Genetic characterization of Theobroma cacao L. in Nicaragua using SSR
Erwin Manuel Aragon Obando
Master’s thesis
University of Helsinki
Department of Applied Biology
Plant Breeding
August, 2009
HELSINGIN YLIOPISTO  HELSINGFORS UNIVERSITET  UNIVERSITY OF HELSINKI
Tiedekunta/Osasto  Fakultet/Sektion  Faculty
Laitos  Institution  Department
Department of Applied Biology
Faculty of Agriculture and Forestry
Tekijä  Författare  Author
Erwin Manuel Aragon Obando
Työn nimi  Arbetets titel  Title
Genetic characterization of Theobroma cacao L Nicaraguan
Oppiaine Läroämne  Subject
Plant breeding
Työn laji  Arbetets art  Level
Aika  Datum  Month and year
August 2009
Master Sciences Thesis
Sivumäärä  Sidoantal  Number of pages
50
Tiivistelmä  Referat  Abstract
Genetic composition of Theobroma cacao L., including 60 Nicaraguan farmers
accessions, was investigated using nine microsatellite (SSR) markers. Fourteen
breeders accessions from Experimental Center “El Recreo”, INTA, Nicaragua, two
Criollos accession from CATIE, Costa Rica, and two accessions from Ecuador
were included as reference material. The average PIC value (0.78) indicated a
high power of discrimination for the nine loci used. A total of 155 alleles were
detected at the nine loci. The number of alleles per marker ranged from 10 to 22
with a means of 17.22 alleles per locus. A heterozygosity deficiency (HExp < HObs)
was registered for all microsatellite loci. The average expected heterozygosity
was=0.68 among Nicaraguan farmers accessions. The Analysis of molecular
variance (AMOVA) showed a low level of differentiation among populations. The
genetic distances determined for the groups of farmers accessions RAAS and
Pacifico Sur are closely similar, while a great genetic distance was observed
between RAAN and RAAS groups. The cluster analysis presented a strong
genetic relation between the Criollo 13 from CATIE and farmers accession
MAT0404. The principal component analysis showed that 7 farmers accession
from Nicaragua are genetically related with the accessions Criollo 13 and Yucatan
from the international accessions. The present study suggested a good possibility
to select farmers accessions to be included in breeding programs, especially
those accessions related to know Criollo accessions.
Avainsanat  Nyckelord  Keywords
Genetic differentiation, Genetic diversity, microsatellites, Farmers and Breeders
accession
Säilytyspaikka  Förvaringsställe  Where deposited
Muita tietoja  Övriga uppgifter  Further information
Supervised by : Helena Korpelainen –- [email protected]
CONTENTS
1. Introduction ...............................................................................................4
1.1 Origin and domestication .................................................................. 4
1.2 Morphology and varieties of Theobroma cacao L...........................6
1.3 Importance of production and use of Theobroma cacao ............... 8
2. Objectives.................................................................................................. 9
3. Materials and methods ........................................................................... 10
3.1 Plant materials .................................................................................. 10
3.2 DNA extraction .................................................................................. 16
3.3 Microsatellite analysis ...................................................................... 16
3.4 Capillary electrophoresis and data collection ................................17
3.5 Data Analysis.....................................................................................18
3.5.1 Genetic diversity ...................................................................... 18
3.5.2 Genetic differentiation .............................................................19
3.5.3 Genetic distances .................................................................... 20
3.5.4 Cluster analysis........................................................................ 20
3.5.5 Principal component analysis................................................. 21
4. Results ..................................................................................................... 21
4.1 Genetic diversity of Nicaraguan farmers accessions ....................22
4.2 Genetic diversity of Nicaraguan farmers accessions at
group level .........................................................................................22
4.3 Genetic distances ............................................................................. 24
4.4 Cluster analysis................................................................................. 25
4.5 Principal component analysis..........................................................27
5. Discussion...............................................................................................29
5.1 Genetic diversity and population structure.................................... 29
5.2 Differentiation among groups and genetic proximities ................ 32
6. Conclusions ............................................................................................34
7. Acknowledgements ................................................................................ 35
8. List of references ....................................................................................36
9. Appendices .............................................................................................44
4
1. Introduction
1.1 Origin and domestication
Sterculiaceae family has 70 genera and 1,500 species of tropical tree, which the
most famous and important species is Theobroma cacao L. (2n = 20) due to its
economic importance. Theobroma cacao L. is native to the tropical rainforest
and considered exclusive of the neotropical area, with a natural distribution in
rainforests, extended from the Amazon basin to southern Mexico (Dias 2001).
However, the origin and domestication have been controversial and widely
discussed due to wide geographic distribution of Theobroma cacao L.
The first propose about origin was done by Vavilov, who has indicated as
Mexico and Andes as centers of diversity, but it was rejected by F.J Pound in
1937/1938, who found an extensive variability in the wild cocoa trees of
Ecuador. According to Chessman (1944), based on Vavilov theory and the
variability of cacao reported by Pound argued that center of origin of
Theobroma cacao was located in 400 km radius of the Amazon River, in the
vicinity of the rivers Napo, Caqueta and Putumayo. Chessman suggested the
possible migration two groups of subspecies of cacao, one towards Venezuela
and other towards Central America, which the isthmus of Panama was a barrier
that helped to evolved independently the differentiation among both groups. The
Venezuelan group was called as Forastero and Central America group was
called Criollo.
More recently, through to biotechnology tools, Motamayor et al (2002)
compared the genetic diversity of cacao populations from Mesoamerica (Mexico
and Central America) and South America population. They concluded that
Criollo populations found in Lacadamia, Mexico, would consider as near to wild
population found in Peru, established as the center of origin. However, Ogata et
al. (2006) suggested include more samples from Oaxaca, southern of Veracruz,
(Mexico), Central America, and South America in molecular studies in order to
definite the real center origin due to previous (De la Cruz et al. 1995; Whitkus et
al. 1998) study have discovered unique cultivars in Yucatan, which suggested
that origin of Theobroma cacao L still a mystery.
Theobroma cacao L. is a relatively domesticated species. According to
Motamayor et al. (2002), McNeil (2006), and Bartley (2005), it has been
5
cultivated in Central America already since pre-Columbian times. Cheesman
(1944) suggested Theobroma cacao L in Central America and South America
have evolved independently. Dias (2001), consider that Criollo populations were
originated and cultivated in central America, from Mexico to the south of Costa
Rica, and Forastero existed in the wild state in South America. Among Criollo,
some populations are classified on geographic basis, such as those from
Central America (Mexican and Nicaraguan Criollos) and those from northern
South America (Colombian and Venezuelan Criollos)
There are many evidences that support Mesoamerica domestication for
Theobroma cacao L. Firstly, the cacao word is a Spanish corruption of Nahuatl
(native language) used for the plant and product, but Indigenous peoples called
to the cacao tree as “Cacahuatl” and “Kakawa”.
Theobroma cacao L was more important in Mesoamerican culture (Maya
and Aztec culture) than Southern American culture; due to Theobroma cacao L
was used in religious ceremony and weeding as special beverage (McNeil,
2006). Also, the preparations, use, and consume of cacao in Mesoamerica was
different than Southern America. In Mesoamerica the husk, pulp, and seed of
cacao was prepared and consumed of different method by native peoples,
nonetheless, southern America only consumed the pulp; also cacao was used
as currency in many places as Nicaragua.
Bartley (2005) suggested that Theobroma cacao L had less importance in
southern America due to existences of many species that were used as
stimulant (e.g., Erythroxylum coca), nonetheless in Mesoamerica was the
principal sources used as stimulant.
In Nicaragua, there are archeological evidences about importance and
cultivation of Theobroma cacao L. during pre-Columbian period, which has
been located to North of Chinandega, Leon, Granada, and Rivas as centers of
cacao production in pre-Columbian time. In addition, the North of Chinandega
was presumably the existences of sporadic tree in areas inhabited by native
peoples; nevertheless, in department of Rivas were cultivated and managed a
precious tree in fertile lands (Stainbrenner, 2006).
6
1.2 The Morphology and varieties of Theobroma cacao L.
The cacao tree seldom exceeds a height of 7.5 m in cultivations and may go up
to 12.5 m or more in wild growing. The main stem is divided in four to six lateral
branches called “Jorqueta” or “fan” with plagiotropic growth. An axillaries bud is
develops with an orthotropic shoot below the Jorqueta, which will again form a
Jorqueta a few feet higher up. Other Jorqueta will develop again, this process
should repeat during four times, producing a leafy canopy.
The mature leaves are dark green, about 37 cm long and 7.5 cm broad,
oblong oval or elliptic oval with prominent veins and veinletss, the short petiole
is provided with two articulations (Kochhar, 1986). The flush (young and
developing leaves) exhibit a range of colors from lightest green to red shades.
The flower is a complex organ and possesses some features that make it
unique and distinctive. The flowers are borne on the bark of the trunk and older
branches, and never on young shoot. Few of many thousand flowers will
develop in fruit. Flowering and fruiting continues through one year.
