SCHOOL OF APPLIED NATURAL SCIENCE DEPARTMENT OF

BIOLOGY POST GRADUATE PROGRAM

ASSESSMENT OF GENETIC DIVERSITY OF GROUNDNUT

(ARACHIS HYPOGAEA L.) IN ETHIOPIA USING INTER SIMPLE
SEQUENCE REPEAT MARKER

BY
MOHAMMED ABDELLA
GSR/0196/09

A Thesis Submitted to the Department of Biology in Partial Fulfillment of the

Requirements for the Degree of Master in Biotechnology

August- 2018

Adama, Ethiopia
 ADAMA SCIENCE AND TECHNOLOGY UNIVERSITY
SCHOOL OF APPLIED NATURAL SCIENCE DEPARTMENT OF
APPLIED BIOLOGY POST GRADUATE PROGRAM

ASSESSMENT OF GENETIC DIVERSITY OF GROUNDNUT

(ARACHIS HYPOGAEA L.) IN ETHIOPIA USING INTER SIMPLE
SEQUENCE REPEAT MARKER

BY
MOHAMMED ABDELLA
GSR/0196/09

Advisor: Dr. Mulugeta Kebede

Co-Advisor: Dr. Tilye Feyisa

A Thesis Submitted to the Department of Biology in Partial Fulfillment of the

Requirements for the Degree of Master in Biotechnology

August- 2018

Adama, Ethiopia

ii
 SCHOOL OF GRADUATE STUDIES
ADAMA SCIENCE AND TECHNOLOGY UNIVERSITY

As project advisor, we here by certify that we have read and evaluated this project, under our
guidance, by Mohammed Abdella entitled as assessment of genetic diversity of groundnut
(Arachis hypogaea L.) in Ethiopia using inter simple sequence repeat marker. I recommend
that it be submitted as fulfilling this project requirement

Dr. Mulugeta Kebede _______________ _______________

Advisor signature Date

Dr. Tiliye Feyissa _______________ _______________

Co-advisor signature Date

As the member of the board of the MSC thesis open defense examination, we certify that we
have read evaluated the project prepared by Mohammed Abdella and examined the candidate.
We Recommended that the project be accepted as fulfilling the thesis requirement for the degree
of Master of Biology in Biotechnology.

Chairman Signature Date

____________ __________ _________

Internal Examiner Signature Date

____________ __________ _________

External Examiner Signature Date

____________ __________ _________

Final approval and acceptance of the thesis is contingent upon the submission of the final copy
of the thesis to the Council of Graduate Studies (CGS) through the Departmental Graduate
Committee (DGC) of the candidate’s major department.

I
 PRIVACY STATEMENT

By my signature below, I declare and affirm that this manuscript is my own work. I have
followed all ethical principles of post graduate guidelines in the preparation, data collection, data
analysis and completion of this project manuscript. All scholarly matter that is included in the
manuscript has been given recognition through citation. I verify that I have cited and referenced
all sources used in this document. Every serious effort has been made to avoid any plagiarism in
the preparation of this manuscript. This project manuscript is submitted in partial fulfillment of
the requirements for MSC degree from the School of Graduate Studies at Adama science and
Technology University. I seriously declare that this manuscript has not been submitted to any
other institution anywhere for the award of any academic degree.

Brief quotations from this project manuscript may be used without special permission provided
that accurate and complete acknowledgement of the source is made. Requests for permission for
extended quotations from, or reproduction of, this document in whole or in part may be granted
by the Head of the Department of Biology or the Dean of the School of Graduate Studies when
in his or her judgment the proposed use of the material is in the interest of scholarship. In all
other instances, however, permission must be obtained from the author of the project.

Name: Mohammed Abdella Signature: _____________________

Place: Adama Science and Technology University, Adama, Ethiopia.

Date of submission: ____________________

II
 DEDICATION

I dedicate this project manuscript to my family and all Ethiopian people who are in need to peace
and development. GOD BLESSES ETHIOPIA!!

III
 ACKNOWLEDGEMENT

First of all I would like to praise the Almighty God for best owing up on me health, strength,
patience and protection throughout the study period.

Secondly, I would like to express my sincere gratitude to my advisor Dr. Mulugeta Kebede
and my co-advisor Dr. Tilye Feyisa (head of post graduate reseach, AAU), for their valuable
inputs, guidance, cooperation, enthusiastic encouragement from beginning to the end of this
thesis, Without their constant guidance, endless efforts and joyful encouragement, this
project would have not been accomplished successful.

I would like to thank ASTU for offering me this precious free scholarship opportunity for second
degree of “MSc”. Next, I would like to thank all the academic staff of Biology Department
for their education support , knowledge and faithfulness at the time of learning would be
remembered lifelong.

I also thankful to Laboratory Assistants, other staff members of post graduate biology
Department, AAU, for their helping hands during carrying out this project.

Special thanks to Ethiopia Biodiversity Institute for their guidance for accessing raw materials,
and Awash Melkasa Agricultural Research Center for permitting me to freely use equipment
and green house area which was available in their research center.

Last but not least, my heartfelt gratitude goes to my colleagues and friends specially Alemayehu
Solomon, Abinet Gezmu, Biniyam Eshetu and Nuredin Mohammed for their generous support
and contribution in the accomplishment of this work. I pray to Lord Almighty to reward
abundantly everybody who made a contribution whether knowingly or unknowingly, small or
big, directly or indirectly and mentioned by name or not.

IV
 ABBREVIATIONS

AFLP - Amplified Fragment Length Polymorphism

Bp - Base pair

CTAB - Cetyl Trimethyl Ammonium Bromide

DNA - Deoxyribonucleic Acid

DNTP - Deoxy Nucleotide Tri Phosphate

EBI - Ethiopian Biodiversity Institute

ICRISAT - International Crops Research Institute for the Semi Arid Tropic

ISSR - Inter Simple Sequence Repeat

Mha - Million hectares

NJ - Neighbor Joining

PCoA - Principal Coordinate Analysis

PCR - Polymerase Chain Reaction

RAPD - Random Amplified Length Polymorphism

RLFP - Restriction Fragment Length Polymorphism

SSR - Simple Sequence Repeats

UPGMA - Unweighted Pair group Method with Arithmetic averages

UV - Ultraviolet

V
 TABLE OF CONTENTS
Contents Page

PRIVACY STATEMENT..........................................................................................................................II
DEDICATION..........................................................................................................................................III
ACKNOWLEDGEMENT.........................................................................................................................IV
ABBREVIATIONS....................................................................................................................................V
LIST OF TABLES..................................................................................................................................VIII
LIST OF FIGURES...................................................................................................................................IX
ABSTRACT...............................................................................................................................................X
1. INTRODUCTION...................................................................................................................................1
1.1 Background of the Study...................................................................................................................1
1.2 Statement of the Problem...................................................................................................................3
1.3 Objectives..........................................................................................................................................6
1.3.1 General Objective.......................................................................................................................6
1.3.2 Specific Objectives.....................................................................................................................6
2. REVIEW OF RELATED LITRATURE..................................................................................................7
2.1 The Groundnut (A. hypogaea L.) Crop Origin and Distribution........................................................7
2.2 Taxonomy and Botanical Description................................................................................................8
2.3 Importance of Groundnut.................................................................................................................11
2.4 Major constraints in groundnut production......................................................................................12
2.5 Groundnut Distribution and Production Areas in Ethiopia..............................................................13
2.6 Genetic Resources of Groundnut.....................................................................................................14
2.7 Genetic Diversity of Groundnut.......................................................................................................16
2.8 Types of Different Markers and Their Application in Genetic Diversity Studies............................18
2.8.1. Morphological Markers...........................................................................................................18
2.8.2 Protein (biochemical) Markers..................................................................................................19
2.8.3 Molecular Markers....................................................................................................................20
3. MATERIALS AND METHODS...........................................................................................................32
3.1 Plant Materials.................................................................................................................................32
3.2 DNA Extraction, Quantification and Purity Checking.....................................................................34
3.3 Primer Selection and Optimization..................................................................................................34
3.4 PCR Amplification and Gel Electrophoresis....................................................................................35
3.5 Data Scoring and Statistical Analysis of Diversity..........................................................................36

VI
4. RESULT................................................................................................................................................37
4.1 DNA Isolation, Purification and Quantification...............................................................................37
4.2 ISSR Polymorphism........................................................................................................................37
4.3 Polymorphism information content (PIC)........................................................................................39
4.4 Genetic Diversity.............................................................................................................................40
4.5 Genetic relationship.........................................................................................................................41
4.6 Cluster analysis................................................................................................................................42
4.7 Principal coordinate analysis...........................................................................................................46
5. CONCLUSION.....................................................................................................................................48
6. RECOMMENDATIONS.......................................................................................................................50
7. REFFERENCES....................................................................................................................................51
8. APPENDIX...........................................................................................................................................56

VII
 LIST OF TABLES

Table Page

Table 1. Regions and sites of Ethiopia from where the samples were collected...........................33

Table 2. List of ISSR primers tested for polymorphism and the reproducibility of the amplified

bands..............................................................................................................................................34

Table 3. PCR cycle parameter.......................................................................................................35

Table 4. DNA Amplification Profile and Polymorphism Generated in A. Hypogaea l................38

Table 5. DNA Amplification Profile and Polymorphism information content Generated in A.

