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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