Theobroma cacao L. flowers are bisexual, regular and pentamerous. The
flower has the variation with regard to their shape and pigmentation. The sepals
are prominent, leathery and fused at the base. The corolla consists of five
petals, smaller than sepals, each having a broad curved, saccate basal and a
much narrower terminal portion expanding terminally into a cup of shaped a
pouch. Ten stamens are arranged in two whorls of five, the outer being
represented by infertile, narrow, ciliate, erect pointed structure forming a fence
around style, while the stamens of the inner whorl have curve filaments. The
stamens and staminoide are fused in the basal region forming a short tube. In
general; apparently there is a relation between color buds and fruit colors
(Kochhar, 1986).
Bartley (2005), the maturity fruit is a berry but commonly called a pod, which
is composed of a fairly thick, leathery, smooth or corrugated pericarp or husk.
The fruit varied in shape from spherical to very narrow elongate, and their colors
ranging from green (immaturity), yellow (mature), and red (immaturity) to purple
(maturity). Generally, a pod has 20 to 40 flat or round seeds, or cacao beans
embedded in a white, pink or brown, aromatic, mucilaginous, sweet or faintly
acid pulp. Each cacao tree produces only one or two pounds of beans every
year (Kochhar, 1986).
7
The period between fertilization of a flower and the initiation of maturity is
around 135 to 180 days. Self-incompatibility is a series problem that some
genotype presents. The discovery of self-incompatibility in cacao was
determinate as consequence of genetic mechanisms. The mechanism by selfincompatibility takes place in the ovary of the flower during fertilization, which is
describes as a rejection by the egg nucleus of a male gamete carrying the same
incompatibility alleles as the female gamete. Bartley & Cope (1973) showed that
a series of alleles were involved of which one had a neutral effect, in which case
the rejection mechanism did not function in the egg cells when the gametes are
identical.
The first classification of variety of cacao was proposed by Chessman
(1944), and it was according to morpho geographical groups designated such
as Criollo, Forastero and Trinitario. According to the geographic origin of the
clones, the Forastero group is subdivided into the lower Forastero and upper
Forastero type. The lower Forastero type was first cultivated in the Amazon
basin and the first to be introduced to Africa, also. The upper Forastero type is
highly diverse and often used in plant breeding (Laurent et al, 1994). The
Forastero group has a green pod when immature, turning yellow at maturity.
They are smooth, melon-shaped with rounded ends and relatively thick walled.
Forastero group are hardier, robust, highly productive and resistance to
diseases. The seed colors are purple due to low Theobromine concentration.
The Criollo group, found from Mexico to Colombia and Venezuela, has
narrow genetic bases (Motamayor, et al. 2002). The Criollo group has a yellow
or a red pod when ripe, usually deeply ten-furrowed and warty with long pointed
end. Enclosed within the thin shell is numerous seed. Criollo group has been
characterized by low productivity, susceptibility to diseases, but pleasant aroma
and white cotyledons as consequence of high Theobromine concentration.
According to Bartley (2005), Forastero and Criollo are words derived from
Spanish. Criollo is translated as “Native” and the word was used to defined to
the first variety cultivated in a geographical unit outside to indigenous range of
species to distinguish it from later introduction. In Nicaragua has reported two
kind of cacao Criollo, Dias (2001) reported the Nicaraguan Criollo, which were
referred as the group of Criollo cacao from Nicaragua, and not to native cacao
of Nicaragua. Bartley (2006) cited to Hart (1893), who described two kind of
8
genotype from Nicaragua, which one of them was called “Alligator cacao” due to
husk characteristics, the another one was called “Nicaraguan Criollo”.
1.3 Importance of production and use of Theobroma cacao L.
According to Lass (2004), cacao cultivation has played an important role in the
transformations of lowland tropical forest landscapes in Latin America, Africa
and Asia over the past centuries and continues to do so today. Cacao is now
grown in some 50 tropical countries, which the most important production take
place in Cameroon (179,239 ton in 2007), Brazil (201,651 ton in 2007), Côte d’
Ivory (1,384,000 ton in 2007), Indonesia (740,006 ton in 2007), Ecuador and
Nigeria (85,891 and 500.000 in 2007). The average per hectare varied between
398.3 kg ha-1 and 800.9 kg ha-1, which Nigeria presented the highest yield ha-1
than others countries.
Currently, in Central America, cacao is produced in Guatemala, Costa Rica,
Honduras and Nicaragua. In the 1990s, Nicaragua had 6,500 hectares
cultivated. However, the Caribbean coast presents larger potential for cacao
cultivars, with its almost 350.000 hectares available to cacao cultivations (INTA,
2005). The yield average is ranged between 100.0 kg ha-1 to 276.1 kg ha-1,
which Nicaragua presented the highest value. However, Costa Rica has more
area harvested in 2007, with 4543 ha (FAOSTAT, 2009). The demand for
chocolate is growing and the question now is whether the world supply of a
product that comes from a strictly tropical, rainforest-inhabiting tree will continue
to meet the demand.
In Nicaragua, the cacao crop is one of the most important crops in the
national politics of agriculture, because the country has a big potential and the
international demands are huge, and it is possible that the national production
does not satisfy the national demand. Therefore the government is promoting
the increase of areas where cacao is grown. This should offer good incomes to
poor farmers and avoiding deforestation in critical areas.
The National Institute of Agriculture and Technology (Instituto Nicaraguense
de Tecnologia Agropecuaria –INTA- in Spanish) has been developing a cacao
breeding program through traditional methods, which is a slow and long
process. The Experimental Center “El Recreo”, situated 285 km from the capital
is part of the INTA. This center has been dedicated to the breeding of cacao
9
tree. There are 180 clones introduced in the 1970s and 1980s from Colombia,
Costa Rica and Mexico.
In addition, in 2003, the INTA began a national collection of cacao Criollo,
including material from the Caribbean and Pacific coast. The principal objective
has been to include them in the breeding program of cacao beans. Currently,
those samples are growing in the Experimental center “El Recreo”.
“El Recreo” has been doing conventional breeding between Trinitario
cultivars; however, the material does not have molecular support for their
genetic
characteristics,
as
those
materials
focuses
on
phenotype
characteristics. This method has resulted in a lot of variability in the new
materials. However, with the use of modern techniques it is possible to improve
some traits, which should be associated such as diseases and poor and the low
quality of the yield. Using molecular markers it should be possible to select
material from the national collection of cacao for diseases resistance and quality
of seed or for breeding before the phenotypic expression. For example, by
identifying a molecular marker associated with pod rot, one could identify
materials for breeding without having to wait until the disease appears in the
fruits. Such measures should greatly reduce the cycle time for the selection of
cacao genotypes.
Currently, breeding programs in cacao are based on crosses between
parents belonging to different groups. However, the classic breeding requires a
long generation time. A more modern technique using genetic markers may be
particularly helpful when used to improve breeding strategies and increase our
knowledge of the genetically important agronomic and quality traits (Lanaud et
al, 1995).
2. Objectives
The present study has the aims to investigate the genetic diversity of the
farmers accessions of Theobroma cacao L. in order to reveal the genetic
relationship among 60 farmers accession and to discover genotypes for
breeding programs.
10
3. Materials and methods
3.1 Plant materials
A total of 60 accessions collected from Nicaraguan’s farms, 14 breeders
accession, and 2 samples from Ecuador, those samples are available in the
national collections of the Experimental Center “El Recreo”, located at 120 07'
North, 820 24' W in Autonomous Region of the Southern Atlantic (“Región
Autónoma del Atlántico Sur –RAAS -” in Spanish), Nicaragua.
Also, two
samples considered as Criollo from CATIE germplasm were included in this
study.
Farmers accessions: The accessions were collected from different farmers
(see figure 1) by National Institute of Agriculture and Technology (Instituto
Nicaraguense de Tecnologia Agropecuaria –INTA- in Spanish). Those sampling
(table 1), it was conducted between 2003 to 2008 in farms around the country
(see figure1). During those collections were considered the parameters related
to cacao Criollo as a trunk with a single plagiotropic branch, leaf with pale green
flush, flowers with white staminodes, an elongate fruits (Pod) with long acute or
attenuate, rough and a ridged surface with a thin or soft husk, and large size
seeds with white cotyledons (Figure 2).
International accessions: In order to use samples with knowing genetic
structure, two samples of international germplasm from CATIE, Costa Rica
were used. Criollo 13 was collected in Panama and Yucatan in Mexico, both
samples considered as pure Criollo due to morphologic characteristic. The
samples of Ecuador were introduced in 2003 and were established in
Experimental Center El Recreo.
Breeders samples: In 1978, INTA introduced a total of 189 clones from
Costa Rica, South America, Africa, Jamaica, and Indonesia. Those clones were
evaluated during 25 years, under tropical rain condition. In 1998, INTA selected
clones with a high yield and tolerance to specific diseases such as frosty pod
and black pod. The clones selected were used as parents in the breeding
program. A total of 14 hybrids (Table 2) were produced and distributed widely
throughout the whole country.
11
Figure 1: Map of Nicaragua with sites of farmer’s collections: 1. RAAS (Región
Autónoma del Atlántico Sur), 2. RAAN (Región Autónoma del Atlántico Norte),
3. RSJ (Rio San Juan), 4. MAT (Matagalpa), 5. Pacifico sur (5.a Masaya, 5.b
Granada and 5.c Rivas), 6. Chi (Chinandega)
12
Figure 2: Characteristics of farmers accessions collected on Nicaraguan farm.
a) Tree habit on farm. b) Pale green flush. c) Long and thin leaf. d) White flower.
e) Fruit shape. f) Long and white seed.