Hypogaea L. Using 4 ISSR Primers..............................................................................................39

Table 6. Overall genetic variability across all studied groundnut (A. hypogaea l.) accessions.....40

Table 7. Overall genetic variability across all studied ISSR primers............................................41

VIII
 LIST OF FIGURES

Figure Page

Figure 1. Longitudinal section of a groundnut flower………………………………………..….. 9

Figure 2. A stylized groundnut plant. …………………………………………… …………….. 10

Figure 3. Groundnut producing areas in Ethiopia based on the data from CSA agricultural sample

survey………………………………………………………………………………………….... 14

Figure 4. ISSR marker amplification region on the genome…………………………………….31

Figure 5. Locations and sites of Ethiopia from where the samples were collected…………….. 32

Figure 6. ISSR fingerprint generated from accessions of A. Hypogaea l. from primers: 810, 841,

857 and 881…………………………………………………………………………….……….. 38

Figure 7. UPGMA based dendrogram obtained for 43 accessins using 4 ISSR primers. ……… 44

Figure 8. Neighbor joining analysis of accessions based on 56 PCR bands amplified by four ... 45

Figure 9. PCoA scatter plot diagram showing relationships among groundnut accessions. …....47

IX
 ABSTRACT

Groundnut (Arachis hypogaea L.) belongs to the family Leguminoseae and genus Arachis.
Groundnut is the 13th most important food crop of the world. It is the world’s 4 th most important
source of edible oil and 3rd most important source of vegetable protein. In Ethiopia their
morphological and biochemical variations have already documented but molecular variations
was not studied for this valuable crop. The main objective of this study is to determine the
genetic diversity of forty three groundnut (A. hypogaea L.) accessions from Ethiopia using ISSR
marker. Using four ISSR primers, 56 reproducible bands were generated of which 41 (73.2%)
were polymorphic. The band size ranges from 100 bp to 1100 bp. The number of amplified
bands varied from 10 in primer 841 to 19 in primer 881. The maximum number of polymorphic
bands (100%) was obtained using primer 857 whereas the minimum number of polymorphic
bands (60%) was obtained in Primer 841. Average number of bands and polymorphic bands per
primer were 14 and 10.25 respectively. The polymorphic information content (PIC) value ranged
between 0.36 (for primer 841) and 0.76 (for primer 857) with an average of 0.53. The mean
Nei’s gene diversity and Shannon’s information index were 0.401 ± 0.213 and 0.586 ± 0.359
respectively. Genetic relationship between groundnut genotypes on the basis of Jaccard’s pair
wise similarity coefficients varies from 44% to 83% with an average value of 63.5% i.e. 0.44 to
0.83 average 0.635. The lowest genetic similarity value (i.e. maximum diversity) was found
between genotypes GOBG-1 and GOB-14 (44%) followed (GOG-1 and GAW-1) and (GOB-17
and GOBG-1) at a similarity value of (46%), while highest similarity coefficient (i.e. minimum
diversity) was found between the genotypes (GOB-10 and GOG-6) and (GOB-7 and GOB-16)
(83%) followed by that between (GOG-12 and GOB-9) at a similarity value of (82%), indicating
that they are belonging to similar genetic background. The dendrogram based on cluster analysis
grouped the forty three groundnut genotypes into five distinct clusters at 0.635 or 63.5%
similarity coefficient, and the principal coordinate analysis revealed similar classification. PCoA
analyses clustered the genotypes into individual groups where most of the landraces were
grouped in separate clusters irrespective of their geographic origins. The result indicates that
there was no association between geographical origin and ISSR patterns. According to similarity
and cluster analysis, it could be inferred that crosses involving between (GOBG-1 and GOB-14)
(GOG-1 and GAW-1) and (GOB-17 and GOBG-1) genotypes are the important ones to
improve groundnut through breeding programs. Results of this study would be promising as a
genetic marker for the identification of groundnut accessions and an important source of
knowledge for subsequent groundnut researches such as genetic conservation and plant
germplasm improvement.

Key Words: Ethiopia, Genetic diversity, ISSR, Nei’s gene diversity, PCoA, polymorphic bands,
Shannon diversity index.

X
XI
 1. INTRODUCTION
1.1 Background of the Study
Groundnut (Arachis hypogaea L.) belongs to the family Leguminoseae and genus Arachis.
Groundnut or peanut is an important oil seed crop, which is cultivated and grown throughout
the tropics and sub tropics between 40° South and 40° North of the equator where the annual
rainfall ranges between 500 to 1200 mm and with average daily temperature of higher than
20 °C (Mastewal et al., 2017). It grows best in temperature range of 25 °C to 30 °C in sandy
loam soil which permits easy entry and growth of pegs in soil and harvest of pods.
Groundnut seeds are valued both for their oil and protein contents. The seeds contain about
48% oil, 25% protein and 18% carbohydrates and are rich source of B-complex vitamins,
minerals, antioxidants, biologically active polyphenols, flavanoids and isoflavones. Being a
legume, groundnut improves soil by fixing nitrogen biologically without consuming non-
renewable energies and without disturbing agro-ecological balance (Jiaramraja and
Fantahun, 2014).

Groundnut is the 13th most important food crop of the world. It is the world’s 4 th most
important source of edible oil and 3rd most important source of vegetable protein. Globally,
groundnut is grown on approximately 42 million hectares with a total production of over 35
million tons. Major groundnut growing countries include China, India, the United States and
Nigeria (Taru et al., 2010). Groundnut is one of the four economically important oilseed
crops including noug, flax and sesame in Ethiopia (Mastewal et al., 2017). Besides, this crop
helps small scale producers in getting significant revenue and also helps Ethiopia in getting
foreign money earnings through export (Jiaramraja and Fantahun, 2014).

In Ethiopia groundnut is grown and covered nearly 80,000 hectares (Fredu et al., 2015) of
arable land per annum and the major producing regions which account for most of
groundnut production in Ethiopia are; Eastern Hararghe, Metekel, Benishangul-Gumuz, and
Eastern Wellega Zones but currently this figure is doubled (Addisu and Ermias, 2017; Fredu
et al., 2015). Despite its importance, the improvement of groundnut productivity is stagnant
in the country. Research result showed that groundnut farmers can produce groundnut yields

1
of 2000 kg/ha or more but the national average yield produced by the farmers in Ethiopia is
considerably low, 1200 kg/ha compared to the world average production 1777 kg/ha,
indicating the need of maximum effort to improve productivity (Addisu and Ermias, 2017).

Groundnut molecular genetics and genomics have remained at the early stage because of
lack of emphasis on molecular genetic researches and project funds, lack of sufficient
materials and fully facilitated advanced molecular laboratories. Assessment of genetic
diversity is an important step in any crop improvement program and it plays an important
role because of hybrids between genetically diverse parent’s manifests greater heterosis
and/or genetic recombination (Bhandari et al., 2017) for potential yield increase and food
production than those between more closely related parents. Evaluation of genetic diversity
based on morphological features may not be efficient as they are highly influenced by
environments.

The molecular markers offer many advantages over morphological markers as they are
phenotypically neutral, occur throughout the genome, neither influenced by environments
nor by pleotropic and epistatic interactions, and expression is not dependent on plant age
(Molosiwa, 2012). Recent achievements in the development of marker protocols such as
RFLP, AFLP, ISSR, SNP and SSRs have revolutionized the genetic analysis by detecting
level of polymorphism/genetic diversity, which suggests that it is possible to use molecular
markers in groundnut marker-assisted cultivar improvement and genetic research (Jaroslava
et al., 2002).

