GENETIC AND MORPHOLOGICAL DIVERSITY IN MONODORA MYRISTICA (GAERTN) DUNAL IN EASTERN NIGERIA

TABLE OF CONTENTS

CHAPTER 1

INTRODUCTION                                                                                                     

1.1    BACKGROUND INFORMATION

1.2    GENETIC DIVERSITY IN PLANTS

1.3    RATIONALE

1.4    OBJECTIVES

CHAPTER 2

LITERATURE REVIEW

2.1    TAXONOMYAND NOMENCLATURE of Monodoramyristica

2.2    GENERAL DESCRIPTION OF Monodoramyristica

2.3    DISTRIBUTION OF Monodora myristica

2.4    CHEMICAL COMPOSITION OF Monodoramyristica

2.5    USES OF Monodoramyristica

2.6    GENETIC DIVERSITY IN ANNONACEAE

2.7    TECHNIQUES IN MOLECULAR ANALYSIS OF GENETIC DIVERSITY

2.7.1 MOLECULAR MARKERS

2.7.2 PRINCIPLE OF THE RAPD TECHNIQUE

2.8    PLANT LEAF PRESERVATION

2.8.   SILICA GEL PRESERVATION

2.9    DNA EXTRACTION

2.9.1 DNA extraction methods

2.10  DNA QUANTITATION/QUALITATION

2.10.1 SPECTROPHOTOMETRIC quantification

2.10.2 Quantificatin using flourescent dyes

2.11  GEL ELECTROPHORESIS

CHAPTER 3

METHODOLOGY

3.1    GERMPLASM IDENTIFICATION ANDSTUDY SITES

3.2    MORPHOLOGICAL AND YIELD STUDIES

3.2.1COLLECTION OF MORPHOLOGICAL AND YIELD DATA

3.2.2ANALYSIS OF MORPHOLOGICAL AND YIELD DATA                       

3.3.   MOLECULARSTUDIES

3.3.1 STUDY SITE

3.3.2  PRESERVATION OF SAMPLES

3.3.3 LYOPHILIZATION

3.3.4 DNA EXTRACTION

3.3.5 SPECTROPHOTOMETRIC ANALYSIS

3.3.6  PROCEDURE FOR GEL ELECTROPHORESIS

3.3.7 RAPD-PCR

3.3.7  AGAROSE GEL ELECTROPHORESIS

3.3.9 ANALYSIS OF MOLECULAR DATA

CHAPTER 4

 RESULTS

4.1    MORPHOLOGICAL AND YIELD ATTRIBUTES

4.1.1 MEANS, STANDARD ERRORS AND ANALYSIS OF VARIANCE

4.1.2 PRINCIPAL COMPONENT ANALYSIS     

4.1.3 CLUSTER ANALYSIS BASED ON MORPHOLOGICAL ATTRIBUTES

4.2    GENETIC DIVERSITY

CHAPTER 5

5.0    Discussion

SUMMARY AND CONCLUSION

Appendix

CHAPTER 1

INTRODUCTION                                                                                                     

1.1 BACKGROUND INFORMATION

Monodoramyristica (Gaertn.)Dunal.,also known as African nutmeg or calabash nutmeg, is a tropical tree of the family Annonaceae (Custard-apple family).  Its seeds are widely used as an inexpensive nutmeg substitute becauseof the similarity between the two in odour and taste. Nowadays, however, it is less common outside its region of production(Celtnet recipes, 2011)

The genus Monodora contains approximately 15 to 20 species includingMonodora borealis, Monodoraclaessensii andMonodoragrandiflora.Monodoramyristica is easily recognizable by its very long and pendulous pedicels, an undulate upper bract, a large globose fruit with a black and smooth but finely ribbed surface (Burkill, 1985).          

The Calabash nutmeg tree is native to tropical West Africa, where it grows naturally in evergreen forests from Liberia to Nigeria and Cameroon. It is also native to Angola,Uganda and West Kenya (Weiss, 2002). Due to the slave trade in the 18th century, the tree was introduced to the Caribbean islands where it was established and became known as Jamaican nutmeg (Barwick 2004). In 1897, it was introduced to Bogor Botanical Garden, Indonesia, where the trees flowered on a regular basis but no fruit could yet be collected (Weiss, 2002).