13
Table 1: Description of Nicaraguan farmers accessions. (See Figure 1 for the
location in the map)
Name
RAAS 0401
RAAS 0402
RAAS 0403
RAAS 0404
RAAS 0405
RAAS 0406
RAAS0407
RAAS0408
RAAS 0409
RAAS 0410
RAAS 0411
RAAS 0412
RAAS0413
RAAS 0414
RAAN 0401
RAAN 0403
RAAN 0404
RSJ 0401
RSJ 0402
RSJ 0403
RSJ 0404
RSJ 0405
RSJ 0406
RSJ 0504
RSJ 0501
RSJ 0503
RSJ 0508
MAT 0401
MAT 0402
MAT 0404
Location
Region Autonoma Atlantico Sur
Region Autonoma Atlantico Sur
Region Autonoma Atlantico Sur
Region Autonoma Atlantico Sur
Region Autonoma Atlantico Sur
Region Autonoma Atlantico Sur
Region Autonoma Atlantico Sur
Region Autonoma Atlantico Sur
Region Autonoma Atlantico Sur
Region Autonoma Atlantico Sur
Region Autonoma Atlantico Sur
Region Autonoma Atlantico Sur
Region Autonoma Atlantico Norte
Region Autonoma Atlantico Norte
Region Autonoma Atlantico Norte
Region Autonoma Atlantico Norte
Region Autonoma Atlantico Norte
Rio San Juan
Rio San Juan
Rio San Juan
Rio San Juan
Rio San Juan
Rio San Juan
Rio San Juan
Rio San Juan
Rio San Juan
Rio San Juan
Matagalpa
Matagalpa
Matagalpa
Group
RAAS
RAAS
RAAS
RAAS
RAAS
RAAS
RAAS
RAAS
RAAS
RAAS
RAAS
RAAS
RAAN
RAAN
RAAN
RAAN
RAAN
RSJ
RSJ
RSJ
RSJ
RSJ
RSJ
RSJ
RSJ
RSJ
RSJ
Matagalpa
Matagalpa
Matagalpa
*RAAS (Región Autonoma del Atlántico Sur), 2. RAAN (Region Autonoma del
Atlántico Norte), 3. RSJ (Rio San Juan), 4.MAT (Matagalpa), 5. Pacifico sur
(Masaya, Granada and Rivas), 6 Chi (Chinandega).
14
Tabla 1: continuation...
Name
MAS 0401
MAS 0402
PACAYITA 0108
SJC 0108
CATARINA 0108
MENIER 0108
EC 0108
MOMBACHO 0108
MOMBACHO 0208
MOMBACHO 0308
RIV 0401
RIV 0405
H-1
H-2
H-3
H-4
H-6
H-5
CHI 0306
CHI 0308
CHI 0304
CHI 0301A
CHI 0407
CHI 0409
CHI 0410
CHI 0411
CHI 0412
CHI 0414
CHI 0415
CHI 0405
Location
Masaya
Masaya
Masaya
Masaya
Masaya
Granada
Granada
Granada
Granada
Granada
Rivas
Rivas
Granada
Granada
Granada
Granada
Granada
Granada
Chinandega
Chinandega
Chinandega
Chinandega
Chinandega
Chinandega
Chinandega
Chinandega
Chinandega
Chinandega
Chinandega
Chinandega
Group
Pacifico sur
Pacifico sur
Pacifico sur
Pacifico sur
Pacifico sur
Pacifico sur
Pacifico sur
Pacifico sur
Pacifico sur
Pacifico sur
Pacifico sur
Pacifico sur
Pacifico sur
Pacifico sur
Pacifico sur
Pacifico sur
Pacifico sur
Pacifico sur
Chi
Chi
Chi
Chi
Chi
Chi
Chi
Chi
Chi
Chi
Chi
Chi
* 5. Pacifico sur (Masaya, Granada and Rivas), 6 Chi (Chinandega).
15
Table 2: Description of breeders accessions and international accessions of
Theobroma cacao L.
Name
Type
ER-012
Ecuador
Criollo 13
Criollo
locatio
n
El
Recreo
El
Recreo
El
Recreo
El
Recreo
El
Recreo
El
Recreo
El
Recreo
El
Recreo
El
Recreo
El
Recreo
El
Recreo
El
Recreo
El
Recreo
El
Recreo
El
Recreo
El
Recreo
Catie*
UF 650 x
POUND 12
UF 667 x Pound
12
EET 400 x Pound
12
UF 296 x Pound
12
EET 399 x SCA
12
IMC 67 x UF 613
Trinitario (Criollo x
Forastero)
Trinitario (Criollo x
Forastero)
Hybrid (Forastero)
Yucatan
Criollo
Catie
Hybrid x Forastero
Hybrid x Forastero
Forastero x Trinitario
UF 613 x Pound
12
UF 668 x UF 613
Trinitario x Forastero
UF 667 x SCA 6
Trinitario (Criollo x
Forastero)
Trinitario (Criollo x
Forastero)
Hybrid x Forastero
UF 29 x Pound 12
EET 95 x SCA 6
IMC 67 x UF 12
UF 654 x Pound
12
UF 676 x Pound
12
ER-011
Criollo x Trinitario
Trinitario (Criollo x
Forastero)
Trinitario (Criollo x
Forastero)
Hybrid x Forastero
Ecuador
Group
Breeders accession
Breeders accession
Breeders accession
Breeders accession
Breeders accession
Breeders accession
Breeders accession
Breeders accession
Breeders accession
Breeders accession
Breeders accession
Breeders accession
Breeders accession
Breeders accession
international
Accession
international
Accession
international
Accession
international
Accession
* Sample provided by CATIE – Centro Agronómico tropical de investigación y
enseñanza, Costa Rica-
16
3.2 DNA extraction
Dried and fresh young plant leaves were used for DNA extractions following the
standard protocol for DNA extraction described by Doyle and Doyle (1990). A 1
cm2 piece of leaf was placed into a 1.5 ml Eppendorf tube and 500 µl of the
CTAB buffer (2% CTAB, 1.4 mM Tris pH 8, 1 % PVP, 0.2 % 2- βmercaptoethanol added before use) and small amount of sterilized sand were
added, after which grinding was conducted with a plastic Kontle Pellet Pestle.
After were incubated at 65 0C for 30 minutes. Then added 500 µl of CIA 24:1
(Chloroform: Isoamylalcohol) and mixed slowly, then was centrifuged at 14,800
g for 5 minutes. After removing supernatant into new 1.5 ml Eppendorf tube and
500 CIA 24:1 was added, and again centrifuged at 14,800 g for 5 minutes. Then
supernatant was removed into new 1.5 ml Eppendorf tube. To precipitate the
DNA, 2/3 of final volume of cold isopropanol was added and it was kept at -20
º
C for 10 minutes. After centrifuging at 14,800 g for 5 minutes was decanted
liquid without disturbed and the pellet was washed added 100 µl of 70 %
ethanol and centrifuged for 5 minutes at 14,800 g for 2 minutes. Then the pellet
was dried. The pellet was re-suspended by adding 50 µl of low salt TE (100 mM
Tris-Cl and 1 mM EDTA pH 8). After was added 0.3 µl of RNAse (100mg/ml),
and then was incubated at 37 ºC for 1 hour. The DNA was electrophoresed on
0.8 % agarose gel, stained with ethidium bromide and visualized under UV
trans-illuminator for quality DNA assessment. DNA quality was estimated from
the clearness of the band, and the amount of DNA was estimate from bands
intensity in comparison with GeneRuler marker (50 bp DNA leader).
3.3 Microsatellite Analysis
DNA polymorphisms were detected by polymerase chain reaction (PCR), using
9 microsatellites markers (Table 3 describes microsatellites used in this study
developed for cacao by Lanaud, 1999).
PCR reaction was performed in a
volume of 20 µl at testing state and 10 µl in the final analysis. The 10 µl reaction
mixture contained 0.5 µl DNA (2-3 ng of total genomic DNA per 1 µl of reaction
volume) as a template, 6 µl of MQ water, 1 µl 10x reaction buffer, 0.2 µl dNTP
mix, 0.3 µl (0.6 U) DynazymeTM II DNA Polymerase, 1 µl (5mM) forward primer
17
and 1 µl (5mM) reverse primer (the forward primers were fluorescently labeled
at 5’ end).
The DNA amplification were performed in a thermocycler (Bio-Rad,
C1000TM Thermal Cycler), with the following programmed; a denaturation step
of 4 minutes at 94 ºC followed by 34 cycles followed by denaturation for 40
second at 94 ºC, annealing for 40 second at 46 ºC or 51 ºC depending on
microsatellite, extension at 72 ºC for 1 minute, and a final extension at 72 ºC for
8 minutes.
3.4 Capillary electrophoresis and data collection
The PCR product was diluted 1/19, using 1µl of PCR product and 19 µl of Mille
Q water. From this solution was used 0.5 µl of each template and mixed with 20
µl of HiDi-formamide and 0.15 µl of size standard (GeneScan 500 ROX).