Genetic diversity in groundnut, based on morphological, biochemical and molecular

markers, has been reported by many researchers worldwide. However, there is a gap in
published works on genetic diversity of the groundnut collections of Ethiopia based on
molecular markers. ISSR marker is more reproducible and cost effective for researchers in
developing countries like Ethiopia. The technique does not need any prior information about
DNA sequence and overcomes many of the technical limitations of RAPD. The ISSR
techniques have been used in Ethiopia to detect genetic diversity and population structure of
Tef, Coffee, Lentils, Rice and sesame (Abate et al., 2015). Therefore, the aim of this study is
to determine the extent of genetic diversity in groundnut accessions found Ethiopian using

2
ISSR markers and identify highly diverse genotypes for the purposes of broadening the
genetic base of groundnut grown in Ethiopia.

1.2 Statement of the Problem

Ethiopia is home to many crop cultivars and landraces. These varieties were developed
through selection based on agronomic traits. This result in a wide spectrum of varieties that
are highly valued both in domestic and foreign market (Gezahagn, 2013). Morphological
diversity is evaluated with reference to yield and quality, using traditional field plot
techniques. However, these techniques are tedious and time consuming. Furthermore, the
morphological characters may be unstable and influenced by environmental conditions.
Therefore, cultivars do not assure the accurate determination for the analysis of genetic
diversity and extent of distribution by morphological markers (Patel and Galakiya, 2014).

Phenotypic/morphological characterizations of genotypes of this plant species were made

(Yusuf et al., 2017), but the molecular genetic diversity of this plant have not been studied
yet although its genetic diversity has long been based mainly on morphological traits.
However, morphological variability is often restricted; characters may not be obvious to
study its genetic diversity since morphological characters may be affected by environment.
Nowadays, a variety of different genetic markers has been proposed to assess genetic
variability as a complementary strategy to more traditional approaches in genetic resources
management (Somasundaram and Kalaiselvam, 2004). The use of molecular marker will be
helpful for the collection of advanced and novel genotypes. Genomic research can provide
new tools and resources to revolutionize crop genetic improvement and production. It also
provides accurate knowledge at gene level which was not possible with phenotypic markers
(johan et al., 2011). However, genomic research in A. hypogaea is far behind in our country
due to the shortage of essential genome infrastructure, tools, and resources. For this reason,
landraces remain the major source of planting materials currently used by farmers.

Existing attention of market oriented agricultural policy of Ethiopian government;

groundnut is promising income sources for producers. However, the improvement of
groundnut productivity is still stagnant in the country. Research result showed that
groundnut farmers can produce groundnut yields of 2000 kg/ha or more but the national

3
average yield produced by the farmers in Ethiopia is considerably low, 1200kg/ha compared
to the world average production which is 1777.33 kg/ha (Gezahagn, 2013). Hence, to meet
the objectives of the national groundnut improvement program, information on genetic
diversity and relationship within and among populations of A. hypogaea in Ethiopia is
important, since a wide range of genetic diversity among parents is essential for breeding
programs. This lack of genetic background information is a major hindrance to exploit the
wealth of groundnut diversity in Ethiopia, which limits the use of the available genetic
resource as a starting material in breeding program.

This large difference in the lack of molecular research has been attributed to several factors
including non-availability of improved varieties, drought, pest and disease, inappropriate
crop management practices. On the other hand, limited knowledge on the inheritance of
importance traits and lack of proper understanding of genetic diversity at the level of inter
and intra-species and population structure among the lines within and between genera. Thus,
molecular genetic research program contributes a magnificent role for its diversity
maintenance, conservation, collection, improvement, cultivation and utilization. Thus,
precise information on the nature and degree of genetic divergence helps plant breeders in
choosing the diverse parents for purposeful hybridization (Beemnet et al., 2011).
Knowledge on molecular level genetic divergence is, therefore, fundamental to identify and
organize the available genetic resources aiming at the production of promising cultivars
(Beemnet et al., 2011). Therefore, this study was conducted to fill this gap by evaluating the
genetic diversity of A. hypogaea accessions based on ISSR molecular marker. This will help
to narrow the wide research gap currently observed between molecular level genetic studies
and improvement programs of A. hypogaea accessions in Ethiopia.

The development of molecular marker based genetic diversity result has become
increasingly important. Molecular characterization using PCR-based ISSR markers provides
a suitable method, which can be used for varietal identification in (A. hypogaea L.) supplies
and to differentiate between the various grades of A. hypogaea. Molecular marker analysis is
a powerful tool for grouping of genotypes based on genetic distance data and for selection of
progenitors that might constitute new breeding populations. This research generated basic

4
information that could be useful for A. hypogaea L. breeding programs, such as breeding for
increasing yield, wider adaptation, desirable quality, pest and disease resistance,
improvement in yield and quality, cultivar identification, for the management of germplasm
collections, choice for developing heterotic hybrids or germplasm preservation and for the
sustainable management of the genetic resources of A. hypogaea for ecological and
economic gains by better understanding the genetic diversity profile at the species and
population level. This would help in the identification and differentiation of various
groundnuts being cultivated, which is especially important for consumption and export.

In addition, the knowledge of the genetic relationships among these accessions is important
to avoid the use of genetically close materials, allowing for a more efficient use of the
genetic heterogeneity. Better understanding on the diversity of this crop is important for
improvement and exploitation of groundnut at a national level. This study will help to
narrow and fill the wide research gap currently observed between molecular genetic studies
and improvement programs of A. hypogaea in Ethiopia.

5
1.3 Objectives

1.3.1 General Objective

- The main objective of this study is to determine the genetic diversity of selected
groundnut A. hypogaea accessions from Ethiopia using ISSR marker.

1.3.2 Specific Objectives

- To evaluate the potential informativeness of ISSR markers for identifying groundnut
accessions.
- To determine the level and pattern of genetic diversity and degree of polymorphism
among selected A. hypogaea accessions.
- To identify accessions and regions with higher diversity for genetic improvement
and conservation of A. hypogaea.

6
 2. REVIEW OF RELATED LITRATURE

2.1 The Groundnut (A. hypogaea L.) Crop Origin and Distribution
The cultivated A. hypogaea is an ancient crop of the New World, which originated in South
America (southern Bolivia/north west Argentina region) where it was cultivated as early as
1000 B.C. Dissemination of the crop to Africa, Asia, Europe and the Pacific Islands
occurred presumably in the sixteenth and seventeenth centuries with the discovery voyages
of the Spanish, Portuguese, British and Dutch (Krapovickas and Gregory, 1994). The center
of origin for the genus Arachis is the Matto Grosso region of Brazil. Wild species are found
in South America, in a large region bound by the Amazon River to the north, the Río de la
Plata to the south, the Andes mountains to the west, and the Atlantic Ocean to the east.
Because of the occurrence of considerable overlaps in distribution between species in
several sections of the genus, species most likely diverged early in the evolutionary history
of the genus (Susana, 2003). Other archeological evidences also suggest, the center of origin
of the cultivated peanut is believed to be on the eastern slopes of the Andes of southern
Bolivia and northern Argentina because its putative progenitor species have been found only
in this region.

Seven primary centers of diversity have been described for cultivated peanut: (1) Guaraní
region (Paraguay Paraná river basins and southwestern Brazil) for var. fastigiata and var.
vulgaris; (2) Goiás and Minas Gerais region of Brazil (Jocantis-São Francisco river basin)
also for var. fastigiata and var. vulgaris; (Susana, 2003) (3) Rondonia and northwestern
Matto Grosso region of Brazil (headwaters of the Amazon River) for var. hypogaea; (4)
Bolivian region (eastern slopes of the Andes) for var. hypogaea; (5) Peruvian region (upper
Amazon and west coast) for vars. hirsuta, fastigiata and peruviana; (6) northeastern Brazil
for var. fastigiata; (Krapovickas and Gregory, 1994) and (7) Ecuadorian region for var.
aequatoriana. Africa has been described as a secondary center of diversity for cultivated
peanut.

Natural hybridization among types introduced to Africa from Brazil followed by selection is
thought to be responsible for the variation in the African collection (Susana, 2003).

