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African Journal of Environmental Economics and Management ISSN 2375-0707 Vol. 2 (2), pp. 181-187, 
February, 2014. Available online at www.internationalscholarsjournals.org © International Scholars Journals 

 

Author(s) retain the copyright of this article. 

 

Full Length Research Paper 
  

A study of the degree of genetic variation among 
populations of cacao grown in Nicaragua  

 

Erielda Corrales, Regina Lacayo and Herman Mariano   
 

Department of Environmental Science, Faculty of Agriculture, Bluefields Indian and Caribbean University, 
Bluefields, Nicaragua. 

 

Accepted 05 January,  2014 
  

The use of local germplasm of THEOBROMA CACAO L. as a basis for developing a breeding program is 
limited by the lack of information on genetic variability and relationships among populations. This study 
covered a sample of 70 cocoa trees in five geographical areas of Nicaragua: El Rama, Nueva Guinea, Muelle 
de los Bueyes, El Castillo and Los Guatusos. The samples were molecularly analyzed using 10 microsatellite 
(SSR) DNA. For the 10 locus was achieved amplification of 95 alleles. Populations showed an average 
observed heterozygosity (Ho) of 0.522, and Nueva Guinea was the population with the value of Ho closest in 
value of expected heterozygosity (He). The latter obtained the lowest inbreeding rate (ƒ = 0.06). The value 
obtained for the average Fixation Index FST was 0.23, which allows you to catalog the cocoa plantations of 
southeastern of Nicaragua with a moderate level of genetic diversity. The populations of Muelle de los 
Bueyes and El Castillo presented the maximum value of genetic distance (0.1784). According to the Principal 
Coordinate Analysis, the population distribution in a geometric plane can be grouped into two blocks. 
Finally, the study phylogenetic populations of Castillo and Guatusos are more closely related to each other, 
than the rest of the population. 

 
Keywords: Theobroma cacao L., Microsatellite markers, Germplasm, Consanguinity, Heterocygosis 

 
INTRODUCTION 
 
When the Spanish conquistadors arrived in the New 
World, indigenous ancestors were using cacao as 
currency. The units of change were a Hand, five seeds, 
Quinque, fifteen seeds or Tzontly forty cocoa beans 
(Rocha, 1976, Palma, 1975). Since then, in Mesoamerica, 
the cultivation of cocoa has a cultural and economic roots 
(Henderson, 2007). Cocoa, Theobroma cacao (2n = 2x = 
20) whose genome is 390 Mega bases (Mb) distributed in 
10 chromosomes, is a neotropical species found in the 
Mesoamerican region between 20 ° N and 20 º S, 
considered the area of domestication of cocoa 
(Henderson, 2007). Commercial cultivars of T. cocoa 
have a narrow genetic base (Schnell, 2005). The 
 
 

 
*Corresponding author email: erieldacorrales@gmail.com 

 
 
 
 

primary types of cocoa are Forastero, Trinitario (hybrid 
between Forastero and Criollo) and Criollo, but 
Motamayor (2008) proposes to classify the species into 
10 clusters or groups: Marañón, Curaray, Criollo, Iquitos, 
Nanay, Contamana, Amelonado, Purus , Nacional and 
Guiana. De la Cruz, (1995) presents divided Theobroma 
into two sub-species: Cocoa and sphaeorocarpum. 
Beside, De la Cruz (1995) suggests that the Criollo and 
Trinitario types should be considered as traditional crops 
and not like genetic groups. In addition, the Amazon 
basin is the region that has greater cacao diversity 
(Gentry, 1988).  

In Nicaragua the cocoa plantations were developed 
hybrid genetic material derived from crosses between 
Trinitario and Forastero, and in addition endogenous 
Criollo cocoa plantations, although it is unknown the 
status of biodiversity of the latter. The producers, through 



Pedro et al.           181
 
 
 

 
social exchange of seeds, are responsible for the spread 
of cacao trees, which were not derived from clonal 
gardens or breeding programs. In Nicaragua the annual 
production of cacao is 1,500 t -2.000 6.500 ha, with an 
approximate yield of 3.5 quintals mz-1 and priced at U$ 
2-6 per kg (Buchert, 2008). According to the International 
Cocoa Organization (2007) Nicaragua is ranked 27 
among cocoa-exporting countries and their production 
represents 0.026% of the global total.  

