon, C., Akaffou,D.S., and Cilas,C., 2005. Assessing genetic diversity in a germplasm collection of Kola trees (Cola nitida (Vent.) Schott and Endl.) Using enzymatic markers PGR Newsletter No., 143: 59-64.

Silva, C.R.S., Albuquerque, P.S.B., Ervedosa, F.R., Mota, J.W.S., Figueira, A. and Sebbenn, A.M., 2011. Understanding the genetic diversity, spatial genetic structure and mating system at the hierarchical levels of fruits and individuals of a continuous Theobroma cacao population from the Brazilian Amazon. Heredity 106: 973-985.

Sonibare, A.M., Soladoye, M.O., Esan, O.O., 2009. Phytochemical and Antimicrobial studies of four species of Cola Schott &Endl. (sterculiaceae). Afr. J. Tradit. Compl.Altern. Med. 6(4):518-522

Takhtajan, A., 1973. The chemical approach to plant classification with special references to higher taxa of Magnoliophyta. In: G. Bendz& J. Santesson (Eds.). Proceedings Of The 25th Nobel Symposium Held August 20-25, Academic Press, New York & London.

Voelcker, O. J., 1935. Cotyledon colour in Kola.Tropical Agriculture 12, 231-234. Weber, W.E and Wrikle, G., 1994. Genetic markers in plant breeding In: Advances in Breeding.

J. Plant Breed, Suppl. Pp: 16.

Whittall, J.B., Hellquist, C.B., Schneider, E.L. and Hodges, S.A., 2004.Cryptic species in an endangered pondweed community (Potamogeton, Potamogetonaceae) Revealed by AFLP Markers.Am JBot 91: 2022– 2029

Wiens, J. J., 2000. Coding morphological variation for phylogenetic analysis: Polymorphism and interspecific variation in higher taxa. In: Phylogenetic analysis of morphological data (J. J. Wiens,). Smithsonian Institution Press, Pp 115-145.

Wilkie, P. C. A., Pennington, R. T. C., Clemens, B., and Wilcock, C. C., 2006."Phylogenetic relationships within the subfamily Sterculioideae (Malvaceae/Sterculiaceae-Sterculieae).Using the Chloroplast Gene Ndhf".Systematic Botany.31 (1): 160–70.

Wilson, E. O., 1992. The diversity of life, Cambridge, Massachusetts Harvard University Press., Pp: 331