7
Groundnut Spread was; through Portuguese in the late 15 th century, carried two -seeded
groundnut varieties from the east coast of South America (Brazil) to Africa, to the Malabar
coast of southeastern India and possibly to the far east. In the early 16 th century Spaniards
took three -seeded Peruvian runner types (including hirsuta types) to Indonesia, China up to
Madagascar from the west coast of South America via the western Pacific (Krapovickas and
Gregory, 1994). It was later in the 1700s that the ‘Spanish’ types were taken to Europe
where they were grown for oil and human consumption (Stalker, 1997). By the nineteenth
century, groundnut became an important food crop in West Africa, Southeast and South
Asia, and USA, generating rich genetic diversity, More than 100 countries now grow
groundnut (Singh and Nigam, 2016).

2.2 Taxonomy and Botanical Description

Arachis hypogaea is a member of the family Leguminosae, subfamily Fabaceae, tribe
Aeschynomeneae, sub tribe Stylosanthenae (Susana, 2003). The genus Arachis has more
than 70 wild species, of which only A. hypogaea L. is domesticated and cultivated. The
taxonomy of the genus Arachis has been well documented and includes 37 named species
and a number of undescribed species. This genus is morphologically well defined and
distinguished from other genera by having a peg and geocarpic reproductive growth (Figure
2) (Prasad et al., 2010). The genus has been divided into nine sections based on
morphology, geographic distribution, and cross-compatibility. Sections Caulorrhizae,
Erectoides, Extranervosae, Heteranthae, Procumbentes, Trierectoides, and Triseminatae
contain only diploid species (2n = 20) (Susana, 2003). The more evolutionarily advanced
tetraploids (2n = 40) have evolved independently only in sections Arachis and Rhizomatosae
(Prasad et al., 2010). Based on differences in branching pattern and in the presence of
reproductive nodes on the main stream, A. hypogaea is subdivided into two sub species: sub
sp. Hypogaea and sub sp. fastigiata Waldron. Sub species hypogaea has alternate branching
pattern, no reproductive nodes on the main stem, spreading or erect growth habit, a longer
maturation period and fresh-seed dormancy (Susana, 2003). This sub species is subdivided
into botanical varieties hypogaea (Virginia and Runner) and hirsute Köhler (but also known
as Peruvian humpback or Chinese dragon type). Sub species fastigiata has a sequential
branching pattern, reproductive nodes on the main stem; erect growth habit, earlier maturity

8
and little or no seed dormancy (Krapovickas and Gregory, 1994). The two botanical
varieties within subspecies fastigiata are var. fastigiata (Valencia type) and var. vulgaris
Harz (Spanish type). Krapovickas and Gregory (1994) later revised the classification of
cultivated peanut to include the two botanical varieties peruviana Krapovicas and W.C.
Gregory (Valencia type) and aequatoriana Krapov. and W.C. Gregory (Zaruma type), which
are classified with vars. fastigiata and vulgaris within subspecies fastigiata (Susana, 2003).

The plant is an annual herbaceous plant, with an undetermined mode of growth and a
number of varieties belonging to either of the subspecies A. hypogaea ssp. hypogaea or A.
hypogaea ssp. fastigiata (Stalker, 1997). The species and varieties are classified according to
the location of the flowers on the plant, patterns of reproductive nodes on the branches,
number of trichomes, as well as pod morphology (Krapovickas and Gregory, 1994).
Cultivated peanut is generally self-pollinating although little out-crossing does occur with
the assistance of bees, which pollinate the flowers. The wild peanut relatives are also
assumed to be self-pollinating, although not much is known about their mating systems. The
geocarpic habit of peanut is a unique characteristic and could be responsible for dispersal
and thus population structure. They further noted that much of the dispersal is by water and
therefore the species distribution matches to a great extend the flow of major rivers
(Holbrook and Stalker, 2003). For this reason, discoverers observed that most ancient
species were found in higher elevations, their immediate descendants occupied the next
lower eroded surfaces, while the distantly evolved species occupied still lower and more
recently eroded surfaces (Singh et al. 2004).

A. hypogaea seed consists of two cotyledons, a stem axis and leaf primordial, hypocotyls
and primary root. Seed germination is of the epigeal mode and the cotyledons tend to
change color to green after emergence. The primary root system is a tap root system and
numerous lateral roots are visible on the third day after germination. A. hypogaea roots do
not have the normal root hair, but tuffs of hair can be seen on the lateral root (Figure 1). The
former is only restricted to a root zone of 35 cm below the soil surface. Although peanut has
a symbiotic relationship with bradyrhizobium, root hairs are not the primary invasive sites in
contrast to most other legumes (Stalker, 1997). Stems are initially solid, but as the plant

9
grows they tend to become somewhat hollow. The main stem develops from a terminal bud
of the epicotyl and two cotyledonary laterals grow on opposite sides near the soil level. The
main stem can be uptight or prostrate and ranges from 12 to 65 cm in length (Holbrook and
Stalker, 2003). Lateral branches can be prostrate and run along the ground or be upright.
Leaflets on the main stem differ in shape and size from those on lateral branches. Branching
patterns of reproductive to vegetative nodes on the cotyledonary laterals is one of the
primary traits dividing the sub sp, hypogaea (alternating pairs of vegetative: reproductive
nodes) and sub sp, fastigiata (sequential patterns of reproductive nodes). However,
intermediate types are commonly observed (Stalker, 1997).

Figure 1. A stylized groundnut plant (Nigam, 2015).

2.3 Importance of Groundnut

A. hypogaea and its products have been a component of the world’s diet for years. The
geographical location, cultivar type and cultivation conditions influence the nutritional
profile of the nuts. A. hypogaea is the 13th most important food crop and 4 th most important
oilseed crop of the world. The A. hypogaea seeds contain oil and protein (Ingale and
Shrivastava, 2011). A. hypogaea contains 18% carbohydrates with a starch content of 0.5 –
5%, and sucrose content of 4 – 7%. A. hypogaea have also been said to contain 3% ash

10
which is composed of 26 inorganic constituents of which phosphorus, potassium,
magnesium and sulfur are high and virtually unaffected by heat while the remaining though
also heat stable are deficient from a nutritional viewpoint (Jasani, 2009). The oil content of
A. hypogaea is 36 - 54%, of which about 76 – 80% is unsaturated fatty acids. Oleic acid
makes up 40 – 45%, and linoleic acid makes up 30 – 35% of the composition of unsaturated
fatty acid. A. hypogaea is an excellent source of mono- and polyunsaturated fatty acids, with
levels exceeding that of soybean. Though the oils have a high caloric value, they have been
shown to have links to improved cardiovascular health. In addition to mono and
polyunsaturated fatty acids, peanuts are a rich source magnesium, fiber, folate, vitamin E,
copper and arginine, all of which have cardiovascular disease risk reducing properties
(Mattes, 2003). It is also reported, the vitamin profile of A. hypogaea to include riboflavin,
thiamin (which is destroyed to a great degree by roasting and blanching), nicotinic acid and
Vitamin E, with appreciable amounts of B complex vitamins and Vitamin K, but practically
no Vitamins A, C or D (Jasani, 2009) .

The uses of A. hypogaea are diverse; all parts of the plant can be used. The nut (kernel) is a
rich source of edible oil, containing 36 to 54% oil and 16.2 to 32% protein. These proteins
are classified into albumin (water soluble), globulins (salt soluble) and glutelins
(acid/alkaline soluble); the globulins constitute about 87% and consists of arachin and
conarachin (Jasani, 2009). The A. hypogaea kernels are consumed directly as raw, roasted or
boiled kernels or oil extracted from the kernel is used as culinary oil. It is also used as
animal feed (oil pressings, seeds, green material and straw) and industrial raw material (oil
cakes and fertilizer). The crop plays an important role in the dietary requirements of
resource poor women and children and haulms are used as livestock feed (Ingale and
Shrivastava, 2011). About two thirds of world production is crushed for oil, which makes it
an important oilseed crop. It is also used in soap making, medicine, pharmaceuticals,
emulsion for insect control, manufacturing cosmetics and lubricants, fuel for diesel engines,
oleic steering and their salts can be made from A. hypogaea. The residual oilcake contains 7
to 8% nitrogen, 1.5% P2O5 and 1.2% K2O and is used as a fertilizer. It is an important
protein supplement in cattle and poultry rations. It is also consumed as confectionary
product. The cake can be used for manufacturing artificial fiber. The haulms (plant stalks)

11
are fed (green, dried or silage) to livestock. A. hypogaea shell is used as fuel for
manufacturing coarse boards, cork substitutes (Jasani, 2009).

