**3. Concept and relevance of genetic diversity**

Although all members of a species have certain traits in common, individual members may vary significantly. While some of these may be environmental, a significant proportion is genetic. Genetic diversity is of fundamental importance in the continuity of a species as it provides the necessary adaptation to the prevailing biotic and abiotic environmental conditions, and enables change in the genetic composition to cope with changes in the environment. The first requisite study in the survival of a species is knowledge about the level of genetic diversity (Van Delden, 1992). This refers to the determination of the number of polymorphic loci, the number of alleles, genetic architecture and spatial distribution of genetic variants. Genetic diversity in a population is preserved when the population is in Hardy Weinberg equilibrium. This means that genotypes are present in expected proportions based on the allele frequencies in the population. However, one or two of the conditions for this may not hold. For instance, introduction of genetic variants through mutation or gene flow from a genetically different population will increase the genetic diversity. Assortative mating will mainly affect genotypic proportions. Directional selection against recessives will eliminate alternative alleles and make the population monomorphic at a particular locus. As a principle, balancing selection in favour of heterozygotes will preserve genetic diversity, while inbreeding and genetic drift, on the other hand, lead to loss of genetic diversity.

#### **4. Utility of genetic markers in defining genetic diversity in cacao**

Genetic markers are inherited variations that can be used to understand genetic events. These include any gene or other DNA variations that are useful for explaining observed genetic event in a population of interest. The use of genetic markers has been useful in the study of population genetics and evolution by providing methods for detecting genetic differences among individuals. The majority of genetic markers are variations in DNA at sites that may or may not be part of a functional gene. However, their transmission to offspring follows Mendelian rules for inheritance. There are three main properties of a genetic marker: It must be locus-specific, polymorphic in the studied population and easily genotyped. The quality of a genetic marker is measured by its heterozygosity in the population of interest, and for a molecular marker, its polymorphism information content (PIC) as described by Botstein *et al*. (1980). Genetic markers that have been used in population genetics are grouped into three main classes: phenotypic or morphological markers, biochemical markers or isozymes and molecular markers utilizing variation at the nuclear DNA level.

### **4.1 Morphological or phenotypic markers**

These are phenotypes for which variation observed in the population of interest can be explained by Mendel's law of inheritance e.g. colour variation, growth habit and fruit shape. Traditionally, morphological markers have been used to characterize varieties based on the assessment of a range of phenotypic characteristics. Several studies have been carried out using morpho-agronomic characteristics of the pods, seeds and flowers to elucidate population structure and genetic diversity of cacao populations (Aikpokpodion, 2010; Bekele and Bekele, 1996; Engels, 1992). Although morphological genetic markers proved useful in several cases, they are subject to several limitations. These include subjectivity in the analysis of character, environmental influences, limited diversity (morphological variants) among cultivars, and restriction of characterization of some useful characters to a particular stage of development, such as flowering or fruit ripening, and limitation to only one locus.

#### **4.2 Allozymes**

Allozymes are allelic variants of enzymes encoded by structural genes. Enzymes are proteins consisting of amino acids, some of which are electrically charged. Due to change in the net electric charge resulting from mutation, allelic variation can be detected by gelelectrophoresis and subsequent specific enzymatic staining. Usually, two or more loci can be distinguished per enzyme, and they are termed isoloci. Therefore, allozyme variation is also referred to as isozyme variation. The study of genetic variation in plant populations was greatly facilitated by the development of protein-based markers (i.e. allozymes). The primary contribution of allozymes to plant population biology has come from their utilization as neutral (or nearly neutral) genetic markers. Allozymes have been employed to characterize patterns of genetic variation within and among populations, and to examine the processes of dispersal and the patterns of mating that influence levels of genetic differentiation. In cacao, several workers such as Ronning & Schnell (1994) and Warren (1994) used isozyme systems to explore genetic diversity among cacao populations.

#### **4.3 Molecular markers**

Use of molecular markers has allowed the complete sampling of the genome, and helped to overcome the limitations of morphological markers and the isozyme markers. DNA markers have been successfully applied in cultivar identification, controlling seed purity of hybrids

differences among individuals. The majority of genetic markers are variations in DNA at sites that may or may not be part of a functional gene. However, their transmission to offspring follows Mendelian rules for inheritance. There are three main properties of a genetic marker: It must be locus-specific, polymorphic in the studied population and easily genotyped. The quality of a genetic marker is measured by its heterozygosity in the population of interest, and for a molecular marker, its polymorphism information content (PIC) as described by Botstein *et al*. (1980). Genetic markers that have been used in population genetics are grouped into three main classes: phenotypic or morphological markers, biochemical markers or isozymes and molecular markers utilizing variation at the

These are phenotypes for which variation observed in the population of interest can be explained by Mendel's law of inheritance e.g. colour variation, growth habit and fruit shape. Traditionally, morphological markers have been used to characterize varieties based on the assessment of a range of phenotypic characteristics. Several studies have been carried out using morpho-agronomic characteristics of the pods, seeds and flowers to elucidate population structure and genetic diversity of cacao populations (Aikpokpodion, 2010; Bekele and Bekele, 1996; Engels, 1992). Although morphological genetic markers proved useful in several cases, they are subject to several limitations. These include subjectivity in the analysis of character, environmental influences, limited diversity (morphological variants) among cultivars, and restriction of characterization of some useful characters to a particular stage of development, such as flowering or fruit ripening, and limitation to only

Allozymes are allelic variants of enzymes encoded by structural genes. Enzymes are proteins consisting of amino acids, some of which are electrically charged. Due to change in the net electric charge resulting from mutation, allelic variation can be detected by gelelectrophoresis and subsequent specific enzymatic staining. Usually, two or more loci can be distinguished per enzyme, and they are termed isoloci. Therefore, allozyme variation is also referred to as isozyme variation. The study of genetic variation in plant populations was greatly facilitated by the development of protein-based markers (i.e. allozymes). The primary contribution of allozymes to plant population biology has come from their utilization as neutral (or nearly neutral) genetic markers. Allozymes have been employed to characterize patterns of genetic variation within and among populations, and to examine the processes of dispersal and the patterns of mating that influence levels of genetic differentiation. In cacao, several workers such as Ronning & Schnell (1994) and Warren

(1994) used isozyme systems to explore genetic diversity among cacao populations.

Use of molecular markers has allowed the complete sampling of the genome, and helped to overcome the limitations of morphological markers and the isozyme markers. DNA markers have been successfully applied in cultivar identification, controlling seed purity of hybrids

nuclear DNA level.

one locus.

**4.2 Allozymes**

**4.3 Molecular markers**

**4.1 Morphological or phenotypic markers**

and checking the genetic relatedness between cultivars. Some of the techniques developed for DNA manipulation in order to detect variations are:
