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QTL Studies Carried out for Agronomic, Technological and Quality Traits of Cocoa in Ecuador
Dominique Crouzillat, Miclie! Rigoreau, Morgan Cabigliera, Milton Alvarez *~ Peter Bucheli and Vincent Pétiard
Centre de Recherche Nestlé, 101, Avenue Gustave Efffel, Notre-Dame D’Oé, liP. 9716, 37097 Tours Cedex 2, France
*Nestlé Agronomia, Via Interoceanica, Castilla postal 17-11-6053, Quito-Cumbaya, Ecuador
An F2 population, obtained by selfing an Ecuadorian hybrid cocoa genotype with Arriba flavour, was the basis for a genetic study on agronomic, technological and qualitative traits. Genetic analyses using RFLP and microsatellite markers indicated that this Ecuadorian hybrid cocoa was the result of a cross between a highly homozygous Nacional genotype and an unknown genotype that probably came from Venezuela. A genetic map was obtained from 171 trees of the F2 segregating population with 113 loci including RFLP and microsatellite markers and covering 815 cM. The average distance between two adjacent Ioci is about 8.4 cM, A major QTL for general agronomic value was found that explained 27.1% of the total phenotypic variation and was co-localised with a OIL for early flowering and trunk diameter. Pleiotropic and epistatic effects were both detected for these traits. QTL for technological traits such as the weight of 100 cocOa beans were found on the genetic map at different locations to the QTL for agronomic traits suggesting that these traits could be selected independently. The majority of the QTL for agronomic and technological traits are favoured by the alleles coming from the Venezuelan parent. Sensory evaluation was initiated on the segregating progeny in order to evaluate the range of variation and the main components of Arriba flavour.
In order to select for Arriba flavour it is important to accumulate knowledge on the genetic components involved in agronomic, technological and sensory characteristics. Even if the technological and sensory traits are the most important from the manufacturer’s point of view, agronomic traits must be taken into account for the selection of elite Arriba clones.
Field and sensory data on one hand and molecular analyses (Lerceteau et al. 1997) on the other hand indicated that the Nacional cocoa types are at the origin of the Arriba flavour. However, their resistance to disease and yield performance are unsatisfactory. Consequently, breeders have introduced accessions from Venezuela and Trinidad to create hybrids with the Nacional type in order to increase productivity. Today these hybrids are predominant in Ecuador but the qualitative aspect of the Arriba flavour has not been taken into consideration in such selection programmes.
Recent developments in bean sample fermentation on a small scale coupled to sensory analysis have supported the observation that flavour potential is a heritable function of the genotype (Lockwood and Eskes 1995). So far, commercial fine flavour cocoas have only been found in Nacional, true Criollos and in some Trinitario genotypes that are often poor yielding and susceptible to diseases. The inheritance of flavour was investigated by Clapperton et at (1994) who found flavour components to be significantly heritable indicating that sensory traits can be manipulated in a conventional breeding programme.
The main objectives of this study are to estimate the genetic influence on Arriba flavour in conjunction with agronomic traits in a supposed hybrid (Nacional x Venezolano). QTL analysis could be used to assess the number of genes involved in flavour determination and allow better management of the development of further elite Arriba clones.
Material and methods
An individual cocoa tree, which was identified as a cross between Nacional and Venezolano Amarillo parents, was selected for its high Arriba flavour score and high heterozygosity level. This cocoa tree was selfed and an F2 progeny was obtained consisting of 171 individuals planted in 1995 at Nestle, Quevedo (Ecuador).
Quantitative traits analysed
Three characteristics were selected to estimate the agronomic value of the segregating progeny:
Trunk diameter was recorded from 1996 to 1999 and is considered a yield predictor. Number of days before flowering; this is important to estimate the precocity of production for the future elite Arriba clones.
The general agronomic value, scored on a scale from 1 (bad) to 10 (excellent) for each segregating tree. This trait shows significant correlation with the two other agronomic traits previously studied. In addition, the number of beans per 100 grams was studied in 1997, 1999 and 2000. Sensory analysis has been initiated on sub-samples of the segregating progeny in order to estimate the range of variation for Arriba flavour.
Molecular markers, genetic mapping and QTL detection
A total of 113 loci (78 RFLP markers and 35 microsatellites) were used. The RFLP probes named gTcCIR or cTcClR and the microsatellites mTcCIR were kindly provided by CIRAD (France). The cocoa genetic map consisting of 113 loci was constructed using the software package MAPMAKER 2.0 (Lander eta!. 1987). QTL mapping was carried out using the approach of ANOVA with the software OGENE 2.24 (Nelson 1997). A p <0.01 value was used as the threshold for the QTL detection. The method of interval mapping (Lander and Botsein 1989) with MAPMAKER/QTL software was also used with a LOD score of 2. The code number of each linkage group was given as described by Lanaud et al. (1995)~
Genetic map and Nacional allele identification
The genetic map obtained from the F2 population type comprised 113 loci covering 815 cM on ten linkage groups (Figure 1). The average distance between two adjacent markers is 8,4 cM. This marker density is sufficient for the QTL study.