In folk medicine, A. hypogaea is used for aphrodisiac purposes, inflammation,

cholecystosis, nephritis and decoagulant. These multiple uses of groundnut plant make it an
excellent cash crop for domestic markets as well as for foreign trade in several developing
and developed countries (Ahmed, 2012). A. hypogaea is a legume crop; therefore it is good
in nitrogen fixation. It is therefore a good option for crop rotation with maize in the case of
the small scale farmers of the province as they will use less fertilizer. Groundnut should be
grown in rotation with cereals (e.g., maize and sorghum), which have been well fertilized,
because A. hypogaea respond well to fertilizer applied to the previous crop rather than to the
groundnuts themselves (Jasani, 2009). It will further reduce the mono-culture of maize and
other crop production in different regions (Ingale and Shrivastava, 2011).

2.4 Major constraints in groundnut production

A. hypogaea suffers from several biotic and abiotic production constraints. Some of them
are global in nature; and others are either regional or local (Singh and Nigam, 2016). A.
hypogaea breeders and physiologists have been working across the world to improve the
yield of the crop under various biotic and abiotic stresses. Biotic stresses include diseases
such as rust (Puccinia arachidis), early leaf spot (Cercospora arachidicola), late leaf spot
(Phaseoisariopsis personata), crown rot (Aspergillus niger), stem and pod rot, stem
necrosis, rosette disease and pests like termites (Odontotermes sp.), whitegrubs
(Lachnosrerna consanguiea), jassids (Empoasca kerri), aphids (Aphis craccivora), leaf
miners (Aproaerema modicella), red hairy caterpillars (Amsacta albistriga), tobacco
caterpillars and thrips etc, whereas among abiotic stresses, drought is predominant (Prasad
et al., 2010). Drought can occur at any stage—early-season, mid-season, end-of-season, and
intermittent. Drought also predisposes groundnut pods to aflatoxin contamination by A.
flavus. Other abiotic constraints include low soil fertility, salinity, iron chlorosis, aluminum
toxicity, cold temperature at germination, and high temperature at podding and harvest
(Singh and Nigam, 2016).

12
2.5 Groundnut Distribution and Production Areas in Ethiopia
After its first introduction to Eritrea in the 1920 and then to Harer, A. hypogaea is grown in
many lowland areas of Ethiopia. A. hypogaea in Ethiopia is produced mainly by small
holder farmers in the lowlands of the country (Fredu et al., 2015). In terms of area
distribution and production in Ethiopia, A. hypogaea is produced in Oromia around 66%
(52, 921.26 ha) (East and West Welega, Illubabor, East and West Hararghe), Benishangul-
Gumuz (Metekel, Assosa, Kemashi, Pawe, Mao Komo), Amhara (Awi, Oromia zone)
(18,592.72 ha), SNNP (South Omo, Gamo Gofa) (376.66 ha), Harari (Harari) (2874.09 ha)
and Gambela regions (Agnuwak). However, Oromia and Benishangul-Gumuz regions are
the major producing regions which account for most of A. hypogaea production in Ethiopia
(Fredu et al., 2015; Addisu and Ermias, 2017). Oromia region is the leading region in both
area cultivation and production in Ethiopia. It accounts for more than 60 percent of area
cultivation and A. hypogaea production. Next to Oromia, A. hypogaea is widely cultivated
in Benishangul Gumuz (CSA, 2015; Fredu et al., 2015).

Though the crop is grown in five of the nine regions in Ethiopia (Figure 2), Oromia and
Benishangul Gumuz regions account for nearly 90% of the production in the country.
Within these regions, Eastern Hararghe zone in Oromia region (28,909.44 ha) and Metekel
zone in Benishangul-Gumuz region are the main centers for A. hypogaea production.
Eastern Hararghe zone located in the Eastern Ethiopia is characterized by plateaus and
rugged mountains. The altitude ranges from 500 to 3,400 meters above sea level. The
lowlands (<1500 m above mean sea level) constitute 62.2% of the total area and is
characterized as dry sub-humid tropics with an annual average rainfall of ranging between
400-820 mm and temperatures above 25 °C. Metekel zone in Benishangul-Gumuz region in
the western Ethiopia is mainly a low altitude area with an altitudinal range of 550 to 2,500
meters above sea level. About 75% of the zone is low lands with less than 1500 meters
above sea level (CSA, 2015). The climate is predominantly sub-humid to humid tropical.
The average annual temperature ranges between 20 and 25 °C. January to May are the
hottest months during which temperature reaches 28 – 34 °C. The annual rainfall amount
ranges from 500 to1800 mm (CSA, 2015; Fredu et al., 2015).

13
Figure 2. A. hypogaea producing areas in Ethiopia based on the data from CSA agricultural
sample survey (CSA, 2015).

2.6 Genetic Resources of Groundnut

The cultivated A. hypogaea got introduced to different parts of the world and evolved
considerable genetic diversity, while the wild Arachis species constitute genetic reservoir of
useful characteristics, particularly resistance to biotic and abiotic stresses. A. hypogaea
genetic resources have been classified into different gene pools (Radhamani and Singh,
2008). The gene pool concept was proposed in order to provide a genetic perspective and
relationship of cultivated plant species to other components of genetic diversity, to the wild
relatives, based on cross-compatibility into (1) primary gene pool (GP-1), (2) secondary
gene pool (GP-2), and tertiary gene pool (GP-3) (Singh and Nigam, 2016). Application of
this principle facilitates clearer understanding of phylogenetic relationships between the
wild and cultivated species and helps to identify appropriate breeding strategies for
incorporating desired genes into conventionally usable form of cultivated species for
designing new cultivars. This helps to facilitate conventional cytogenetic manipulations to
establish fertile hybrids, improve genetic recombination for incorporation of desirable genes

14
into a usable form and hybridization using pre- or post-fertilization manipulations to
establish hybrids (Singh and Nigam, 2016).

Alternatively, biotechnological approaches may be applied to access genes through sexual

or parasexual means of genetic transformation. This approach has been used in groundnut
classifying the genetic diversity into four gene pools: (1) The primary gene pool consists of
landraces and traditional cultivars of groundnut from primary and secondary centers of
genetic diversity in South America and other groundnut-growing countries and wild A.
monticola found in northwest Argentina having free crossability with A. hypogaea
producing normal segregants, (2) The secondary gene pool consists of diploid species from
section Arachis, cross-compatible with A. hypogaea, despite ploidy differences, producing
sterile to partially fertile hybrids, (3) The tertiary gene pool includes species of section
Procumbentes, which have crossed with diploid species of section Arachis and probably
coevolved with series perennes of section Arachis; Erectoides, whose species are weakly
cross-compatible with diploid species of section Arachis and A. hypogaea; and
Rhizomatosae, whose tetraploid species can be crossed both with diploid species of section
Arachis and A. hypogaea, (4) The quaternary gene pool of the remaining Arachis species
that are cross-incompatible or very weakly cross-compatible to species of section Arachis
and are classified into five other sections (Radhamani and Singh, 2008). Besides the
variability of primary gene pool of cultivated A. hypogaea, the wild Arachis species have
attracted groundnut workers because of their resistance to diseases and insect pests for
which the genetic variation in primary gene pool is limited. The most accessible variability
of primary and secondary gene pools have been successfully utilized in A. hypogaea
improvement and their potential value is now much more predictable and productive. The
exploitation of tertiary and quaternary gene pools waits for advancement in the
biotechnological techniques and policy decision with regard to release of transgenic
varieties at global level (Singh and Nigam, 2016).

2.7 Genetic Diversity of Groundnut

Genetic diversity is the sum of genetic characteristics within any species or genus. Analysis
of genetic relationships in crop species is an important component of crop improvement

15
programs, as it serves to provide information about genetic diversity and is a platform for
stratified sampling of breeding populations (Rao and Hodgkin, 2002). Exploration for
collecting seeds and living plants of cultivated groundnut varieties and wild Arachis species
started in mid-twentieth century by USDA and CSIRO scientists. The first exploration
dedicated to collection of germplasm was conducted in Argentina in 1945 with the initiation
of plant breeding program at the Manfredi Agricultural Experiment Station (Cordoba) and
with the organization of the Department of Plant Exploration and Introduction (DEIP) of the
Ministerio de Agricultura de la Nación. Since then to early 70s, extensive explorations were
made by Krapovickas (CONICET) and Gregory (USDA) collecting live specimens of wild
species and samples of cultivated A. hypogaea. It was followed with introductions of these
collections to other parts of the world (Singh and Nigam, 2016).