One of the main concerns of the world cocoa market is 
the ability of producing countries to supply the market 
continuously with fine cocoa quality, value added, as high 
oil content of cocoa, polyphenol groups (antioxidants) and 
terpenes (flavor and aroma) (Othman, 2007; Niemenak, 
2006). In this regard it is important to understand the 
genetic diversity of cacao plantations in Nicaragua due to 
its impact on production sustainability. Therefore, the 
objective of this study was to establish the degree of 
genetic variation among populations of cacao grown in 
Nicaragua and to determine the genetic diversity of cacao 
and extrapolate cocoa elite populations of the 
Mesoamerican region. 
 
 
MATERIALS AND METHODS 
 
Plant material 
 
Between February and June 2010 took place sampling 
plant material in Nueva Guinea, El Rama, Muelle de los 
Bueyes, El Castillo and Los Guatusos. From different 
trees, 70 samples were collected of immature leaves and 
not young. The tree selection was based on its productive 
potential and physical condition. The collection of each 
sample was accompanied with the raising of ecological 
agriculture tab of the plot where the tree stood. Moreover, 
each tree was referenced satellite and labeling. Each leaf 
was wrapped in paper towels and packed in a Ziploc bag 
and preserved at -20 º C until processing. 
 
 
DNA extraction 
 
Prior to DNA extraction leaf was rinsed several times with 
distilled water and drilled to obtain 200 mg leaf discs were 
placed in a 1.5 mL Eppendorf tube which was immersed 
in liquid nitrogen and crushed using a mortar Kontes. 
Then we used a commercial medium for DNA extraction, 
DNeasy Plant Mini Kit (Qiagen). We proceeded with the 
protocol recommended by the commercial company with 
the variant that AP1 extraction solution was added 10 mg 
mL-1 of Polyvinylpolypyrrolidone (Sigma). The DNA was 
quantified using Biospectofotómetro (Shimadzu UV-1200) 
and all DNA samples were diluted to a concentration of 
about 5 ng mL-1 (Saunders et al., 2004; Zhang et al., 
2006). 

 
 
 
 
 

Microsatellite markers 
 
For molecular characterization was implemented 10 
microsatellite (SSR) reported by Saunders (2004) and 
considered the comments of Zhang (2006). The name of 
microsatellite markers, the sequence of the primers, 
temperature, alignment and size are reported by 
Saunders (2004). The PCR reaction was performed in a 
volume of 50 µL, containing 5 ng of genomic DNA. All the 
PCR reactions contained: 1.0 U Platinum Taq DNA 
polymerase (Invitrogen), 0.2 mm dNTPs (Invitrogen), 
0.2µM of each primer (Invitrogen), 1.5 mM MgCl 2, 1X 
buffer (20 mM Tris-HCl, pH 8.0, 40 mM NaCl, 2 mM 
sodium phosphate, 0.1 mM EDTA, 1 mM DTT, 50% 
glycerol). The conditions of the thermocycler (Eppendorf) 
were: 4 min denaturation at 94°C, followed by 33 cycles 
of denaturation at 94 °C for 30 s, 1 min at a temperature 
of alignment of 46 °C or 51°C and 1 min extension at 
72°C, with a final extension of 72°C for 7 min. 
 
 
Electrophoresis 
 
Amplified products were separated on a vertical 
electrophoresis apparatus (T-Rex Aluminum Backed 
Sequencer, Model FB-SEQ-3545-FISHER) in a 
denaturing gel (6 M urea) 6% polyacrylamide (Fisher) 
running at 55W, 1700V by 2 or 3 h in 1X TBE buffer 
(Qiagen) and detected by silver staining. The size of the 
amplified bands was determined with a commercial DNA 
marker, 125bp DNA Ladder (Invitrogen). 
 