Skewed segregation was detected for seven loci (6.2%), five being significant at a threshold of p = 0.06 and two at p = 0.01. They are located on linkage groups 1. 2, 3, 6 and 7, five of them are situated in the distal part of the linkage group (Figure 1). Generally at these seven loci the Venezolano Amarillo allele predominates.
Due to the genetic status of the parental F1 clone (Nacional X Venezolano Amarillo) and due to the genetic specificity of Nacional clones (Crouzillat et a!. 2000), it was possible to assess Nacional alleles on 50 of the 113 loci mapped. For all these loci covering the ten linkage groups, the Nacional alleles were always in the same coupling phase suggesting that the Nacional parent was highly homozygous. This allelic identification allowed the determination of the genetic effect for each QTL analysed.
The objective of the QTL analysis was to study the agronomic and technological traits of interest. Analysis of trunk diameter, assessed annually between 1996 and 1999, allowed the detection of three QTL for 1996, two for 1997 and one QTL for 1998 and 1999 (Table 1). The QTL located on chromosome I was found for all four years and the other three QTL detected on linkage groups 3 and 10 only once. The two QIL detected on chromosome 10 in 1996 and 1997 are not considered as the same because their map locations and their genetic effects are different. The QTL detected on the linkage group 3 in 1996 appeared to be related to the Nacional allele which appears to be favourable for this trait (Table 1). These results indicate that only the QTL on chromosome I detected in each of the four years studied has a significant and durable effect on trunk diameter. Two QTL for early flowering were detected on linkage groups 2 and 10 and each explained 6.6 and 6%, respectively, of the phenotypic variance.
One major QTL was found for general agronomic value on linkage group 10, explaining 27.1% of the phenotypic variance. In this case, the favourable allele comes from the Venezolano parent. This QTL appears to be the major component of the agronomic value for this cross. It is significantly correlated with early flowering (r = 0.28, p<0.01) and trunk diameter assessed in 1997 (r = 0.64) explaining the overlapping of these three QTL on chromosome 10.
One technological trait, the number of beans per 100 g, was analysed in 1997, 1999 and 2000. Two QTL for this trait were detected each year. These QTL were at different map locations, except for a shared QTL detected on chromosome 3 for 1997 and 2000 as shown in Table 2. These five different QTL do not overlap with other agronomic QTL and the Venezolano alleles were generally found to be favourable for this trait.
Pleiotropic and epistatic effects
The map location coincidence of QTL for early flowering, trunk diameter in 1997 and general agronomic value on chromosome 10 could be considered as pleiotropy since the Venezolano allele is found to be favourable for the three traits and because the same genetic effect (dominance) was detected (Table 1).
An epistatic effect was found for the QTL detected on chromosome 10 for general agronomic value. The ANOVA study indicated that the region near the locus CCG 1328 on chromosome 6 (Figure 1) has a highly significant (p=0.000005) impact on the QTL detected on chromosome 10. The favourable alleles are coming from the Venezolano parent for these two loci (Table 3), indicating the influence of the genetic background on QTL expression.
Field performance, including disease resistance and yield, is the major trait of interest to cocoa farmers and breeders. As 90% of the world’s cocoa is produced by smallholders and trade is by country of origin and bean weight, most individual growers have, however, only a limited opportunity to add value through quality.
Cocoa trees are faced with a wide spectrum of climatic conditions during their lifetime. Modifications in QTL expression over diverse environments have been reported in crop plants (Paterson eta!. 1991; Hayes eta!. 1993). More recently QTL analyses were performed on fruit trees (Asins eta?. 1994) and on forest trees such as poplar (Bradshaw and Stettler 1995), pine (Plomion et at 1996) and Eucalyptus (Verhaegen et at 1997) indicating an influence of environmental effects on the stability of QTL expression. In our study, according to the year analysed, several different QTL were found for trunk diameter and average bean weight. These data suggest a possible influence of environment on QTL expression. Nevertheless, a cocoa yield study over 15 years (Crouzillat et at 2000) permitted the identification of some major QTL, consistently detected over the years, that could be useful in a breeding programme. With regard to the present data, the QTL detected on chromosome 10 for general agronomic performance appears to be a major component of this trait.
The average bean weight is considered to be partly under the influence of the environment (Toxopeus and Wessel 1970). Diallel crosses in Costa Rica (Engels 1985) and factorial mating designs (Tan 1990; Lockwood and Pang 1995) have demonstrated high broad sense heritability of bean weight and a large additive component of the genotypic variance. The data obtained on this trait in our study demonstrated that only one common QTL was found for two of the three years studied. Consequently, further confirmation of these QTL is required before possible use in selection.
In parallel to these agronomic and technological analyses, the sensory evaluation of this segregating population has started. A detailed protocol has been described from bean harvest to liquor tasting using single tree sampling (Bucheli et at 2000). The first sensory data obtained indicate that a range of variability for Arriba flavour exists. Further quantitative studies are necessary to estimate the role of environmental factors, the fermentation process and sensory evaluation on the determination of Arriba flavour.
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