In the last few decades, particularly after the establishment of International Crops Research
Institute for Semi-Arid Tropics (ICRISAT), Hyderabad, a significant progress has been
made in the collection of A. hypogaea germplasm from various centers of genetic diversity.
The genetic diversity in relation to certain botanical varieties, such as fastigiata, peruviana
and hirsuta still remains unrepresentative in the world collections at ICRISAT (Radhamani
and Singh, 2008). Additionally, cultivated A. hypogaea accessions were collected from A.
hypogaea -growing areas of various countries, included landraces, farmers’/traditional
varieties, material developed by the breeders and/or released varieties, and the genetic
stocks identified with special features or sources of resistance to biotic and abiotic stresses,
representing different botanical varieties and cultivar groups. A. hypogaea germplasm is
conserved as pods or seeds, except for wild Arachis species belonging to section
Rhizomatosae, which rarely produce seed and if produced, progenies are highly
heterogeneous and therefore are conserved as live plants under controlled conditions
providing an environment close to their habitat (Singh and Nigam, 2016).

Globally, centers maintaining major collection of A. hypogaea genetic resources includes:

Argentina – IBONE/INTA (3534 accessions); Brazil - CENARGEN/EMBRAPA, Brasilia
(3340 accessions); China - Oil Crops Research Institute, Wuhan / National Gene Bank,
Chinese Academy of Agricultural Sciences, Beijing (8349 accessions); ICRISAT - RS

16
Paroda Gene Bank, ICRISAT, Patancheru, India (15,418 representing 95 countries); India –
ICAR - Directorate of Groundnut Research, ICAR, Junagadh (9024 accessions); National
Bureau of Plant Genetic Resources, ICAR, New Delhi (14,585 accessions); USA - Southern
Regional Plant Introduction Station, Griffin, GA (USDA collection - 9917 accessions);
National Seed Storage Laboratory, Ft. Collins, Co (duplicate collection under long - term
storage); NC State University, Raleigh, NC (740 accessions). Other centers are: Senegal -
Senegalese Institute of Agricultural Research (ISRA), Bambey and USA - Texas A&M,
College Station, TX (Nigam, 2015). One of the most significant outcomes of this broad
collecting effort was the publication of a new monograph of the genus Arachis (Krapovickas
and Gregory, 1994) in which 69 species are described, including six botanical varieties of
Arachis hypogaea L. The monograph represents a major milestone for the study and
conservation of peanut genetic resources, and provides a sound scientific foundation upon
which future work can be based (Krapovickas and Gregory, 1994).

Constraints with genetic improvement for assessment of genetic variability and

identification of genetic resources with desired features, the A. hypogaea germplasm
assembled at various places have been characterized and evaluated based on the common
groundnut descriptors developed by IBPGR/ICRISAT (1992). Variability analysis has
shown greater variation in landraces and in the accessions collected from the primary and
secondary centers of diversity in South America, particularly for resistance to biotic and
abiotic stresses and agronomic features like seed mass (Nigam, 2015). An assessment of
genetic diversity on world collection at ICRISAT for 16 morphological and 10 agronomic
traits has shown vast diversity in size and shape of pods and seeds. Principal Component
analysis using 38 traits and clustering on first seven PC scores produced three clusters,
consisting North America, middle East, and East Asia in the first cluster, South America in
the second cluster, and West Africa, Europe, Central Africa, South Asia, Oceania, Southern
Africa, Eastern Africa, Southeast Asia, Central America, and Caribbean in the third cluster.
The means for different agronomic traits differed significantly among regions, while the
variances for all the traits among regions were heterogeneous (Singh and Nigam, 2016).

17
2.8 Types of Different Markers and Their Application in Genetic Diversity

Studies

2.8.1. Morphological Markers

The morphological method is the oldest and considered the first step in description and
classification of plants (Molosiwa et al., 2015). Morphological markers are the earliest
classical methods of genetic markers for assessment of variation based on highly heritable
phenotypic traits such as flower color, seed shape, growth habits and pigmentation (Tadele,
2014). These measures have the advantage of being readily available, do not require
sophisticated equipment and are the most direct measure of phenotype, thus they are
available for immediate use, an important attribute (Praneet and Pankaj, 2015). Phenotypic
traits have the advantage that they may be directly related to the fitness of the populations
and usefulness for plant breeding (Tadele, 2014). The use of morphological and agronomic
traits is a standard way of assessing genetic variation for many species, especially under-
researched crops such as groundnut (Molosiwa et al., 2015). These analyses are carried out
by raising germplasm lines, purelines, improved varieties etc. in a particular experimental
design. This involves morphological characterization of different entries grown in the field
as the morphological characteristics are the strongest determinants of the agronomic value
and taxonomic classification of plants (Bhandari et al., 2017). If phenotypic observations are
based on adequately large sample sizes and the physical traits measured show significant
differences among populations, they can provide a reasonable representation of overall
genetic performance (Tadele, 2014).

However, morphological determinations need to be taken by an expert in the species, they

are subject to changes due to environmental (show continuous variation) factors and may
vary at different developmental stages and their number is limited (Praneet and Pankaj,
2015). They are mostly dominant/recessive, have some biological effect and some
morphological variants cannot survive. Different sets of morphological traits are taken into
consideration for different group of crop plants (Bhandari et al., 2017). In addition to these
shortcomings they show lower polymorphism, complex inheritance, epistatic interaction,
pleotrophic effect, dominant intra-allelic interactions and need extra time as well as

18
resources for evaluation in field and greenhouse. This limits their application in taxonomy
and breeding, can be overcome by the application of biochemical and molecular markers.
But it does not mean that any of the biochemical or molecular or both replace morphological
markers, rather they are used in combination (Tadele, 2014).

2.8.2 Protein (biochemical) Markers

To overcome the limitations of morphological traits, other markers have been developed at
both the protein level (phenotype) and the DNA level (genotype). Protein markers are
usually named biochemical markers but, more and more; they are mistakenly considered as
a common class under the so-called ‘molecular markers (Praneet and Pankaj, 2015).
Biochemical markers (seed storage proteins and isozymes) are markers based on protein
polymorphisms through electrophoretic separation of protein molecules. The assays require
the extraction of proteins from plant tissues, followed by their separation using gel
electrophoresis based on their net charge and size (Tadele, 2014) taking advantage of the
migrational properties of proteins and enzymes, and revealed by histo-chemical stains
specific to the enzymes being assayed (Praneet and Pankaj, 2015). Detecting polymorphisms
i.e. detectable differences at a given marker occurring among individuals in protein markers
is a technique that shares some of the advantages of using morphological ones. However,
protein markers are also limited by being influenced by the environment and changes in
different developmental stages. Even so, isozymes are a robust complement to the simple
morphometric analysis of variation (Praneet and Pankaj, 2015).

Therefore, depending up on number of loci, state of homo-zygosity or hetero-zygosity, and

enzyme configuration, from one to several bands are visualized. The positions of these
bands can be polymorphic and considered as informative loci. Isozymes have provided a
valuable tool for the study of genetic variation in crop populations and to investigate species
relationships (Johan et al., 2011). Seed protein and isozyme profiles as revealed by
polyacrylamide gel electrophoresis are successfully used to resolve taxonomic problems and
describing the genetic diversity within many crops. Seed proteins are considered as practical
and reliable methods because seed storage proteins and nucleotide sequences are largely
independent of environmental fluctuations. The main advantages of protein markers are

19
their co-dominant inheritance, very little environmental influence and low cost of assay.
While disadvantages include restricted number of suitable allozyme (isozyme) loci in the
genome, highly biased genomic sampling, requirement of fresh tissue, and sometimes
limited variation. Therefore, their major shortcomings are lower polymorphism, lower
genome coverage, technical difficulties and the need for specific developmental stage for
certain enzymes (Tadele, 2014).