Statistical data processing 
 
The statistical program Genetic Analysis in Excel v6.1 
(GenAlEx) (Peakall, 2006) was used to determine the 
average number of alleles per locus, allele frequency, the 
percentage of polymorphism loci, the value of observed 
and expected heterozygosity, the pattern of alleles in the 
population, and the Principal Coordinates Analysis (PCA). 
The statistical program Genetix 4.02 (Belkhir, 2001) was 
implemented for statistical determination of Nei genetic 
diversity and the calculation of the F-statistics (F-Statistic 
of Wright). The phylogenetic tree was constructed using 
Phylogeny Inference Package (Phylip v3.6) program and 
based on molecular data. Other support programs used 
were Arlequin v3.1 and Convert (Glaubitz 2004). 
 
 
 
RESULTS 
 
With the implementation of 10 microsatellite (SSR) was 
determined the variation at the level of allele. This was 
amplified 95 alleles with an average of 9.5 alleles per 
locus. In our study, Y16988 locus has the highest earning 



182         Afr. J. Environ. Econ. Manage. 
 
 
 

Table 1.  General statistics of the 10 microsatellites used in cocoa samples 
 

 Locus SSR
†
  Na

††
 Alleles (bp)

‡
  Ho

§
 He

¶
  

 Y16980 mTcCIR6 10   230, 235,240, 250, 275, 280, 285, 0.504 0.609   

    290, 295, 300     

 Y16981 mTcCIR7 9 155, 160, 165, 170, 173, 175, 180, 0.684 0.653   

    185, 190      

 Y16982 mTcCIR8 8 275, 280, 285,290, 295,300, 305, 0.611 0.507   

    310      

 Y16985 mTcCIR11 11  210, 280, 285, 290, 295, 300, 303, 0.399 0.620   

    305, 310, 320, 470     

 Y16988 mTcCIR15 15   220, 225, 230, 235, 240, 245, 250, 0.514 0.702   

    255, 260, 270, 275, 280, 290, 300,     

    310      

 Y16991 mTcCIR18 9 305, 310, 315, 320, 325, 330, 340, 0.512 0.624   

    345, 350      

 Y16991 mTcCIR22 7 270, 275, 280, 285, 290, 295, 300 0.395 0.419   

 Y16998 mTcCIR26 7 280, 285, 290, 295, 300, 305, 310 0.492 0.498   

 AJ271943 mTcCIR40 9250, 255, 260, 265, 270, 275, 280, 0.631 0.670   

    285, 290      

 AJ271958 mTcCIR60 10   180, 185, 190, 195, 200, 205, 210, 0.476 0.670   

    215, 220, 225     

 Total / Average 95   0.522 0.597   
 

† microsatellite representation; † † alleles number; ‡ base pare; § observed heterozygosity; ¶ expected heterozygosity. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
Figure 1. Silver staining 6% polyacrylamide gel of amplified molecular marker (mTcCIR18). 
MW: molecular weight of DNA marker (Invitrogen, 125 bp DNA Ladder). The numbers 
represent the samples of cacao analyzed 

 

 
a total of 15 alleles (Table 1). This locus has a better 
chance of change in the frequency of any his alleles.  
The Figure 1 shows the homozygous or heterozygous 
condition for a particular locus identified using molecular 
markers. Viewing a single band determines the  
. 

 

 
homozygous condition; otherwise two bands mark the 
state of heterozygosity.  

An important parameter in genetic diversity of a 
population is the relationship between values for 
observed heterozygosity (Ho) and expected 



Pedro et al.           183 
 
 
 
Table 2. Parameters of genetic diversity of the populations under study 
 
        Parameters genetic diversity 

Population  N¶ K¶¶ Ho He ƒ§  HS
†
 HT

††
 Dm

‡
 DST

Þ
    GST

ÞÞ
 

El Castillo 23 6.3 0.609 0.733 0.19240       
Los Guatusos 10 4.3 0.548 0.619 0.16758       

Nueva Guinea 11 4.3 0.555 0.562 0.06082       

El Rama 17 4.1 0.448 0.553 0.21885       

Muelle de los 9 3.6 0.450 0.520 0.19245       

Bueyes             

Average 4.5 0.522 0.600 0.16642 0.5974 0.7781 0.2258 0.1807 0.2322  
 
¶ alleles number; ¶¶ average of alleles by locus; § Coefficient of Consanguinity; † Genetic Diversity inside of 
population; †† Total Genetic Diversity ; ‡ Absolute Genetic Differentiation; Þ Genetic Diversity Between Populations  
; ÞÞ Relative Magnitude of Gene Differentiation among Subgroups. 
 