Before the run the samples were briefly denatured (5 min, 95 0C). Finally the
samples were loaded with Applied Biosystems 3730 DNA Analyzer into 96-well
plates. Fragment sizes were determined by Peak Scanner™ Software v1.0.
18
Table 3: Description of the microsatellite loci developed by Lanaud et al. (1999),
with primer sequences used. The fluorescent labels used in this study are
shown.
SSR
EMBL
Accession
Linkage
group
MtcCIR1
Y16883
8
MtcCIR7
Y16981
7
MtcCIR8
Y16982
9
MtcCIR
10
Y16984
5
MtcCIR
11
Y16985
2
MtcCIR
12
Y16986
4
MtcCIR
15
Y16988
1
MtcCIR
18
Y16991
4
MtcCIR
21
Y16995
3
5’
Forward
primer
GCAGGG
CAGGCT
CAGTGA
AGCA
ATGCGA
ATGACAA
CTGGT
CTAGTTT
CCCATTT
ACCA
3’
Reverse
primer
Annealing
0
Temp C
Lab
el
Repeat
HEX
Size of
cloned
allele (bp)
143
TGGGCAAC
CAGAAAACG
AT
51
GCTTTCAGT
CCTTTGCTT
51
HEX
160
(GA)11
TCCTCAGCA
TTTTCTTTC
46
TET
301
CCCAAGCAA
GCCTCATAC
TC
46
FAM
208
(TC)5T
T(CT)1
7TTT(C
T)4
(TG)13
CCGAATT
GACAGA
TGGCCT
A
CATTGC
GGATTAC
GGTTTTT
TTTCTGA
CCCCAA
ACCTGTA
A
CAGCCG
CCTCTTG
TTAG
GCTAAG
GGGATT
GAGGAA
GC
GTCGTT
GTTGATG
TCGGT
TGATTAAGC
ACACGAGCA
CTG
TTCCAGTTA
AAGCACATG
AGGA
46
FAM
298
(TC)13
46
FAM
188
(CATA)
4N18(T
G)6
TATTTGGGA
TTCTTGATG
46
TET
254
(TC)19
TGGGTTGCA
GTCAATGTC
TC
46
FAM
345
(GA)12
GGTGAGTGT
GTGTGTTTG
TCT
46
TET
157
(TC)11
N5(CA)
12
(CT)14
3.5 Data Analysis
3.5.1 Genetic diversity
For farmers selection, the following genetic diversity parameters were estimated
using POPGENE software v 3.2. (Yeh and Boyle, 1997)
1. Number of allele per locus. It was accounted for each amplified locus.
Effective number of allele. It was estimated with GENALEX v.6.2 software
program.
2. The mean of observed and expected heterozygosity (HObs and HExp). The
Heterozygosity was calculated at each locus using the formula proposed by
Nei, 1987.
For observed heterozygosity:
HObs= 1- ∑Pij,
(1)
19
Where Pij= ∑wiPij at each locus, which wi is a relative size of sub population and
∑wi=1 and Pij is a genotype frequency
For expected heterozygosity:
HExp =1-∑p2j
(2)
Where p2j= ∑wip2ij, Pij is the frequency of the jth allele at ith locus summed at
across all alleles of the locus.
The polymorphism information content value is a value used to measure the
polymorphism for a marker locus (Botstein et al. 1980). It depends only on the
number of alleles and the frequency of each allele at the marker locus (Guo and
Elston, 1998).
The PIC estimator was calculated for each locus using the formula proposed by
Anderson et al., 1993:
PICi= 1-∑p2ij
(3)
Where: pij is the frequency of the jth microsatellite allele.
The Shannon index is once the most commonly measurement used to compare
diversity among individuals. It has been widely used in studies as ecological
diversity and diversity of social relationship among adult primates and female
primates (Di Bitetti, 2000). The Shannon diversity index is tipically used to
measure the number of species in a community (the richness) and the relative
frequency of these species.
The Shannon index was calculated for each locus and groups using
software Arlequin ver. 3.1. (Excoffier et al. 2005)
3.5.2 The genetic differentiation
Genetic differentiation was quantified by the analysis of molecular variance
(AMOVA) and F-statistics estimator (FST), as described by Weir and Cockerha
(1984), Excoffier et al. (1992) and Weir (1996) using software Arlequin ver. 3.1.
(Excoffier et al. 2005)
20
3.5.3 Estimation of Genetic Distance
The genetic distance is estimation between two entities that can be described
by allelic variation (Nei´s, 1973).
The genetic identity (Di) and genetic distance (D) between groups were
calculated based on Nei´s (1987) unbiased genetic distance.
According to Nei´s (1978), the genetic distance is defined as:
D= -ln [GXY/√GXGY]
(4)
Where: GX, GY and GXY are the means of ∑pi2, ∑qj2 and ∑piqi over all loci in the
genome, respectively.
For genetic identity, GX, GY and GXY, by sample gene identities, JX, JY, and J XY,
which are the means of ∑xi2, ∑yi2, and ∑xiyi over r loci studied. However, from D
was obtained by substitution the unbiased estimates of GX, GY for J X and JY:
Ď= -ln[ĞXY/√ĞXĞY]
(5)
Where: ĞX and ĞY are the means of (2nXJ X-1)/ (2nX-1) and (2nYJ Y-1)/ (2nY-1)
over r loci studied, respectively, and ĞXY = JXY.
Nei (1978), suggested the used of unbiased when D bias has a large
magnitude, because bias is negligible. The unbiased estimator of Di and D were
obtained using POPGEN software.
3.5.4 Cluster analysis
From the original microsatellite data, a bootstrap values were computed over
1,000 replications with Seqboot program. After, the Mix program was used to
cluster the accessions according to the Wagner parsimony method (Eck &
Dayhoff, 1996; Kluge & Farris, 1969) for discrete character data. Then, the
Consense program was used to find the consensus tree applying the extended
majority rule. The Seqboot, Mix, and Consense program are from Phylip version
3.68 (Felsentein, 2004). The tree was drawn using the Treeview software
version 1.6.6 (Page, 1996).
21
3.5.5 The Principal component analysis
In order to know the relationship between different individuals was carried out
the Principal Component Analysis (PCA), using GENALEX v.6.2 software
program.
The PCA allows identifying differences among the individuals and identifying
possible group. PCA derives a 2 or 3-dimensional scatter plot of individuals,
making geometrical distances among individuals in the plot reflect the genetic
distances among them with minimal distortion (Mohammadi & Prasanna, 2003).
This analysis used a variance-covariance matrix, considering absolute changes
between individuals. The farmers accessions were compared again breeders
materials and internationals accessions in order to know the relationship
between them.
4. Results
4.1 Genetic diversity of Nicaraguan farmers accessions
A total of 155 alleles were identified in the 60 farmer’s accessions with a mean
of 17.2 alleles per locus (Table 4). The microsatellites MtcCIR8 detected high
polymorphism with 22 alleles per locus. Moreover, the lowest detection was
identified by MtcCIR1 with 10 alleles per locus. (See appendix 1 for allele
frequency).
The effective number of alleles per locus had a mean of 3.7. However, the
highest effective number of allele per locus was found by MtcCIR15 with 6
alleles per locus and most of rest microsatellites were ranged between 2.9 and
4.2 effective number alleles per locus.
Heterozygote deficiency (HExp < HObs) was registered for all microsatellite
used in this study. The lowest observed Heterozygote valued was presented by
MtcCIR7 with 0.28 and the highest value was presented MtcCIR21 with 0.53,
respectively. The highest value for the heterozygote expected was 0.82 for the
microsatellite MtcCIR15 and 0.61 was the lowest value for the microsatellite
MtcCIR10.
22
The PIC values presented were generally high for all loci (PIC > 0.5). PIC
values varied between 0.73 and 0.91, with a mean 0.78. The microsatellite
MtcCIR15 shown the highest value with 0.909 and MtcCIR8 presented the
lowest value with 0.73. The Shannon diversity index showed a mean of 1.97.
The microsatellite MtcCIR1 presented lowest value with 1.53 and MtcCIR15 the
highest value with 2.63, respectively.
Table 4: Genetic parameters for microsatellite markers L. based on Farmer’s
accession from Nicaragua using 9 microsatellites : number of alleles per locus
(N), effective number of alleles per locus (Ne), heterozygosity observed (Hobs)
and expected (HExp), PIC (Polymorphism information content), and Shannon
genotypic diversity index, based on Farmer’s accession from Nicaragua.
Microsatellite
N*
Ne*
Hobs
Hexp
PIC
MtcCIR1
10
2.9
0.52
0.62
0.75
MtcCIR7
12
2.9
0.28
0.63
0.75
MtcCIR8
22
3.3
0.45
0.61
0.73
MtcCIR10
18
4.2
0.49
0.64
0.85
MtcCIR11
20
3.4
0.43
0.65
0.75
MtcCIR12
15
2.9
0.54
0.65
0.70
MtcCIR15
20
6.0
0.47
0.82
0.91
MtcCIR18
18
4.2
0.41
0.70
0.81
MtcCIR21
20
3.6
0.53
0.68
0.76
Mean
17.22 3.7 0.47±0.06 0.67±0.08 0.78±0.07
*Ne = Effective number of alleles [Kimura & Crow (1964)]
I*
1.53
1.69
2.03
2.22
1.91
1.54
2.63
2.14
1.97
1.97±0.35
***I = Shannon Index [Lewontin (1972)]
HObs and HExp were computed using Levene (1949)
4.2 Genetic diversity of Nicaraguan farmers accessions at group level
The genetic parameters used were a number of alleles per locus (N), Number of
different alleles per locus (Na), number of effective alleles per locus (Ne), and
heterozygosity observed (Hobs) and expected (HExp), and Shannon information
Index (I) (Table 5). Those parameters were evaluated for farmer accession,
international accession and breeder accessions.