2.8.3 Molecular Markers

Diversity based on phenotypic and morphological characters, usually varies with
environments and evaluation of traits requires growing the plants to full maturity prior to
identification, but now the rapid development of biotechnology allows easy analysis of large
number of loci distributed throughout the genome of the plants. Molecular makers have
proven to be powerful tools in the assessment of genetic variation and in elucidation of
genetic relationships within and among species (Johan et al., 2011). Understanding the
molecular basis of the essential biological phenomena in plants is crucial for the effective
conservation, management, and efficient utilization of plant genetic resources (PGR) (Linda
et al., 2009). DNA-based molecular markers have acted as versatile tools and have found
their own position in various fields like taxonomy, plant breeding, and genetic engineering
etc. Collecting DNA marker data to determine whether phenotypically similar cultivars are
genetically similar would therefore be of great interest in crop breeding programs (Johan et
al., 2011).

Molecular markers are fixed marks in the genome found at specific locations of the genome,
there are used to identify specific genetic differences. In order to precisely identify traits of
interest, the marker must be close to the gene of interest so that the allele of both the marker
and the gene could be inherited together (Molosiwa, 2012). DNA markers are passed on
from one generation to another through the laws of inheritance. Several markers are
available to choose for genetic diversity studies. The selection criteria could be based on
cost, technical labour, level of polymorphism, reproducibility, locus specificity and genomic
abundance (Molosiwa, 2012). DNA polymorphisms can be detected in nuclear and organelle
DNA, which is found in mitochondria and chloroplasts. Molecular markers concern the

20
DNA molecule itself and, as such, are considered to be objective measures of variation
(Praneet and Pankaj, 2015). Molecular markers may or may not correlate with phenotypic
expression of a genomic trait (Linda et al., 2009).

Molecular markers are the method of choice for genetic diversity assessment and offer
numerous advantages over conventional, phenotype-based alternatives on account of their
hyper variability, high reproducibility, amenability to automation, neutral, stable, (Bhandari
et al., 2017), not subject to environmental pleiotropic and epistatic effects, tests can be
carried out at any time during plant development regardless of growth, differentiation,
development, or defense status of the cell and best of all, they have the potential of existing
in unlimited numbers, covering the entire genome (Praneet and Pankaj, 2015). Molecular
markers can be applied in the identification of cultivars and clones, genetic mapping, marker
assisted selection (MAS), population genetics, molecular systematics, etc. (Ijara, 2015).
Molecular markers can be used for dissecting polygenic traits into their Mendelian
components or Quantitative Trait Loci (QTL) and this increasing understanding of the
inheritance and gene action for such traits allows the use of markers - selection procedures
(Johan et al., 2011).

Molecular markers are also useful in the development of genetic and physical maps, and
have increased the efficiency of indirect selection of marker linked traits (Molosiwa, 2012).
These techniques provide opportunities to obtain high amplification of genetic traits for the
development of genetic maps, variety identification and for the analysis of important
morphological and agronomic traits. In addition, these markers reveal a high level of
polymorphism on plant materials (Bhandari et al., 2017). An ideal molecular marker should
possesses the following features: (1) be polymorphic and evenly distributed throughout the
genome; (2) provide adequate resolution of genetic differences; (3) generate multiple,
independent and reliable markers; (4) be simple, quick and inexpensive; (5) need small
amounts of tissue and DNA samples; (6) link to distinct phenotypes; and, (7) require no
prior information about the genome of an organism. Nevertheless, no molecular marker
presents all the listed advantages (Linda et al., 2009). Different marker systems such as
Restriction Fragment Length Polymorphisms (RFLPs), Random Amplified Polymorphic

21
DNAs (RAPDs), Sequence Tagged Sites (STS), Amplified Fragment Length
Polymorphisms (AFLPs), Simple Sequence Repeats (SSRs) or microsatellites, inter simple
sequence repeat (ISSR), Single Nucleotide Polymorphisms (SNPs) and others have been
developed and applied to a range of crops. The DNA based marker systems are generally
classified as hybridization-based (non-PCR) markers and (PCR)-based markers (Ijara,
2015).

2. 8.3.1 Hybridization (Sequence dependent) Non-Polymerase Chain Reaction (PCR)

method
Molecular markers based on restriction- hybridization techniques were employed relatively
early in the field of plant studies and combined the use of restriction endonucleases and the
hybridization method (Ijara, 2015).

2.8.3.1.1 Restriction Fragment Length Polymorphism (RFLP)

A Restriction Fragment Length Polymorphism, or RFLP, (commonly pronounced "rif lip"),
is a technique that exploits variations in homologous DNA sequences. It refers to difference
between samples of homologous DNA molecules that come from differing locations of
restriction enzyme sites (Tharachand et al., 2012). This was the first molecular marked
technique developed and used in MAS for plant breeding. Saturated molecular genetic maps
based on RFLP markers have been developed for several crops. The technique centers
around the digestion of genomic DNA digested with restriction enzymes (Mishra et al.,
2014). The possibility of comparing band profiles generated after restriction enzyme
digestion in DNA molecules of different individuals (Praneet and Pankaj, 2015). Restriction
endonucleases are bacterial enzymes able to cut DNA, identifying specific palindrome
sequences and producing polynucleotidic fragments with variable dimensions. Any changes
within sequences (i.e., point mutations), mutations between two sites (i.e., deletions and
translocations), or mutations within the enzyme site, can generate variations in the length of
restriction fragment obtained after enzymatic digestion (Linda et al., 2009). These
differences in fragment lengths resolved by gel electrophoresis and western blotting,
Specific banding patterns are then visualized by hybridization with labeled probe (Linda et
al., 2009).

22
The main advantages of RFLP markers are co-dominance, high reproducibility, no need of
prior sequence information, and high locus-specificity (Lateef, 2015). RFLP analysis is a
well-accepted method in plant breeding and is used for many different purposes: e.g. the
selection of traits of agronomic importance linked to RFLP markers, quality testing of seeds
segregation analysis of progenies, evaluation of diversity in a germplasm collection
(Jaroslava et al., 2002), also for the construction of linkage maps and gene tagging in many
crop species. It has been successfully applied for genetic diversity assessments, particularly
in cultivated plants. In self-pollinating legumes such as chickpea, lentil, peanut and soybean,
a very low level of polymorphism has been reported (Tadele, 2014). However, RFLP
technique has a problem of detecting low polymorphism and few loci per assay; it is also not
amenable to automation (Molosiwa, 2012), requires relatively large amounts of purified
DNA, time consuming, laborious and uses probe that is difficult to handle (Ijara, 2015). The
limitations in terms of routine use of RFLP lead to the development of other markers such as
RAPDs (Molosiwa, 2012).

2.8.3.2 Polymerase Chain Reaction (PCR)-based molecular markers

The Polymerase chain reaction (PCR) is a molecular biology technique for enzymatically
replicating (amplifying) small quantities of DNA and useful in studying DNA sequence
variation without using a living organism. It is used to amplify a short (usually up to 10 kb),
well-defined part of a DNA strand (between two specific priming sites in the genome) from
a single gene or just a part of a gene (Tharachand et al., 2012). Polymerase chain reaction
based markers require less DNA per assay than RFLP and are higher throughput (Molosiwa,
2012). The process involves two oligonucleotide primers that flank the DNA fragment of
interest and amplification is achieved by a series of repeated cycles of heat denaturation of
the DNA, annealing of the primer to their complementary sequences, and extension of the
annealed primers with a thermophilic DNA polymerase. Since the extension products
themselves are also complementary to primers, successive cycles of amplification
essentially double the amount of the target DNA synthesized in the previous cycle and the
result is an exponential accumulation of the specific target fragment (Johan et al,. 2011).

23
After the invention of polymerase chain reaction (PCR) technology, a large number of
approaches for generation of molecular markers based on PCR were detailed, primarily due
to its apparent simplicity and high probability of success. Usage of random primers
overcame the limitation of prior sequence knowledge for PCR analysis and facilitated the
development of genetic markers for a variety of purposes mainly in genetic diversity studies
(Milee et al., 2008).

2.8.3.2.1 Random Amplified Polymorphic DNA (RAPD)

In the last decade, the RAPD technique based on the polymerase chain reaction (PCR) has
been one of the most commonly used molecular techniques to develop DNA markers (Firas
and Abdulkareem, 2015). RAPDs were the first PCR-based molecular markers to be
employed in genetic variation analyses (Linda et al., 2009). The principle of RAPD is that, a
single, short oligonucleotide primer (mostly ten bases long), which binds to many different
loci, is used to amplify random sequences from a complex DNA template. This means that
the amplified fragment generated by PCR depends on the length and size of both the primer
and the target genome (Firas and Abdulkareem, 2015). The use of short primers is necessary
to increase the probability that, although the sequences are random, they are able to find
homologous sequences suitable for annealing. DNA polymorphisms are then produced by
rearrangements or deletions at or between oligonucleotide primer binding sites in the
genome. As this approach requires no prior knowledge of the genome analyzed, it can be
employed across species using universal primers (Linda et al., 2009).