 

Table 3. Estimation of Fixation Index (F-Wright) 
Þ

 for the 10 loci studied 
 

Locus SSR FIT
†
 FST

‡
 FIS 

Y16980 mTcCIR6 0.36338 0.23398 0.16892 
Y16981 mTcCIR7 0.39264 0.24157 0.19919 
Y16982 mTcCIR8 0.39278 0.23493 0.20633 
Y16985 mTcCIR11 0.35616 0.23794 0.15514 
Y16988 mTcCIR15 0.36169 0.24308 0.15671 
Y16991 mTcCIR18 0.35986 0.23041 0.16821 
Y16991 mTcCIR22 0.36392 0.22175 0.18268 
Y16998 mTcCIR26 0.37466 0.23081 0.18702 
AJ271943 mTcCIR40 0.38277 0.24788 0.17934 
AJ271958 mTcCIR60 0.37068 0.24939 0.16159 
Average  0.37234 0.23708 0.17770 

 
Þ calculated with a confidence interval 95% Bootstraps based on 1000 replications per locus; † Total 
Fixation Index; ‡ Fixation Index Among Populations; § Fixation Index Within Populations 

 
heterozygosity (He). Heterozygosity is determined by the 
number of times (frequency) repeated the different alleles 
for a particular locus. Table 1 show that in 8 of the 10 loci 
analyzed, the value of Ho is less than the value recorded 
for He.  

The values for the Fixation Index also known as the 
Coefficient of Inbreeding (ƒ) is showed in table 2. This 
parameter determines the reduction of heterozygosity or 
outcrossing level. Values can range from -1 to +1. The 
study did not yield any negative value for ƒ indicating that 
the populations under study there is no excess of 
heterozygosity.  

The distribution of variability within and between the 
populations studied is determined by the Total Genetic 
Diversity, Total Heterozygosity (HT), Heterozygosity for 
each Population (HS) and Genetic Diversity among 

 
 
populations (DST). In this study the value of the HT is the 
result of the sum of (HS) and DST. The value reported for 
DST (0.1807, Table 2) indicates that loci for the five 
populations studied has few heterozygotes and several 
types of homozygotes.  

The Coefficient of Genetic differentiation among 
populations (GST) is calculated by the relationship DST / 
HT, in the present study, the GST value of 0.2322 (Table 
2) and FST of 0.23708 (Table 3), very close together, 
indicating that diversity among populations is supported 
by the presence of two alleles at a locus.  

The parameter that determines the absolute magnitude 
of Genetic Divergence (Dm) expresses the variability 
between interpopulations with respect to the 
intrapopulations. This ratio is calculated by NDST / n-1, 
where n represents the number of populations. 

. 



184         Afr. J. Environ. Econ. Manage. 
 
 

 
Table 4. Genetic Distance (Nei, 1972) among the five 

populations
§
 under study south-eastern of Nicaragua 

 
 CAGUNG RAMB 
 CA ( 23) 0.000     

 GU ( 10)   0.507   0.000    

 NG ( 11) 1.285 1.864  0.000  
 RA ( 17) 1.674 1.633 0.180 0.000 
 MB ( 9) 1.784 1.386 0.360 0.110 0.000 

 
§ El Castillo, CA ; Los Guatusos, GU; Nueva Guinea, NG ;El 
Rama, RA; Muelle de los Bueyes, MB 

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
Figure 2. Dimensional array of association (distance) between individuals from different 
populations (El Castillo, CA; Los Guatusos, GU; Nueva Guinea, NG; El Rama, RA; 
Muelle de los Bueyes, MB). 

