View
4
Download
0
Category
Preview:
Citation preview
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
Recommended