In this reaction, a single species of primer anneals to the genomic DNA at two different sites
on complementary strands of DNA template. If these priming sites are within an amplifiable
range of each other, a discrete DNA product is formed through thermo cyclic amplification.
On an average, each primer directs amplification of several discrete loci in the genome
(relatively small number of primers can be used to generate a very large number of
fragments) (Jaroslava et al., 2012), making the assay useful for efficient screening of
nucleotide sequence polymorphism between individuals (Ijara, 2015). With this technique,
there is no specific target DNA, so each particular primer will adhere to the template DNA
randomly. As a result, the nature of the obtained products will be unknown. The DNA

24
fragments generated (usually within the 0.5–5 kb size range) are then separated and detected
by gel electrophoresis. RAPDs can be detected by running PCR products through
electrophoresis on an agarose or acrylamide gel (Praneet and Pankaj, 2015) and view under
ultraviolet light and presence and absence of band was observed (Ijara, 2015).

The power of RAPD is that it is a fast technique, easy to perform and comparatively cheap.
It is immediately applicable to the analysis of most organisms because universal sets of
primers are used without any need for prior sequence information (Jaroslava et al., 2012); it
is viewed as having several advantages compared to RFLP and fingerprint (Firas and
Abdulkareem, 2015). RAPD markers offers many advantages such as higher frequency of
polymorphism, use of fluorescence, requirement of only a few nanogram of DNA, no
requirement of prior information of the DNA sequence and feasibility of automation. The
use of such techniques for germplasm characterization facilitates the conservation and
utilization of plant genetic resources, permitting the identification of unique accessions or
sources of genetically diverse germplasm. The technique is widely used to analyze the
genetic relatedness in several crop species including groundnut. It is also used in the
identification and classification of accessions, identification of breeds and genetic diversity
analysis (Mishra et al., 2014). Nevertheless, disadvantages of RAPD markers are the fact
that it predominantly provides dominant markers, and incapability to detect allelic
differences in heterozygotes. Polymorphisms are detected only as the presence or absence of
a band of a certain molecular weight, with no information on heterozygosity. Additionally,
because of their random nature of amplification and short primer length, they are not ideal
for genome mapping. Moreover, these markers do not exhibit dependable amplification
patterns and differ with the experimental conditions (Lateef, 2015).

2.8.3.2.2 Amplified Fragment Length Polymorphism (AFLP)

AFLP was developed to overcome some of the shortcomings of reproducibility of RAPDs as
the technique combines the digestion of DNA with some specific restriction endonucleases
with a PCR-based technique (Molosiwa, 2012). It is a combination of RFLP and RADP
methods (Ijara, 2015). In this approach the sample DNA is enzymatically cut up into small
fragments (as with RFLP analysis) (Johan et al., 2011), but Only DNA fragments with

25
nucleotides that flank the restriction sites that match the selective nucleotides of the primer
are amplified during PCR . The technique is amenable to high-throughput analysis which is
an added advantage. It is also more efficient and reproducible as compared to the RAPD.
AFLP analysis first uses the restriction digestion of genomic DNA with a combination of
rare cutting (EcoRI or PstI) and frequent cutting (MseI or TaqI) restriction enzymes digest
genomic DNA (Molosiwa, 2012), followed by ligation of double-stranded adaptors to the
sticky ends of the restriction fragment to generate template DNA. The nucleotide sequence
of the adapters and the adjacent restriction sites serve as primer binding sites during
selective PCR amplification of some of these fragments. The amplified fragments are
separated by electrophoresis on polyacrylamide gels and visualized either through
autoradiography or fluorescence methods (Tadele, 2014). Scoring AFLP data is easy since
polymorphisms are recognized in the form of presence or absence of data rather than
determination of sizes at various loci.

The primer pairs used for AFLP usually produce 50-100 bands per assay. The number of
amplicons per AFLP assay is a function of the number selective nucleotides in the AFLP
primer combination, the selective nucleotide motif, GC content, and physical genome size
and complexity (Ahmed, 2012). The origins of AFLP polymorphisms are multiple and can
be due to: (i) mutations of the restriction site which create or delete a restriction site; (ii)
mutations of sequences flanking the restriction site, and complementary to the extension of
the selective primers, enabling possible primer annealing; (iii) insertions, duplications or
deletions inside amplification fragments. These mutations can cause the
appearance/disappearance of a fragment or the modification (increase or decrease) of an
amplified- restricted fragment (Linda et al., 2009).

AFLP represents dominant marker system among the different marker systems available at
present and also provides multi-locus and genome wide marker profiles. These features
make the AFLP technology more suitable for molecular characterization and DNA
fingerprinting of any germplasm collection. In addition, no prior sequence information is
needed for amplification. As a result, AFLP has become extremely beneficial in the study of
taxa including bacteria, fungi, and plants, where much is still unknown about the genomic

26
makeup of various organisms (Tharachand et al., 2012). AFLPs can be applied in studies
involving genetic identity, phenotyping, population genetics, quantitative trait loci (QTL)
mapping, parentage and identification of clones and cultivars, and phylogenetic studies of
closely related species because of the highly informative fingerprinting profiles generally
obtained. Their high genomic abundance and generally random distribution throughout the
genome make AFLPs a widely valued technology for gene mapping studies (Ijara, 2015),
these include establishing linkage groups in crosses, saturating regions with markers for
gene landing efforts and assessing the degree of relatedness or variability among cultivars
(Milee et al., 2008). However, there are some limitations of AFLP includes: (1) It requires
more number of steps to produce the result, (2) It requires template DNA free of inhibitor
compounds that interferes with the restriction enzyme, (3) The technique requires the use of
polyacrylamide gel in combination with AgNO3 staining, radioactivity, or fluorescent
methods of detection, which will be more expensive and laborious than agarose gels, (4) It
involves additional cost to purchase both restriction and ligation enzymes as well as
adapters, and (5) Most AFLP loci are dominant, which does not differentiate dominant
homozygotes from heterozygotes. This reduces the accuracy of AFLP markers in population
genetic analysis, genetic mapping, and marker assisted selection (MAS) (Mishra et al.,
2014).

2.8.3.2.3 Simple Sequence Repeat (SSR)

Microsatellites, also known as simple sequence repeats (SSR), variable number tandem
repeats (VNTR) and short tandem repeats (STR) are tandem repeats motifs of 1-6
nucleotides found at high frequency in the nuclear genomes of most taxa. It provided a
choice for many genetic researches since they are amenable to low, medium and high-
throughput approaches (Lateef, 2015). It consist of tandemly repeated 2-7 base pair units
arranged in repeats of mono-, di-, tri-, tetra and penta-nucleotides (A,T, AT, GA, AGG,
AAAG etc.) with different lengths of repeat motifs. These repeats are widely distributed
throughout the plants and animal genomes that display high level of genetic variation based
on differences in the number of tandemly repeating units of a locus (Johan et al., 2011).
PCR amplification protocols used for microsatellites employ either unlabeled primer pairs or
primer pairs with one of the primers being radiolabelled or fluorolabelled. Electrophoresis of

27
unlabelled PCR products can be carried out on smaller vertical polyacrylamide gels or on
horizontal agarose gels (Molosiwa, 2012). If nucleotide sequences in the flanking regions of
the microsatellite are known, specific primers (generally 20–25 bp) can be designed to
amplify the microsatellite by PCR. Polymerase slippage during DNA replication, or slipped
strand mispairing, is considered to be the main cause of variation in the number of repeat
units of a microsatellite, resulting in length polymorphisms that can be detected by gel
electrophoresis (Ijara, 2015). Basically there are two strategies used for microsatellite
development: microsatellites markers can be sourced based on DNA sequence information
deposited in the databases (mining in public libraries/databases) or through screening of
genomic DNA libraries specifically constructed for discovery of repeated sequences in the
genome (Molosiwa, 2012). The development of microsatellite markers involves several
distinct steps from obtaining the library to developing a working set of primers that can
amplify polymorphic microsatellite loci. These include: (1) Microsatellite library
construction, (2) Identification of unique microsatellite loci, (3) Identifying a suitable area
for primer design,(4) Obtaining a PCR product, (5) