Cladistics groupings of the active breeding cocoa genetic resources of Nigeria for physicochemical and nutraceutical traits

Preference for functional and nutritious food capable of meeting consumers ’ demand and health is on the increase. The present preliminary study seeks to assess physico-chemical and nutraceutical diversity in the cocoa bean powder of 77 genotypes present in four Nigerian cocoa field banks. Twenty ripe pods/genotypes in each of the four active breeding field banks at the Cocoa Research Institute of Nigeria (CRIN), Ibadan, Nigeria were utilized. Composite beans from the 20 pods of each genotype were singly fermented, sun-dried and milled. Duplicate samples of the powder of each genotype were analysed for physico-chemical and nutraceutical components. Twenty-one polymorphic variables distinguished the 77 cocoa genotypes. Grouping by dendogram identified four clusters, three differently and uniquely captured 100% of the genotype membership in the local clone, international clone and the regional varieties field bank but 86% of the genotypes in the hybrid trial field bank were grouped in cluster I. Prominent traits with highest values in each clusters were: protein, p H, Ca, K and Fe (Cluster I), Zn and Mg (Cluster II), crude fat and P (Cluster III) and crude fibre, ash, theobromine, flavonoids and caffeine (Cluster IV). Exploitable diversity for nutritional quality improvement is present in the active breeding and working collections of Nigerian cocoa field banks.


Introduction
Cocoa (Theobroma cacao L.), of the family Malvaceae sensu lato, a perennial cash crops (Marita et al., 2001) predominantly grows in the tropics of Central and South America, Asia and Africa.It had an estimated world output of 3.5 million tons in 2006 (de Almeida and Valle, 2007) and 5.2 million metric tons in 2020 (WorldAtlas, 2021).The economic value of cocoa beans exported as whole, broken, raw or roasted was 8.6 billion US Dollars (USD) in 2017 (Eghbal, 2018).It has remained the foremost non-oil source of foreign exchange to the major cocoa-producing countries in West and Central Africa.It is cultivated by more than five million growers in 50 countries and 40-50 million people derive their livelihood from it (CacaoNet, 2012;World Cocoa Foundation, 2012).The chocolate and cocoa powder from cocoa beans are good and nutritional sources of minerals especially potassium, magnesium, copper and iron (Afoakwa et al., 2007;Torres-Moreno, et al., 2015;Adeyeye, 2016).Aikpokpodion et al. (2010) presented the historical chronology of the introduction schemes of cocoa genetic resources into Nigeria as follows: (i).Upper Amazon and 'Trinitario' populations, which were introduced from Trinidad in 1944 (Posnette and Todd, 1951), (ii).1965-1967 large-scale introduction of Upper Amazon cacao materials from Trinidad and Tabago (Aikpokpodion et al., 2010), (iii).Cacao Introduction Scheme sponsored by the Cocoa Alliance that included quite a large number of intra-Nanay, intra-Parinari, intra-Iquitos and inter-Pound selections (Atanda, 1977;Olatoye and Esan, 1992) and (iv).some cocoa genetic resources from Costa Rica, Indonesia, Fernando Po, Kew Garden, Wageningen and Miami (Jacob et al., 1971).These four introductions have long remained the primary cocoa genetic resources for cultivar development in the breeding programmes and planting materials generation at the Cocoa Research Institute of Nigeria (CRIN), Ibadan, Nigeria.Cocoa genetic resources in the list of the various introductions have greatly dwindled; their erosion has been attributed to: old age of plants and plantations.Furthermore, their neglect has been obvious due to poor record keeping and inconsistent data on extant plants.Urbanization and pressure on land for alternative uses have also contributed to genetic erosion, especially in the out-station breeding field banks of CRIN.For example, the out-station field bank at Ibule-soro, Akure, Ondo state, Nigeria has been lost to urbanization.
CRIN has remained the only agricultural institute in Nigeria with the mandate for cocoa improvement, it therefore hosts cacao germplasm collections of Nigeria.Currently, there are four ex situ cocoa breeding field germplasm collections at CRIN headquarters in Ibadan, Nigeria.The four field banks include: (i) introduced cocoa genetic materials established at the International Clone (IC) field bank, (ii) hybrids generated from crosses among some local clones and established in the hybrid trial (HT) field bank, (iii) hybrids generated for regional (central and west Africa) variety evaluation trial (RVT) field bank and (iv) some old 'C' and 'T' clones that were established at the local clones trial (LC) field bank.The C-clones were mixtures of local Amelonado and red podded local Trinitario (Lockwood and Gyamfi, 1979) and T-clones were the approved Upper Amazon populations whose open pollinated progenies formed the F3-Amazon (Aikpokpodion et al., 2009).
High bean yield and disease (especially black pod) resistance had long been the primary breeding goal of cocoa in Nigeria.A large proportion of the cocoa genetic resources in the Nigerian cocoa field genebanks have remained unutilized for food quality improvement because their potentials for nutritional and functional food values have not been unveiled.The eight cocoa varieties released for Nigerian farmers in 2011 (CRIN, 2011) were promoted for high bean yield and resistance to black pod and pod rot diseases.Record of effort(s) for cocoa bean quality improvement in Nigeria is scarce.The present investigation for physico-chemical and nutraceutical diversity among 77 genotypes could present a platform for the initiation of a bean quality improvement programme for cocoa in Nigeria.
Significant variation exists among West African cocoa genotypes at phenotypic and genomic levels (Opoku et al., 2007;Sonwa et al., 2007;Pokou et al., 2009;Aikpokpodion, 2010;Adewale et al., 2013;Olasupo et al., 2018).The nutraceutical properties of the cocoa germplasm in Trinidad and Tobago and Cameroun have been documented (Bekele and Phillips-Mora, 2019).Quite recently, Adeigbe et al. (2021) noted that genotypes within each of the four cocoa field banks in Nigeria showed variation for some physicochemical properties.Utilization of plant genetic resources in breeding programmes is dependent on the understanding of their potentials (CacaoNet, 2012;Bekele and Phillips-Mora, 2019).Cocoa beans have complex raw ingredients.More than 500 flavour compounds have been identified in cocoa products; to the manufacturing industries, understanding the source of each is a science on its own (Reed, 2010).There are documented proximate and mineral composition in cocoa bean in Nigeria (Adeyeye et al., 2010;Ndife et al., 2013), however, the number of genotypes employed in such studies were less than three.The unavailability of nutritional and quality information of the already characterized (phenotypic - Aikpokpodion et al., (2009) and genomic -Olasupo et al., (2018)) Nigerian cocoa germplasm is an evident gap, thus substantiating the essence of the present investigation.
Improved consciousness about health among consumers has greatly increased global promotion of functional foods (Gaikwad et al., 2020).Furthermore, the cocoa-based product manufacturers now seek cocoa with enhanced nutraceutical value (Bekele and Phillips-Mora, 2019).Naturally occurring plant nutraceuticals (which are consumed as food or food parts) deliver benefits to consumers beyond basic nutrition, such as protecting organisms against oxidative stress, and playing other roles, such as: antimicrobial, anti-oxidant, anti-inflammatory and anti-cancer properties (Aguilar et al., 2017;Rosa et al., 2022).Identifying physicochemical and nutraceutical status of the different cocoa genotypes in the active Nigerian cocoa breeding germplasm has become evidently necessary.The present investigation, therefore seeks to identify diversity for some biochemical and bioactive traits among genotypes within the germplasm and superior groups of genotypes for specific nutritional, phytochemical, mineral and physical properties.

Materials and methods
The Active cocoa breeding collections in CRIN The Breeding and Improvement Division of CRIN, Ibadan, Nigeria, has four active cocoa breeding field banks.They are: LC trial field bank, containing 13 clones; HT field bank containing 23 hybrids; RVT field bank which hosts nine hybrids from Ghana, four from Cote d'Ivoire, three from Cameroon, two common crosses from Nigeria and six local control crosses; and the IC field bank which contain 17 introduced cocoa clones.The genotypes were the treatments in each of the field banks.They were laid out in a randomized complete block design with six replicates.Each field banks contained two rows of five plants.List of the genotypes held in the various active breeding field banks is presented in Table 1.

Materials for the study and analytical protocols
Pods were harvested during a cropping season and bulked from all the trees of a genotype.Twenty uniform fruits were then selected from the bulked pool of fruits for each genotype.Total number of pods/genotypes in the harvest was within 250-450.Pods were broken seeds were extracted and pooled on genotype basis.Pooled seeds of each of the 77 genotypes were separately fermented in trays.Fermented samples were sun-dried and milled into powder using standard procedures (ICCO, 2021).Duplicate samples, each of the 77 genotypes were determined following the recommendations of Fitzpatrick (2013) for powdered food samples.Twenty-one biochemical traits were analysed, including: physical properties (bulk density, dispersibility, re-hydration time, oil and water absorption capacity) and proximate (moisture, crude ash, fat, fibre and protein) contents which were determined following the procedures in Ooi et al. (2012).The pH was determined following the procedure in Vijayakumar and Adedeji (2017).After high pressure digestion, macro-elements (P, K, Ca and Mg) were analysed by atomic absorption spectrometer and micro-elements (Fe and Zn) were determined by sensitive atomic spectrometric techniques and the results were obtained using the working standards of AOAC (1990); 1000 ppm for each of the genotypes (Poitevin, 2016).Caffeine and theobromine were determined following the procedure in Thomas et al. (2004) and flavonoid was analysed following the procedure of Lin and Tang (2007).Variability and character association of 17 of the 21 traits for each of the four breeding field banks have been earlier determined by Adeigbe et al. (2021).

Data analysis
Basic descriptive statistics analysis including coefficient of range was carried out on the data and Pearson correlation analysis was conducted on the mean matrix table containing the 77 genotypes and the 21 variables.The replicated data on the 77 treatments and 21 variables was subjected to multivariate analysis of variance using the PROC GLM procedure of SAS version 9.4 (SAS, 2011).A mean data matrix comprising 77 genotypes by 21 variables was submitted to SAS using PROC DISTANCE to generate pairs of Gower genetic distances among the 77 genotypes Plant Genetic Resources: Characterization and Utilization (Gower, 1971).The paired genetic distances were then submitted for principal component analysis (using PROC PRINCOMP) and clustering analysis (using PROC TREE, WARD minimum variance hierarchical clustering method (Ward, 1963)) in SAS.Mean genetic distances were further generated for each cluster and the mean performances for the 21 traits in each cluster were determined.Significant differences between pairs of clusters were tested by paired t-test using SAS.Moreover, inter-and intra-cluster variability was further verified using the cluster means, standard error (SE) and coefficient of variation (CV) for each trait.

Correlations among the various biochemical traits
In online Supplementary Table S1, 121 pairwise correlations were significant, of which 74 were positive and 47 were negative.Protein content in the cocoa bean had a positive and significant correlation with crude fat, dispersibility, bulk density, calcium, potassium, phosphorus, iron, magnesium, water and oil absorption capacity, but it had a significant negative correlation with crude fibre and theobromine content (online Supplementary Table S1).A significant positive correlation existed between all the mineral elements, except for zinc with phosphorus and potassium, then potassium with magnesium.Furthermore, all the mineral elements had a positive significant correlation with dispersibility (online Supplementary Table S1).The relationships between crude ash, moisture content, theobromine and flavonoid were positive and significant.Moreover, the correlation of bulk density with water and oil absorption capacity was positive and significant (online Supplementary Table S1).Each of the following traits: ash, moisture content, theobromine, flavonoid, caffeine and rehydration time had a negative and significant correlation with crude fat.Moreover, each of the six minerals except potassium equally had a negative and significant correlation with crude fibre (online Supplementary Table S1).

Variance contribution to principal components and genotypes' grouping pattern
In online Supplementary Table S2, the eigenvalues of the first three PC axes were higher than 2.5.The highest variance contribution (37.5%) was in PC1.The first three PC axes accounted for and explained 70% of the total variation among the 77 genotypes (online Supplementary Table S2).The Ward clustering method separated the 77 genotypes into four main clusters at the similarity coefficient above 0.10 (Fig. 1).The grouping pattern was very specific: cluster I contained all genotypes in the hybrid trial field bank and five others: APA 4, Playa Alta, RVTG18, RVTG19 and RVTG20.Clusters II, III and IV, respectively, had 100% members of the genotypes in the international clone field bank, regional variety field bank and local clone field bank (Fig. 1).From the cluster history (data not shown), HYB14 and HYB15 were the first pair of genotypes to unite at 0.000 point of similarity.
Cluster II was most compact; the 15 genotypes it captured formed a single cluster at a similarity coefficient of 0.0082, with IMC47 and UF676 as the first pair of genotypes to unite at 0.0003 (Fig. 1).Cluster IV was equally highly compact; the 13 genotypes it contained formed a unit (a cluster) at 0.0232 similarity coefficient, whereas all the 28 genotypes formed Cluster I at 0.0286 similarity coefficient (Fig. 1).The 21 genotypes in Cluster III were loose, their grouping into a cluster was at a lower similarity coefficient (Fig. 1).

Intra and inter-cluster variability
In Table 4, the cluster with the highest (0.933) intra-cluster similarity was Cluster II, the group of genotypes with the least (0.816) intra-cluster similarity was Cluster III (Table 4).Furthermore, similarities between pair of clusters revealed that cluster I and III had the highest inter-cluster similarity (0.63), whereas cluster III and IV had the least similarity (0.261).The six possible paired comparisons of the four clusters by t-test statistics revealed significant differences between each pair of the four clusters at P ≤ 0.05 (Table 4).
The mean performance of the grouped genotypes in each cluster for the 21 biochemical traits was presented in Table 5. Group of genotypes with the highest mean for crude protein, dispersibility, water absorption capacity, rehydration time, calcium, potassium and iron existed in Cluster I (Table 5).Genotypes in Cluster II had the highest mean for zinc and magnesium.Highest values for crude fat, butterfat, bulk density, oil absorption capacity and phosphorus occurred among genotypes in Cluster III, whereas the genotypes in Cluster IV had the highest mean values for crude fibre, ash, moisture content, theobromine, flavonoid and caffeine (Table 5).

Discussion
Existence of significant difference(s) among genotypes for the measured traits reveals the availability of exploitable variation for nutritional improvement of cocoa.Although the present study was based on a seasonal harvest, the result presents inferential platform for subsequent investigation of the same genotypes.

Plant Genetic Resources: Characterization and Utilization
The 21 studied variables were significantly useful in discriminating the 77 cocoa genotypes in the Nigerian field bank, hence they are polymorphic.The use of polymorphic variables for characterization according to Kaufman and Rousseeuw (1990) do enhances clarity in the grouping pattern of genotypes.
The noted high phenotypic diversity in the studied population for the various biochemical traits makes the cocoa field bank collections of Nigeria a promising valuable resources for nutraceutical breeding and improvement programmes.Aikpokpodion et al. (2009) had much earlier reported high morpho-agronomic diversity in the Nigerian cocoa genetic resources.Identification of differences among the various genotypes agrees with the known axiom that different genotypes have unique genetic identities and hosting unique potentials which are primary resources in plant breeding.
Low CV is a good measure of similarity within sample and hence the reliability of generated descriptive statistics from the data, as such values does not imply diversity.In our study, percentage differences (between the minimum and the maximum values) among the 77 cocoa genotypes for the 21 traits ranged from 5.2% in moisture content to 81% in theobromine.Crude fibre, bulk density, water absorption capacity, caffeine, theobromine, iron, magnesium and zinc hosts wider variabilities having coefficient of range values ≥50%.The large variation observed in this study was expected because the different genotypes did not share the same genetic origin as earlier reported for Nigeria cocoa germplasm by Aikpokpodion et al. (2009) and Olasupo et al. (2018).These arrays of diversity among the extant cocoa genotypes in the field banks of CRIN, Nigeria offer great promises for subsequent cocoa bean nutritional and quality improvements.
Among the 77 genotypes in this study, significant positive correlations existed between some biochemical compounds.This promises to ease independent selection of genotypes for different traits and enhance faster advancement in genetic development of better bean quality in Nigerian cocoa.Summarily from the study, calcium strongly and positively associated with zinc, iron and magnesium, protein improvement in cocoa will positively affect the improvement of calcium, potassium, phosphorus, iron, magnesium and butterfat.In consonance with the report of Jiang et al. (2007), phosphorus and potassium were strongly correlated.
Among the phytochemicals, selection for caffeine will simultaneously and positively affects flavonoid and theobromine, Meng et al. (2009) noted similar relationship.
West African cocoa has long been known to be non-acidic (Wood, 1987), the pH range in the fermented beans in our study was 6.21-7.03,and this is in agreement with Jayeola and Oluwadun (2010) who reported near alkaline to alkaline pH of 6.4-7.4 for some cocoa genotypes in Nigeria.However, our result differed from that of Ndife et al. (2013) who obtained pH range of 5.65-6.15, the variation could be due to genotype, processing and component composition.The low acidity to alkalinity status of cocoa bean reported in this study hinted on the inherent health benefit in consuming cocoa products.The nutritional quality of cocoa beans was further revealed in this study, crude protein ranged between: 12.5-15.1%.This is higher than the reported crude protein content of 8.14% in cocoa powder by Ndife et al. (2013) for unspecified cocoa genotype(s) from Akure, Ondo state, Nigeria.However, in the review by Rawel et al. (2019), a mean crude protein of 11-13% was recorded, however, the work further stated that 11.8-15.7%could be possible depending on the geographical origin of cocoa genotypes.The identified variation among the 77 cocoa genotypes for crude protein in this study present opportunity for genetic improvement of the trait as intercrossing among different genotypes could lead to the evolvement of heterotic progenies.
Diversity is critical to ensuring the future of the world cocoa production and to meeting the challenging and changing climate changes and consumer preferences (CacaoNet, 2012).The classification of the 77 genotypes to the four different groups revealed that there is similarity among genotypes within the same cluster.The hybrids had the best and outstanding performances for 65%  Plant Genetic Resources: Characterization and Utilization of the traits.T63/967 × T17/524 was the most alkaline genotype, T65/7 × T53/8 had the highest protein, MAN 15-2 × T85/799 had the highest fat, GU147A × NA33 had the highest oil content, T82/27 × T16/17 structurally was most dispersible, T60/887 × ICS89 had both the highest bulk density and highest iron content, highest calcium, potassium, phosphorus and zinc was obtained in T86/2 × T53/8, C77 × C67, T65/7 × T79/501 and P7 × T60/887 respectively.The present study re-emphasizes the importance of cross breeding, a method which mostly result in heterosis among hybrids.Individual direct selection can be made on these genotypes for advancement or use as parent in di-hybrid cross breeding programme.For each of the clusters, the highest biochemical traits of significance were: high pH (i.e.low acidity), protein, Ca, K and Fe (cluster I, containing 82% hybrids, 7% introduced clones and 11% regional varieties), high zinc and magnesium (cluster II, containing introduced clones from international sources), high crude fat and phosphorus (cluster III, containing regional varieties) and high crude ash, fibre, theobromine, flavonoid and caffeine (cluster IV, containing the local clones).This provides a hint that the group of genotypes in each of the various field banks have specific uniqueness to which selection can be directed.The 13 genotypes in the local clone field bank constitute the earliest introduction of cocoa genetic resources to Nigeria, comprising very few lower Amazon (e.g.N38), mostly upper Amazon and some Trinitario sub-species.The overall superiority in performances of the different genotypes for the various traits was across four field banks.The observed strict grouping of the thirteen cocoa clones in the local clone field bank with the highest content of theobromine, flavonoids and caffeine hints that some percentage of the earliest cocoa germplasm introduced to Nigeria are still intact and further reveal that the Nigerian cocoa germplasm still hold some genetic materials with strong chocolate flavour that is characteristic of the West African Amelonado.The other clusters where the content of theobromine, flavonoids and caffeine were lower must have undergone selections against the quality traits.Aikpokpodion (2010) informed that Nigerian cocoa farmers hosts significant genetic variability in their fields, genomic divergent within Nigerian cocoa genetic resources is wide (Olasupo et al., 2018), and the present report identified diversity for

Figure 1 .
Figure 1.The grouping of the seventy seven cocoa genotypes by Ward clustering method.

Table 1 .
List of cocoa genotypes in the four active cocoa breeding field banks at the Cocoa Research Institute of Nigeria (CRIN), Ibadan, Nigeria

Table 3 .
Descriptive statistics for the 21 biochemical traits employed for the diversity study of the 77 cocoa genotypes a Genotypes are in parenthesis: RVTG1

Table 4 .
Level of similarity within each cluster (in parenthesis), similarities coefficients by Gower genetic distance method between pairs of cluster (lower diagonal) and probability showing significance in the paired comparison among the four clusters (upper diagonal) *, ** and *** denote significance at 0.05, 0.01 and 0.001 probability level, respectively.

Table 5 .
Mean and variability of the 21 biochemical traits within each cluster (I to IV)