https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3492346/
Insight into the Wild Origin, Migration and Domestication History of the Fine Flavour Nacional Theobroma cacao L. Variety from Ecuador
Randall P. Niedz, Editor
This article has been corrected. See PLoS One. 2013; 8(2): 10.1371/annotation/2357f0f1-7dc3-4781-afb0-29a8ce56b3f0.
This article has been cited by other articles in PMC.
Abstract
Ecuador’s economic history has been closely linked to Theobroma cacao
L cultivation, and specifically to the native fine flavour Nacional
cocoa variety. The original Nacional cocoa trees are presently in danger
of extinction due to foreign germplasm introductions. In a previous
work, a few non-introgressed Nacional types were identified as potential
founders of the modern Ecuadorian cocoa population, but so far their
origin could not be formally identified. In order to determine the
putative centre of origin of Nacional and trace its domestication
history, we used 80 simple sequence repeat (SSR) markers to analyse the
relationships between these potential Nacional founders and 169 wild and
cultivated cocoa accessions from South and Central America. The highest
genetic similarity was observed between the Nacional pool and some wild
genotypes from the southern Amazonian region of Ecuador, sampled along
the Yacuambi, Nangaritza and Zamora rivers in Zamora Chinchipe province.
This result was confirmed by a parentage analysis. Based on our results
and on data about pre-Columbian civilization and Spanish colonization
history of Ecuador, we determined, for the first time, the possible
centre of origin and migration events of the Nacional variety from the
Amazonian area until its arrival in the coastal provinces. As large
unexplored forest areas still exist in the southern part of the
Ecuadorian Amazonian region, our findings could provide clues as to
where precious new genetic resources could be collected, and
subsequently used to improve the flavour and disease resistance of
modern Ecuadorian cocoa varieties.
Introduction
Theobroma cacao L. is the most economically important species of the Theobroma genus which is a member of the Malvaceae family. It is a diploid perennial species (2n = 2× = 20) with a small genome ranging in size from 411 to 494 Mb [1]. Most of the T. cacao accessions are self incompatible due to a gameto-sporophytic incompatibility system [2]–[4].
However, some varieties, domesticated long ago, as the varieties
Criollo from central America, Comun from Brazil, or Nacional from
Ecuador, and some of their hybrid forms are self compatible.
Traditionnaly,
two main genetic groups have been defined to classify cocoa accessions,
based on morphological traits and geographical origins [5]:
Criollo and Forastero. This classification also reflects the first
commonly cultivated cocoa varieties: Criollo, first domesticated in
Central America more than 2000 years ago, and a Lower Amazon Forastero
variety (Amelonado type) domesticated in Brazil.
Trinitario, a third group, corresponding to hybrids between Criollo and
Forastero, was also recognised. However, the Forastero group also
includes many other populations from all the Amazonian and Orinoco
regions and presents a high diversity, as revealed by many genetic
studies [6], [7], [8]–[16]. A new classification, identifying 10 genetic clusters, was more recently proposed [15].
The domestication of Criollo, originally cultivated by the Mayas in Central America was previously studied [17].
The authors concluded that the genetic base of the ‘ancient’ Criollo
variety, cultivated in Central America before foreign cocoa
introductions occurred during the 18th century, was very narrow.
Cocoa
cultivation in Ecuador is very ancient, dating back to the
pre-Columbian age before the Spanish colonization of this territory.
Pizarro, during his first voyage in 1526 along the South American coasts
(nowadays Ecuador), found evidence of small plantations of an
apparently native cocoa tree [18], [19].
It is very likely that the ’Nacional’ Ecuadorian cocoa population
existed for several centuries prior to the arrival of the Europeans [20],
but its origin in the Ecuadorian coastal region has never been
clarified. Two hypotheses have been put forward: according to Allen and
Lass [21],
the Nacional cocoa variety could have originated from a local wild
population, that has nowadays completely disappeared along with the
original forest cover of the region, or Nacional could have been
introduced in the coastal region from the Amazonian area of Ecuador
where wild cocoa is common [22]–[24]. There is no evidence that T. cacao
and its products played any part in the lives of Ecuadorian coastal
inhabitants during the Chorrera phase of the pre-Columbian Valdivia
culture (2000 BC), contrary to the extensive amount of information
available on this phenomenon with respect to Mesoamerican people, most
notably the Mayas and Aztecs. In Ecuador, available information related
to the history of Nacional cocoa cultivation is linked with the Spanish
colonization history in this country.
Since the time
when the first Spanish colonists began deforestation of the Ecuadorian
coastal region, a large number of native cocoa trees have been reported [21]–[25],
principally along the Guayas basin. Apparently, these colonists began
to sow seeds of these native trees approximately 100 years after the
discovery of America, and when the native Mesoamerican populations
started to decline. The cocoa cultivation areas expanded, and the native
Nacional variety rapidly became known worldwide due to its strong
floral so-called “Arriba” aroma, exclusively generated by Nacional cocoa
beans. The fine flavour quality of chocolate products obtained from
Nacional cocoa beans has always been highly appreciated by chocolate
manufacturers.
The native Nacional variety was the only
one planted in Ecuador until the early 1890s, when foreign germplasm was
first introduced in this country. In 1890, due to the quality traits of
Nacional cocoa beans, Ecuador had a privileged position in the markets
of Hamburg and London [26].
From 1910, foreign germplasm introductions progressively increased due
to the appearance of two fungal diseases known as witches’ broom (Moniliophthora perniciosa) and frosty pod (Moniliophthora roreri), which together devastated the native plantations.
A
large genetic admixture between the native variety and foreign
germplasm is currently found in modern Ecuadorian cocoa plantations [27].
The fine flavour cocoa aroma has decreased in this hybrid complex, and
25% of the Ecuadorian cocoa production was recently classified as ‘bulk’
cocoa by the International Cocoa Organization (ICCO). There is nowadays
increasing demand for fine flavour cocoa, which presently represents
6.8% of the world cocoa production and for which Ecuador remains the
main supplier [28].
In
a previous work, a few non-introgressed Nacional types were identified
within this hybrid population as potential representatives of the native
Nacional variety [27].
Here we analyze their relationships with a wide range of wild cocoa
genotypes covering a broad geographical range from upper and lower
Amazonian regions [21], [22], [29], [30], [31],
in order: a) to identify the putative centre of origin of Nacional, and
b) to trace the domestication history of the Nacional cocoa variety.
Materials and Methods
Plant Material
A
total of 176 individuals from different geographical origins were used
for simple sequence repeat (SSR) analyses in this study (Table 1 and Table S1):
Molecular Markers
Eighty SSR markers were chosen for these analyses (Table 2). Seventy four SSRs were isolated from expressed sequence tags (EST) and six from genomic sequences, and mapped [33]–[35]. They are distributed on all of the 10 cocoa chromosomes.
DNA Isolation and PCR Amplification
Statistical Analysis
Amplified
SSR fragments were scored as alleles. The following genetic parameters
were calculated for each population using GENETIX software [36]: unbiaised expected heterozygosity [37]
(HE), observed heterozygosity (HO), proportion of polymorphic loci at
which the frequency of the most common allele does not exceed 95%
(P0.95) and 99% (P0.99), and mean number of alleles per locus.
We also estimated allelic richness (Rs) for each population, using a rarefaction approach to correct for unequal sample sizes [38] with the program FSTAT V. 2.9.3 [39].
Wright’s F-statistics were estimated according to Weir and Cockerham [40] using the GENETIX software: FIS, an estimate of heterozygote deficiency or excess, calculated for the whole population sample and for each population, and FST,
the proportion of variance due to population differenciation,
calculated for the whole population sample and between pairs of
populations; their significance were assessed using a 1000-fold
permutation test.
As most mutations in SSR involve the
addition or subtraction of a small number of repeat units, according to
the stepwise mutation model population, genetic differentiation was also
estimated by RST
[41]. RhoST, an unbiased version of RST
in which allele sizes are transformed to standardize variances, was
estimated for pairs of populations using GENEPOP software, WEB version
4.0.10 [42]–[44].
Analyses of genetic distance among pairs of populations [45]
were carried out to determine the genetic similarity between the
Nacional pool and the wild and cultivated genotypes using GENETIX.
In
addition, a dendrogram was constructed using a dissimilarity index
(simple matching) and the neighbour-joining (NJ) method with 500
bootstrap replicates. Neighbour-joining cluster analyses were carried
out using DARWIN software 5.0 [46].
Finally, a paternity analysis was performed using the software CERVUS, version 2.0 [47],
to identify the most likely ancestral population of the Nacional
variety. An 80% confidence threshold was used for paternity assignment.
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