 
 

 
The probability that two alleles of the same locus in an 

individual, are identical from the same offspring is 
denoted as the Fixation Index or Coefficient of 
Differentiation (F) and in the context of the present study, 
this index corresponds to the values F (Table 3), where 
the subscripts representing the level of hierarchy, which 
are being compared.  

It is instructive to determine the proximity or the Genetic 
Distance between populations, to achieve this we 
determined the Coefficient of Genetic Distance (Table 4). 
This parameter allows compared pairs of populations of 
the five populations under study.  

The Principal Coordinates Analysis (PCA) can 
transform a number of variables correlated with a small 
number of uncorrelated variables, facilitating visual 
interpretation (graph) of the similarity or dissimilarity that  
. 

 
 

 
exists between individuals or populations. This 
relationship is embodied in a plane where the geometric 
distance between points reflects the genetic distance. 
The representation does not take into account the 
evolutionary processes, there is a phylogenetic tree and 
the relationship between two points is determined by 
calculating the frequency of alleles. In the Figure 2 shows 
the distribution in a geometric plane of each sampled 
tree, represented by a point.  

If a pair of genes from different species or populations 
is developed in parallel and whether the degree of 
divergence between the two genes / populations implies 
that they have originated from the generation t, we can 
infer that these genes diverged from an ancestor 
common at time period t / 2. This reasoning allows us to 
construct the phylogenetic tree based on genetic 



Pedro et al.           185 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
Figure 3. Dendrogram for the phylogenetic tree of the five 
geographical cacao populations included in this study. 
Molecular phylogenetics tree is aimed at relationships for 
populations based on molecular data (allele and haplotype 
frequencies) 

 

 
distance. Figure 3 shows the phylogenetic tree 
representing the relationships of five populations under 
study, based on the degree of similarity or number of 
shared characteristics. 
 
 
DISCUSSION 
 
The genetic variation between individuals or populations 
may manifest at different levels. The first level comprises 
a set of indicators that measures the change internally in 
the population (frequency of alleles, polymorphism, 
observed and expected heterozygosity, mean number of 
alleles per locus, effective number of alleles per locus, 
fixation index, etc. ) and the second level includes 
another set of indicators to measure the variation of a 
population relative to other populations (genetic distance, 
inbreeding coefficient, geometric correlation between 
genetic distance, cluster analysis, phylogeny, etc.). These 
indicators were identified in this study  

In Brazil’s cacao plantation Sereno (2006) used 11 
microsatellite and report the amplification of 49 alleles  
. 

 

 
with an average of 4.45. A higher average (5.6) reported 
by Zhang (2009) a study developed in the international 
collection of cocoa held in CATIE, Botanical Garden 
(Costa Rica). This study reports an average of 9.5 alleles 
per locus. Value obtained due to the size of the area 
sampled. Populations of trees sampled in this study are 
scattered and are not under the care of a germplasm 
collection.  

This study reported a deficiency of heterozygosity. It is 
very likely that the sampling would have developed 
among related trees (consanguinity). This means that a 
percentage of the sampled trees, have arisen from a 
facultative selfing or almost total, what makes the union 
of gametes not random. A second explanation could be 
the result of the Wahlund effect, which occurs when the 
samples have different allele frequencies from two mixed 
populations.  

In relation to the divergence between Ho and He values 
(Table 2) for population El Castillo and El Rama, they are 
far apart, it is likely that one or more considerations for 
the Hardy-Weinberg (HW) are not being met (being 
diploid, sexually reproducing, not this cross generational, 



186         Afr. J. Environ. Econ. Manage. 
 

 
gene with two alleles, the allele frequency is identical 
male and female gametes, random crossover, mutation 
and migration is ignored and, finally, that natural selection 
does not affect the alleles). The relationship between Ho 
and He makes the link between alleles and genotypes 
frequency, also determines the proportion of genotypes in 
a population remain constant from generation to 
generation.  