The total means of number alleles per locus in whole groups was 5.7. In
addition, the groups RAAS and Pacifico sur obtained a high number of alleles
per locus with 8.4 and 7.8, respectively. Moreover, Matagalpa and RAAN
presented lowest value for number alleles per locus with 2.8 and 3.4.
23
On other hand, the heterozygote presented a deficit in all groups (HObs >
HExp). The total means of HObs for whole groups studied was 0.46; the highest
values were presented by Chinandega and Rio San Juan with 0.55 and 0.51,
respectively. Pacifico Sur and RAAS presented lowest values with 0.34 and
0.42. The highest value for HExp was 0.79 for RAAS and the lowest 0.48 for
Matagalpa.
Shannon information Index (I) presented variation between 0.82±0.15
(Matagalpa) and 1.81±0.14 (RAAS).
Table 5: Genetic diversity of Theobroma cacao L. L. based on Farmer’s
accession from Nicaragua, international accession and breeders accessions,
using 9 microsatellite: mean number of alleles per locus (Na), Number of
different alleles per locus, number of effective alleles per locus (Ne), and
heterozygosity observed (Hobs) and expected (HExp), and Shannon information
Index (I).
Group
RAAS
RAAN
Rio San Juan
Pacifico sur
Chinandega
Matagalpa
Mean for Famer
accessions
Group
HObs
0.42
0.44
0.51
0.34
0.55
0.48
0.46±0.17
HExp
0.79
0.65
0.69
0.76
0.64
0.48
0.67±0.08
I
1.81
1.14
1.47
1.66
1.32
0.82
1.37±0.12
International
accessions
Breeder accessions
Mean
0.26±0.06
0.75±0.1
1.26±0.17
0.66±0.08
0.46±0.07
0.78±0.32
0.77±0.21
1.66±0.12
1.46±0.15
The Analysis of molecular variance (AMOVA) showed that 6.1 % of genetic
variation was due to variance among groups, whereas 93.9 % was divided
within groups which show statistic different in AMOVA for both components
among groups and within group (Table 6). The mean FST presented for all group
was 0.06.
24
Table 6: Component of Analysis of molecular variance of Theobroma cacao L.
L. based on Farmer’s accession from Nicaragua using 9 microsatellites
Source of
variation
Among groups
Within groups
Total
Mean FST
d.f
5
114
119
Sum of
squares
36.76
376.77
413.53
Variance
components
0.22 Va
3.31 Vb
3.52
0.06
Percentage of
variation
6.1
93.9
The pair comparison between groups (Table 7), shown that group RAAS has
not presented statistic difference between Matagalpa groups, but has statistic
significance with rest groups. RAAN has only statistic difference with
Chinandega and Matagalpa, Rio San Juan has only statistic difference with
Chinandega.
Table 7: Pairwise (FST) comparisons of Theobroma cacao L. for groups of
farmers accession from Nicaragua using 9 microsatellites
RAAS
RAAN
Rio San
Juan
Pacifico sur
Chinandega
Matagalpa
RAAS
RAAN
Rio San
Juan
0
0.10490*
0.06058*
0
0.09236
0
0.03716*
0.08957*
0.07369
0.09309
0.14122*
0.20582*
0.02645
0.04623*
0.07564
Pacifico
Sur
0
0.04606*
0.07711
Chinandega
Matagalpa
0
0.08247
0
*significant at P< 0.05
4.3 Genetic distances
Nei’s distances and genetic identity matrices are shown in table 8. Those
results were based on analysis of six groups including all individuals. The
ranged presented for Nei’s distances was between 0.1374 for group
Chinandega and 0.9096 for Matagalpa. The genetic distance between RAAN
and Matagalpa was the highest, and Rio San Juan with Pacifico Sur was the
lowest.
Genetic identity values varied between 0.4027 and 0.8716. Pacifico Sur and
Rio San Juan obtained the highest value and Matagalpa with RAAN the lowest
value, respectively.
25
Table 8: The Genetic identity and genetic distance of Theobroma cacao L.
based on Farmer’s accession from Nicaragua using 9 microsatellites
RAAS
RAAN
Rio San
Juan
Pacifico sur
Chinandega
Matagalpa
RAAS
RAAN
Pacifico Sur
Chinandega
Matagalpa
0.4438
****
0.4999
Rio San
Juan
0.7438
0.6066
****
****
0.8124
0.2960
0.7992
0.5631
0.8716
0.7002
0.5330
0.8464
0.7437
0.4027
0.7624
0.2242
0.3564
0.2961
0.5744
0.6293
0.9096
0.1374
0.1667
0.2713
****
0.1620
0.2800
0.8505
****
0.2580
0.7558
0.7726
****
*Nei's genetic identity (above diagonal) and genetic distance (below diagonal).
4.4 Cluster analysis
The cluster analysis classified whole accessions into 9 groups and three distinct
individuals. The distinct individuals were RAAS0405, RAAS0401, and
RAAN0404. The first group was divided into two subgroups and one distinct
individual. The first subgroup was formed for two hybrids (UF676xP12 and
UF29xP12), the second subgroup were included three individuals (ER012,
Chi0304, Chi0411). A hybrid (IMC67xUF613) was clustering as distinct
individual into this group. The second group was formed by four individuals
(RAAS0409, Chi0407, Chi0308, and RAAN0401); all of them presented a
strong clustering relationship (96%)
The third group was formed for one hybrid (UF668xUF613) and one farmer
accession (RSJ0404). In the fourth group were included two hybrids
(UF654xP12 and EET95xSCA6) and two farmers accession (RAAS0407 and
RSJ0503); they were exhibited a strong clustering relationship. The fifth group
present bootstrap values below 80%. However, this group was divided into two
subgroups. The first subgroup presented a strong relationship and was formed
for 5 individuals Pacayita0108, Mombacho0108, UF650x P12, Chi0410 and
Chi0301a), the second subgroup presented a weak clustering relationship
(below 80%).
The sixth group presented a strong relationship (100%) and was formed
from three individuals (RAAS0408, RAAS0404, and Chi0415). The seventh
group presented a weak clustering relationship value (below 80%). However,
into this subgroup was presented a strong bootstrap value. The eighth group
was formed for 25 individuals with a strong clustering relationship (100 %). In
this group was included the CRIOLLO 13, which presented a strong clustering
relationship with Mat0404. (Figure 3)
26
100
99
99
100
100
96
100
100
100
100
100
100
100
100
96
100
100
94
100
100
100
98
100
99
99
100
82
98
95
100
100
95
100
93
100
96
100
100
100
100
100
100
96
100
100
100
100
100
100
92
100
100
93
100
100
100
100
R aas0405
R aas0401
R aan0404
Imc67xUf613
U f676xP12
U f29xP12
Er 012
C hi0304
C hi0411
R aas0409
C hi0407
C hi0308
R aan0401
U f668xUf613
R sj0404
R sj0503
R aas0407
U f654xP12
Eet95xSca6
R aas0403
C hi0301a
C hi0410
U f650xP12
Mombacho0108
Pacayita0108
R sj0504
U f613xP12
Eet00xP12
C hi0412
C hi0306
R aas0408
R aas0404
C hi0415
R sj0406
C hI0414
U f667xP12
R aas0413
Yucatan
H6
C hi0409
H1
H2
H4
C hi0405
Mat0402
U f296xP12
R iv0405
Sjc0108
Imc67XUf613
Mombacho0208
Ec0108
C atarina0108
Mas0402
R aas0410
Mombacho0308
Eet399xSca12
R aas0414
R iv0401
U f667xSca6
R aan0403
R sj0401
R aas0402
R aas0406
H3
H5
Mas0401
R sj0508
R sj0402
R aas0411
Mat0404
C riollo13
R aas0412
Er 011
Menier0108
R sj0501
R sj0405
R sj0403
Mat0401
1
2
3
4
5
6
7
8
Figure 3: Cluster analysis bootstrapped over 1,000 replicates, based on the
genetic distances between 60 farmer accessions from Nicaragua, 4
internationals accessions and 14 improve samples of Theobroma cacao.
Bootstrap values above 80 % were marked.
27
4.5 Principal component analysis
The principal component analysis distributed all individuals in four different axes
(Figure 4). The axis one was found 24 individuals. In this axis was included
only three improved individuals, which were dominated by the cross Forastero x
Trinitario. Also, three pairs individuals were found with equally genetic alleles:
MAT0402 with CHI0301A, RSJ0504 with RSJ0404, and the last pair were
RAAN0404 with RAAS0409.