1.2 GENETIC DIVERSITY IN PLANTS

Genetic diversity refers to any variation in the nucleotides, genes, chromosomes, or whole genomes of organisms. At its most elementary level, it is represented by differences in the sequences of nucleotides (adenine, cytosine, guanine, and thymine) that form the DNA within the cells of the organism. Nucleotide variation is measured for discrete sections of the chromosomes, called genes. Thus, each gene comprises a hereditary section of DNA that occupies a specific place of the chromosome, and controls a particular characteristic of an organism (Harrison et al, 2004).

Diversity enhances the chances of populations’ adaptation to changing environments. With more variation, it is more likely that some individuals in a population will possess variations of alleles that are suited for the environment. Such individuals are more likely to survive to produce offspring bearing that allele. The population can thus continue for more generations because of the success of these individuals (NBII, 2011).

Most organisms are diploid, having two sets of chromosomes, and therefore two copies (called alleles) of each gene. However, some organisms can be haploid, triploid, tetraploid or more (having one, three, four or more sets of chromosomes respectively) (Harrison, et al, 2004). Within any single organism, there may be variation between the two (or more) alleles for each gene. This variation or polymorphism is introduced either through mutation of one of the alleles, or as a result of reproduction processes,especially if there has been migration or hybridization of organisms, so that the parents may come from different populations and gene pools. Harmless mutations and sexual recombination may allow the evolution of new characteristics which increases diversity(Andayani,et al.,2001).

Each allele codes for the production of amino acids that string together to form proteins. Thus differences in the nucleotide sequences of alleles result in the production of slightly different strings of amino acids or variant forms of the proteins.These proteins code for the development of the anatomical and physiological characteristics of the organism, which are also responsible for determining aspects of the behavior of the organism (Harrison, et al, 2004).

Plant diversity is part of the biological diversity and contributes towards achieving food security, poverty alleviation, environmental protection and sustainable development(Frankel 1984). It is being eroded rapidly in important spice plants and other crops mainly because of replacement of traditional landraces by modern, high yielding cultivars, natural catastrophes (droughts, floods, fire hazards, etc.), as well as large scale destruction and modification of natural habitats harboring wild species(Frankel 1984, Bramel-cox and Chritnick, 1998).M. myristica population is threatened by urbanization which damages its natural habitat, and leads to the cutting of most of the trees without replanting. Additionally, the plant is listed under Kew’s difficult seeds due to its inability to easily grow outside its natural habitat(Burkill, 1985). Genetic variation in traditional landraces and wild species is essential to combat pests and diseases and to produce cultivars better adapted to constantly changing environments(FAO, 1994).

Molecular tools such as have been found to be more useful and accurate in the study of inter-species and intra-species genetic diversity in several plants. Randomly amplified polymorphic DNA (RAPD) markers have been successfully employed for determination of intraspecies genetic diversity in several plants. These include Phaseolus vulgaris (Razviet al., 2013),Ocimumspp (Sairkaret al., 2012), Chrysanthemum (Martin et al., 2002), Annonacrassiflora( Cotaet al.,2011), Prosopis ( Goswami and Ranade, 1999), date palm (Corniquel and Mercier, 1994), papaya (Stiles et al., 1993), poplars (Bradshaw, et al., 1994) and amaranths (Ranade, et al. 1997). No such attempt has so far been reported for Monodoramyristica

1.3RATIONALE

M. myristica is largely harvested from the wild and greatly affected by wild fires, urbanization, reckless and uncontrolled felling of trees for timber and firewood without replanting. There is need, therefore, to initiate breeding programs for this orphan crop by first documenting available genetic and phenotypic variations in this crop. The present report was done with this in mind, and should provide the much needed baseline for further studies.

1.4 OBJECTIVES

The general aim of the project was to characterize accessions of African nutmeg inSouth eastern Nigeria and estimate the range and distribution of genetic diversity.

             The major objectives of this work were:                            

v  To determine the level of genetic diversity among 21 accessions of Monodoramyristica using RAPD technique

v  To compare morphological and yield related traits among the accessions using analysis of variance tests

v  To confirm the efficiency of RAPD technique in genetic diversity studies of this important plant.

v  To identify traits contributing significantly to variation in this species.

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