Obtain positive values for Coefficient of Consanguinity 
(ƒ) 0.06 to 0.21 denote different levels of heterozygote 
deficiency, probably due to the effect of consanguinity. 
This coefficient (ƒ) can also be considered as decreasing 
the proportion of heterozygosity at the crossing between 
related species. Inbreeding tends to expose harmful and 
recessive alleles, a situation that may be affecting the 
productivity of cocoa farms in the study area. This 
phenomenon is referred as biparental inbreeding that 
occurs when genetic information of the progeny comes 
from two different parents but related to each other, this 
could be explain the level of abortions in cacao tree, 
inducing by incompatibility between gametes (Hartl, 
2007).  

The GST value 0.2322 indicates that most of the total 
variation is distributed among populations and the 
variation or diversity is not exclusive to any of the 
populations studied. Beside a value of Dm 0.225 suggest 
that variability present in the populations is supported by 
the diversity of the individuals in the population and not 
by the variations among populations.  

Thus, FST is the probability that any particular locus in 
an inbred population have alleles identical by descent. 
The reported value for FST equal to 0.23 indicates that 
the populations studied have a moderate genetic 
diversity. FIS index is a critical parameter in measuring 
genetic variation. This parameter has a minimum value of 
0 (indicating absence of genetic divergence) and the 
maximum value of 1 (indicating fixation for alternate 
alleles in different subpopulations). Also set 
heterozygosity deficiency in individuals within the 
population.  

Based on data obtained from Table 2 and 3, it shows 
that the cacao trees are showing an overlap of 
generations and fertilization preferably between related 
plants that grow close together and the place occupied by 
the mother plant. This situation reduces the genetic 
diversity of cacao plantations. However, the trend of loss 
of heterozygosity can be reversed by providing, in just a 
one generation the random crossing between no related 
trees.  

Thus Muelle de los Bueyes (MB) and El Castillo (CA) 
are the populations with the highest distance value 
0.1784, being reported as the most diverse populations 
analyzed. Conversely, the closest are the Rama (RA) and 
Muelle de los Bueyes with a distance value 0.110, 
providing a lower limit differentiation. This value 
determines the proportion of the genetic elements 
(alleles, genes, gametes, genotypes) that share the two 

 

 
populations compared. A value equal to zero indicates 
that population are absolutely identical.  

Regarding to the PCA analysis, the points that cross 
from one quadrant to another indicate genetic similarity 
between individuals (Figure 1). Also seen in certain 
quadrants as there are points that fit perfectly one on 
another as if they were a single point. Observe how 
population tend fall into two main sections. One block 
represented by the populations of Los Guatusos and El 
Castillo, and a second block consisting of El Rama, 
Muelle de los Bueyes and Nueva Guinea. Within this 
second group, the population of El Rama, has a 
heterogeneous profile of individuals closely linked to 
populations of Muelle de los Bueyes and Nueva Guinea.  

The phylogenetic tree permit grouped populations in 
relation who submit the smallest genetic distance. Each 
branch represents the most recent common ancestor. In 
our study, less connected populations or "neighbors" from 
the point of view common ancestor belong to Los 
Guatusos and El Rama. 
 
 
CONCLUSION 
 
In conclusions, the present study generated information 
on the genetic diversity of cacao grown in the five villages 
in Nicaragua and is able to generate information 
regarding the degree of relatedness between populations, 
genetic distance between populations, establish the level 
of genetic diversity and heterozygosity. Based on 
determined parameters we can conclude that the genetic 
diversity of cacao in the study area is moderate. All this 
information would provide the baseline for establishing a 
breeding and conservation program of local biodiversity 
of cocoa. Breeding programs focused at the selection of 
cocoa tree with a particular property (flavor and aroma). 
 
 
 
ACKNOWLEDGEMENTS 
 
Our appreciation go to the producers and field 
technicians who actively participated in the taking of 
samples. Thank categorically to IICA-Nicaragua and the 
Austrian Development Cooperation-ProDeSoc by the 
financial support provided in the study. To all of them our 
gratitude. 
 
 
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