A total of 17 individuals were found in axis 2. The Criollo 13 and Yucatan
were included in this axis. In addition, three samples were found related to
Criollo 13 such as SJC0108, Catarina0108 and MAS0402. Also, Yucatan was
related
with
RAAS0406,
RAAS0410.
RAAS0414,
and
Menier0108.
Furthermore, RAAN0403 was found related with ER-012 (Ecuador). In addition,
most materials found in this axis were homozygote.
In the Axis three were found 25 individuals. Moreover, the majority of
improved materials were found in this axis. In additions, two pairs of individuals
were found close related with RAAS0403 with UF296xP12, and RAAS0401 with
EET95xSCA6. The rests of individuals (12) were distributed in axis 4. Further,
H5 and H6 had the same genetic structure.
28
Principal Coordinates
RIV0401
CHI0308
SJC0108
MAS0402
RSJ0403
Catarina0108
Criollo13
Pacayita0108
RSJ0503
RAAS0413
H2
CHI0301A
RSJ0402
RAAS0411
RAAS0410
MAT0401
MAT0402
Yucatan
RAAS0406
RAAN0401
Menier0108
CHI0415
CHI0410
RSJ0504
RSJ0404
ER-012
RSJ0401
Mombacho0308
RAAN0403
RAAS0414
Coord. 2
RAAN0404
MAT0404
RAAS0412
Pop1
CHI0409
RSJ0501
IMC67xUF613
UF613xP12
RAAS0409
UF29xP12
UF668XUF613
Pop3
Mombacho0108
RSJ0508
CHI0306
H5
EET399xSCA12
Pop2
H6
RAAS0408
Pop4
Pop5
Pop6
CHI0304
RAAS0404
RSJ0405 RSJ0406
H3
H4
EC0108
ER-011
UF667xSCA6
UF654xP12
CHI0412
UF676xP12
RAAS0402
RAAS0407
Mombacho0208
UF650xP12
IMC67xP12
MAS0401
UF296xP12
EET95xSCA6
Pop7
Pop8
CHI0405
CHI0411
RAAS0403 RAAS0401
UF667xP12
EET400xP12
RAAS0405
RIV0405
H1
CHI0407
CHI0414
Coord. 1
Figure 4: Principal component Analysis (PCA) plot for 78 individuals using 9
microsatellites, based on the genetic distances between 60 farmer accessions
from Nicaragua (Pop1= RAAS, Pop2 = RAAN, Pop3 = RSJ, Pop4 = Pacifico
Sur, Pop5 = Chi, Pop7 = Mat), 4 internationals accessions (Pop6) and 14
breeders accessions (Pop7) of Theobroma cacao.
29
5. Discussion
In the present study were used microsatellites genotyping to examine the
genetic diversity, population structure, and the relationship between farmers
accession, breeders accession and internationals accessions.
5.1 Genetic diversity and population structure
Studies of genetic diversity have been carried out using isozymes, RAPD,
AFLP, RFLP, and SSR (microsatellite) markers in Theobroma cacao L. due to
the importance of this crop in chocolate industry. AFLP has been used to
distinguish cultivars and create a linkage map in cacao (Davey et al. 1998;
Risterucci et al. 2000). In previous studies, Motamayor et al. (2002) and
Risterucci et al. (2000) have shown that microsatellites are more efficient than
RFLP in
genetic diversity study for
Theobroma cacao L., because
microsatellites have allowed detected more alleles per locus as well as higher
level of polymorphism and are equally powerful tools for estimation of
heterozygosity.
Microsatellites have been widely used to study genetic diversity in
Theobroma cacao (Zhang et al. 2009; Cryer et al. 2006; Efombagn et al. 2006;
Sereno et al. 2006; Takrama et al. 2005; Fossati et al. 2005; Saunders et al.
2004; Motamayor et al. 2003; Motamayor et al. 2002; Marita et al. 2001;). The
main purpose of those analyses has been to identify differences between Criollo
groups and Forastero groups.
The polymorphic information content (PIC) was used to measure the ability
of a marker to detect polymorphism in cacao samples of Nicaraguan farmers
accession. In the present study, the PIC value showed a high power of
discrimination (PIC>0.78), of the loci used in Nicaraguan famers accessions,
which is an indicator of a high genetic diversity for nine loci studied. This
corroborated the use of these microsatellite loci in the estimation of genetic
diversity among each famer accession collected in Nicaragua. This result has
similarity with the result found by Zhang et al. 2006 and Efombagn et al. 2006
(0.58 and 0.69, respectively).
In this study, the overall allelic richness per locus presented was (17.2
overall allelic per locus and 3.7 effective allelic per locus). The level of
30
polymorphism detected in the present study would be affected by the mutation
rate of microsatellite and the small size population in the present studied.
According to Jin et al. (1996), Thuillet et al. (2002), and Wilkinson (2000),
mutation rate of microsatellite can be originated by replication slippage, which
can create high similarity in allele’s sizes and differences between few repeat
units.
The allelic diversity (relatively low Ne= 3.7), presented in this study was
similar to results presented by Smulder et al. (2009), that reported a low allelic
diversity (Ne= 3.54) for cacao populations from South America, and by Zhang et
al (2006) with an allelic diversity of 3.68 for semi natural population from
Huallaga, Peru, while Alves et al. 2007 presented an average 3.37 of alleles per
locus for a natural population of Theobroma grandiflorun. Previous studies, in
Theobroma cacao, using microsatellite found averages of 5.7 allelic per locus
for managed populations (Zhang et al. 2006). Studies realized separately in
Brazil and French Guiana presented averages of 4.45 and 4.48, respectively.
Those values were considered moderately high for richness allelic (Sereno et
al. 2006; Lachenaud & Zhang, 2008). In additions, low allelic diversity could be
explain by the fact that cacao is an endemic plant from Central America or
genetic drift, which would have reduced allelic diversity or allelic loss in that
accessions.
The heterozygote deficit (HExp<HObs) showed an inbreeding in all Nicaraguan
famers’ accessions analyzed. The results presented in study coincided with
results presented in previous studies by Zhang et al. (2009); Lachenaud &
Zhang, (2008); Cryer et al. (2006); Efombagn et al. (2006); Sereno et al. (2006);
Takrama et al. (2005); Fossati et al. (2005); Saunders et al. 2004; Motamayor et
al. 2003; Motamayor et al. 2002; Marita et al. (2001). The inbreeding could be
related to the biology of the reproductive system in cacao plant and the
selection that famers have been managed with cacao plantation through time.
Nonetheless, the inbreeding was unexpected, as cacao is an outcross species,
which it would be assumed an absence of inbreeding, which indicate that most
individuals into Nicaraguan farmers accessions are homozygosis and have selfcompatibility. In that sense, RAAS and Pacifico Sur presented more inbreeding,
compare to places where that have been considered as places where
indigenous tribes have managed and selected the cacao trees since the preColumbian period. In that way, Dias (2002), have supported the idea that
31
Indigenous people started the cacao selection based on the white beans, by
doing so, they indirectly selected self-compatibility, promoting inbreeding.
Breeder accession was expected to present high level of heterozygosis
(HObs= 0.66) as they are product of strong human intervention, Chi and RSJ
groups, also presented relative value with breeder accession (HObs = 0.55 and
0.5, respectively), which was expected the presence of high heterozygosis in
those areas where cacao crops has been promoting with breeding accession by
national government and nongovernmental organization.
The gene diversity (HExp= 0.67) presented in this study was similar to
results presented by Sereno et al. (2006) for population form the Brazilian
Amazon (HExp= 0.5), also Motamayor et al. (2002) presented a similar gene
diversity for modern Criollo (HExp= 0.52). Motamayor et al. (2002) defined as
"Modern Criollo" those samples that have presented the morphological trait that
were described by Chessman (1944), but collected on farms. Farmers
accessions presented less gene diversity than breeders accession, and it was
expected, because Breeder accessions are the product of intensive genetic
recombination in hybridization realized. This result coincides with previous
comparison between farmers selection and breeders accession realized in
Cameroon by Efombang et al. (2006), also, Laurent et al (1993) found a high
level of polymorphism among Criollo group and low level in Forastero group.
The study found that RAAS and Pacifico sur of farmers accessions are more
close to International accessions (Criollo group) than the rest of groups studied
(Table 7), and RSJ and Chi are more related to Breeders materials. Several
studies have coincided that during pre-Columbian time, cacao Criollo was
distributed along the Pacific Coast in Nicaragua (Steinbrenner, 2006; Bartley,
2004; Ciferri & Ciferri, 1957; Bergmann, 1969), which result presented in this
study support previous hypothesis about Criollo distribution.
As cacao
exchange and the human migration until jungle as consequence of the
conquest, indigenous carry out cacao samples, for this reason samples of
RAAS are similar to Pacifico Sur.
Moreover, RAAS and Pacifico Sur presented heterozygosis similar to
international accession (Criollo reference). Moreover, RSJ and Chi groups were
more close to breeders accession, due to the recent promotion of cacao crops
in Nicaragua, which indicate that farmers have obtained breeders materials and
32
this action have allowed the genetic introgression between Criollo group and
breeder accession.
5.2 Groups differentiation and genetic proximities
The analysis of molecular variance (AMOVA), shown that most variation was
found within groups (Table 6) while low variation among groups. This was
expected due to the endemic nature of Thebroma cacao in the region (Central
America and South America). According to Silvertown & Charlesworth (2001),
endemic species have a low variation among group as they have a unique
genetic structure.
Additionally, the pairwise comparison (Table 7) shown that RAAS and
Pacifico Sur have low genetic variability (0.037), this result coincided with
genetic distance between both groups, which was more near among the others
groups. It seems that exchange of materials due to human migration, have
reduced the genetic distances between both group (RAAS and Pacifico Sur).
The Cluster and PCA analyses allowed identify the relationship between
individuals. The cluster analysis showed a strong clustering relationship among
individuals (Figure 3). Four undistinguished farmers accessions were indentified
as well as some stronger genetic relation between farmers accessions and
international accessions (Criollo). This was expected, as researcher such as
Dias (2002), Bartley (2005) and Laurent et al. (1993) have considered the
existence of Criollo sample in Nicaragua. Nonetheless, Motamayor (2002)
considered that most cacao samples in Nicaragua are Trinitario type, as
consequence of outcrossing among Forastero and Criollo. The cluster analysis
found many farmers accession and breeders accession with a strong
relationship.
An important finding in the cluster analysis was the strong relationship
among ER011 from Ecuador and Nicaraguan farmers accession (Menier0108,
Criollo 13, RAAS0412, RSJ0405, RSJ0403, and Mat0401). According to Dias
(2002), the Nacional accession from Ecuador is more genetically related to
Criollo than forasteros, also Bartley (2006) reported that first gene flow of
Theobroma cacao from Nicaragua occurred in 1893, when Harts carried out a
total of 150 plants with Criollo characteristic toward Trinidad. This event could
33
be considered as trigger genetic origin of the relationship between Ecuador's
and Nicaraguan's accession.
In addition, PCA allowed identification of duplicated samples and relation
between Criollo 13 and Yucatan samples (Figure 4). The samples duplicated
were found in Matagalpa and Chinandega (Mat0402 and Chi0301a) and
Pacifico sur (H5 and H6). Also, most Trinitarios group was finding in axis 3,
which were included most breeders accessions that were classified as
Trinitario. In this axis was predominated farmers accession from RAAS.
The PCA also indicated that most samples, genetically related with
Criollo 13 were collected in pacific Sur (SJC0108, Catarina0108 and MAS0402).
Those samples had morphologic characteristic similar to the samples described
by Chessman (1944) and Hart 1893 (in Bartley, 2002) for cacao Criollo.
Additionally, it was found that Nicaraguan farmers accessions (RAAS0406,
RAAS0410. RAAS0414 and Menier0108) were genetically related to Yucatan,
that relationship would indicate that Nicaraguan indigenous was related to
Mexican indigenous, especially indigenous for the groups located in the Pacific
Coast, Nicaragua.
From the PCA included in axis 3 (Figure 4), it can be seen that most
breeders accession (Trinitario), as well as 25% of all farmers accessions were
genetically related to breeder accessions. this result could be caused by the
high promotion of cacao and renovations of old plantations for new breeders
plantations
with specific characteristic as diseases resistance to Black pod
(Phytophthora spp) and Frosty pod (Moniliophthora roreri) and high yield.
34
6. Conclusions
The present study has revealed the existence of a genetic diversity of 60
Nicaraguan farmers accessions based on nine microsatellites. This study
provides a basic knowledge about genetic structure of cacao farmers in
Nicaragua and differences between cacao accessions from different area when
were collected those samples. The values of the genetic parameter obtained in
this study, mainly the values of heterozygosis indicate that Nicaraguan farmers
accessions are inbreed with high genetic diversity. Also, the allelic richness
suggests the presence of potential unique and rare alleles.
The analysis molecular of variance (AMOVA) was highly significant and
showed a low variance among groups but high variance within groups. Based
on this study (cluster and PCA analyses), it has been found that some cacao
farmers accession are genetically related with Criollos genotypes, but most
farmers accessions have Trinitario characteristic as consequence of promoting
improved materials. Also, was found a few farmers accessions and Ecuadorian
accession with the same genetic origin. Those results would suggest a good
possibility for selection and improvement of cacao. Those promising accession
should be used in a breeding program.
In the future, will be necessary collect and increase the collections of
farmers accessions and the molecular data generated will be correlated with
morphologic characterization.
35
7. Acknowledgements
To Finnish Government –specially Finnish Embassy in Nicaragua and Foreigner
Ministry of FinlandTo University of Helsinki –specially Department of Applied BiologyTo NIFAPRO Staff – Jari Valkonen, Helena Korpelainen, Paula Elomaa, Aldo
RojasINTAs’ Authorities and all cacaos Workers -present and not presents- that have
contributed and rescued the knowledge about cacao.
To 71 years of foundation of Experimental Center “EL Recreo” and 30 of cacao
research
To CATIE –Costa Rica- for facilities the Criollo genotypes
36
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9. Appendices
Appendix 1: Allele frequencies of microsatellites loci in groups of Theobroma
cacao L. from Nicaragua
Locus
Allele/n RAAS RAAN RSJ
P.S
Chi
I.A
B.A
Mat
mTcCIR 1
N
14
3
10
18
12
4
14
3
127
0.107 0.333 0.250 0.333 0.250 0.500 0.179 0.000
128
0.357 0.000 0.100 0.083 0.042 0.000 0.107 0.667
131
0.036 0.000 0.000 0.000 0.000 0.000 0.000 0.000
133
0.000 0.000 0.000 0.028 0.000 0.000 0.000 0.000
134
0.000 0.000 0.000 0.000 0.000 0.000 0.071 0.000
135
0.000 0.000 0.000 0.000 0.000 0.125 0.000 0.000
139
0.036 0.000 0.000 0.028 0.000 0.000 0.000 0.000
140
0.107 0.667 0.250 0.111 0.208 0.250 0.179 0.000
141
0.357 0.000 0.400 0.389 0.500 0.125 0.464 0.167
150
0.000 0.000 0.000 0.028 0.000 0.000 0.000 0.167
mTcCIR 7
N
14
3
10
18
12
4
14
3
149
0.000 0.000 0.000 0.000 0.000 0.000 0.036 0.000
150
0.000 0.000 0.000 0.000 0.000 0.250 0.000 0.000
153
0.000 0.000 0.000 0.000 0.000 0.000 0.036 0.000
155
0.071 0.333 0.100 0.167 0.042 0.125 0.036 0.000
156
0.321 0.500 0.700 0.389 0.375 0.000 0.393 0.667
157
0.071 0.000 0.000 0.222 0.250 0.250 0.000 0.333
158
0.286 0.000 0.000 0.000 0.000 0.250 0.000 0.000
159
0.000 0.167 0.050 0.167 0.000 0.000 0.250 0.000
160
0.250 0.000 0.000 0.000 0.125 0.000 0.071 0.000
161
0.000 0.000 0.150 0.028 0.208 0.000 0.107 0.000
162
0.000 0.000 0.000 0.000 0.000 0.000 0.036 0.000
163
0.000 0.000 0.000 0.028 0.000 0.125 0.036 0.000
mTcCIR8
N
14
3
10
17
12
4
14
3
250
0.000 0.000 0.000 0.029 0.000 0.000 0.000 0.000
251
0.107 0.000 0.000 0.000 0.000 0.000 0.000 0.000
257
0.000 0.000 0.000 0.000 0.000 0.250 0.000 0.000
270
0.000 0.167 0.000 0.000 0.000 0.000 0.000 0.000
286
0.071 0.000 0.000 0.000 0.000 0.000 0.000 0.000
288
0.071 0.000 0.000 0.176 0.000 0.000 0.000 0.000
289
0.393 0.333 0.600 0.324 0.708 0.250 0.393 0.833
291
0.107 0.000 0.050 0.029 0.000 0.000 0.214 0.000
292
0.071 0.000 0.050 0.000 0.000 0.000 0.000 0.000
293
0.036 0.000 0.000 0.088 0.000 0.000 0.214 0.000
294
0.000 0.000 0.000 0.000 0.042 0.000 0.000 0.000
295
0.000 0.000 0.050 0.000 0.000 0.000 0.000 0.000
45
Appendix 1: Continue…
Locus
Allele/n
mTcCIR8
N
297
299
300
302
303
304
305
307
308
389
mTcCIR 10
N
194
212
213
214
215
216
217
218
219
220
221
222
225
226
228
229
230
303
mTcCIR11
N
196
197
198
200
202
203
205
209
RAAS
14
0.000
0.000
0.000
0.036
0.000
0.036
0.000
0.071
0.000
0.000
14
0.000
0.000
0.036
0.071
0.071
0.000
0.000
0.393
0.036
0.179
0.000
0.000
0.071
0.000
0.071
0.071
0.000
0.000
14
0.036
0.036
0.000
0.000
0.000
0.000
0.036
0.107
RAAN
3
0.000
0.333
0.000
0.000
0.000
0.000
0.167
0.000
0.000
0.000
3
0.000
0.333
0.000
0.167
0.000
0.000
0.000
0.000
0.500
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
3
0.000
0.000
0.000
0.000
0.000
0.333
0.000
0.333
RSJ
10
0.100
0.000
0.000
0.000
0.000
0.000
0.150
0.000
0.000
0.000
10
0.000
0.000
0.000
0.000
0.050
0.050
0.050
0.200
0.000
0.050
0.250
0.050
0.000
0.100
0.150
0.000
0.050
0.000
10
0.000
0.100
0.000
0.000
0.050
0.000
0.000
0.050
P.S
17
0.000
0.000
0.088
0.000
0.059
0.029
0.118
0.000
0.000
0.059
18
0.000
0.000
0.000
0.000
0.000
0.083
0.000
0.306
0.000
0.278
0.056
0.000
0.000
0.000
0.167
0.056
0.000
0.056
18
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.028
Chi
12
0.000
0.000
0.000
0.000
0.000
0.000
0.208
0.000
0.042
0.000
12
0.000
0.000
0.000
0.250
0.000
0.000
0.000
0.250
0.042
0.167
0.083
0.000
0.000
0.000
0.125
0.083
0.000
0.000
12
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
I.A
4
0.000
0.250
0.250
0.000
0.000
0.000
0.000
0.000
0.000
0.000
3
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
1,000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
4
0.000
0.000
0.000
0.250
0.000
0.000
0.000
0.000
B.A
14
0.143
0.000
0.000
0.000
0.000
0.000
0.036
0.000
0.000
0.000
14
0.036
0.000
0.000
0.107
0.036
0.036
0.036
0.071
0.036
0.214
0.107
0.036
0.036
0.000
0.214
0.036
0.000
0.000
14
0.000
0.429
0.071
0.000
0.000
0.036
0.000
0.000
Mat
3
0.000
0.000
0.000
0.000
0.000
0.000
0.167
0.000
0.000
0.000
3
0.000
0.000
0.000
0.000
0.000
0.000
0.000
1,000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
3
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
46
Appendix 1: Continue...
Locus
Allele/n
mTcCIR11
N
210
211
212
216
217
219
220
221
223
224
225
295
N
mTcCIR 12
163
189
197
202
204
205
206
207
210
213
214
215
223
224
238
N
mTcCIR 15
214
234
235
236
237
239
242
245
RAAS
14
0.250
0.000
0.071
0.000
0.000
0.000
0.000
0.000
0.179
0.000
0.214
0.071
14
0.000
0.179
0.000
0.036
0.000
0.000
0.000
0.000
0.000
0.250
0.000
0.536
0.000
0.000
0.000
14
0.000
0.214
0.071
0.071
0.000
0.036
0.000
0.036
RAAN
3
0.167
0.000
0.000
0.000
0.000
0.167
0.000
0.000
0.000
0.000
0.000
0.000
3
0.000
0.000
0.000
0.000
0.000
0.167
0.000
0.333
0.333
0.000
0.000
0.167
0.000
0.000
0.000
3
0.000
0.000
0.167
0.000
0.000
0.000
0.000
0.000
RSJ
10
0.250
0.050
0.000
0.000
0.100
0.000
0.000
0.000
0.000
0.000
0.400
0.000
10
0.000
0.200
0.000
0.050
0.000
0.050
0.050
0.000
0.000
0.000
0.000
0.650
0.000
0.000
0.000
10
0.000
0.000
0.000
0.000
0.300
0.000
0.000
0.000
P.S
18
0.361
0.000
0.000
0.028
0.083
0.000
0.000
0.028
0.028
0.056
0.389
0.000
18
0.000
0.306
0.000
0.111
0.028
0.000
0.028
0.000
0.000
0.056
0.000
0.472
0.000
0.000
0.000
17
0.000
0.000
0.059
0.000
0.206
0.029
0.000
0.029
Chi
12
0.083
0.000
0.000
0.042
0.000
0.000
0.083
0.000
0.000
0.000
0.792
0.000
12
0.000
0.375
0.000
0.208
0.000
0.000
0.000
0.000
0.000
0.125
0.000
0.292
0.000
0.000
0.000
12
0.000
0.042
0.083
0.000
0.125
0.000
0.000
0.042
I.A
4
0.250
0.000
0.000
0.125
0.000
0.000
0.000
0.000
0.000
0.000
0.375
0.000
4
0.250
0.000
0.250
0.000
0.125
0.000
0.000
0.000
0.000
0.125
0.125
0.000
0.125
0.000
0.000
4
0.000
0.000
0.000
0.125
0.500
0.000
0.000
0.000
B.A
14
0.071
0.000
0.036
0.071
0.000
0.000
0.000
0.000
0.000
0.000
0.286
0.000
14
0.000
0.036
0.000
0.143
0.036
0.000
0.107
0.000
0.000
0.000
0.000
0.643
0.000
0.036
0.000
14
0.000
0.000
0.107
0.000
0.107
0.036
0.000
0.071
Mat
3
0.167
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.167
0.000
0.667
0.000
3
0.000
0.333
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.333
0.000
0.000
0.333
3
0.167
0.000
0.000
0.333
0.167
0.000
0.167
0.000
47
Appendix 1: continue…
Locus
Allele/n
N
mTcCIR 15
246
248
250
251
252
253
254
255
256
257
258
259
N
mTcCIR18
192
199
200
201
202
203
204
205
207
209
210
212
213
214
227
236
243
255
N
mTcCIR 21
142
143
144
145
147
150
RAAS
14
0.000
0.036
0.000
0.107
0.143
0.000
0.143
0.071
0.036
0.036
0.000
0.000
13
0.038
0.000
0.231
0.000
0.038
0.038
0.038
0.077
0.000
0.000
0.077
0.154
0.077
0.077
0.038
0.038
0.038
0.038
14
0.000
0.000
0.107
0.071
0.000
0.000
RAAN
3
0.000
0.000
0.000
0.000
0.000
0.000
0.333
0.333
0.000
0.000
0.000
0.167
3
0.000
0.000
0.000
0.000
0.167
0.000
0.000
0.000
0.000
0.333
0.000
0.000
0.000
0.500
0.000
0.000
0.000
0.000
3
0.000
0.000
0.167
0.000
0.000
0.000
RSJ
10
0.100
0.000
0.000
0.000
0.100
0.000
0.000
0.200
0.150
0.000
0.100
0.050
10
0.000
0.100
0.150
0.000
0.100
0.000
0.050
0.000
0.000
0.000
0.000
0.050
0.000
0.550
0.000
0.000
0.000
0.000
10
0.100
0.000
0.000
0.000
0.000
0.000
P.S
17
0.000
0.000
0.059
0.059
0.000
0.118
0.176
0.265
0.000
0.000
0.000
0.000
18
0.000
0.111
0.167
0.083
0.028
0.000
0.000
0.000
0.056
0.000
0.000
0.111
0.167
0.278
0.000
0.000
0.000
0.000
17
0.000
0.000
0.029
0.000
0.000
0.029
Chi
12
0.000
0.000
0.125
0.042
0.083
0.208
0.083
0.125
0.042
0.000
0.000
0.000
12
0.000
0.000
0.000
0.042
0.000
0.000
0.042
0.000
0.000
0.000
0.000
0.292
0.000
0.625
0.000
0.000
0.000
0.000
12
0.000
0.042
0.083
0.042
0.000
0.000
I.A
4
0.000
0.000
0.250
0.000
0.000
0.000
0.125
0.000
0.000
0.000
0.000
0.000
3
0.000
0.333
0.333
0.000
0.167
0.000
0.167
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
4
0.000
0.000
0.125
0.000
0.250
0.000
B.A
14
0.000
0.000
0.000
0.107
0.036
0.107
0.000
0.321
0.036
0.036
0.000
0.036
14
0.000
0.000
0.107
0.000
0.179
0.036
0.286
0.000
0.000
0.000
0.000
0.179
0.000
0.214
0.000
0.000
0.000
0.000
14
0.071
0.000
0.036
0.250
0.000
0.000
Mat
3
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.167
0.000
0.000
0.000
3
0.000
0.167
0.167
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.167
0.500
0.000
0.000
0.000
0.000
3
0.000
0.000
0.000
0.000
0.000
0.000
48
Appendix 1: Continue...
Locus
Allele/n RAAS RAAN RSJ
mTcCIR
N
14
3
10
21
151
0.000 0.000 0.000
153
0.071 0.000 0.100
154
0.143 0.000 0.100
155
0.286 0.500 0.350
157
0.000 0.000 0.150
158
0.000 0.000 0.000
159
0.179 0.167 0.000
160
0.000 0.000 0.000
164
0.143 0.167 0.150
165
0.000 0.000 0.000
169
0.000 0.000 0.000
173
0.000 0.000 0.050
180
0.000 0.000 0.000
181
0.000 0.000 0.000
P.S
17
Chi
12
I.A
4
B.A
14
Mat
3
0.029
0.000
0.118
0.500
0.000
0.000
0.000
0.029
0.206
0.000
0.029
0.000
0.029
0.000
0.083
0.000
0.000
0.542
0.000
0.000
0.000
0.000
0.125
0.083
0.000
0.000
0.000
0.000
0.000
0.250
0.000
0.250
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.125
0.000
0.000
0.000
0.393
0.000
0.071
0.071
0.000
0.000
0.000
0.107
0.000
0.000
0.000
0.000
0.000
0.000
0.667
0.000
0.000
0.000
0.000
0.333
0.000
0.000
0.000
0.000
0.000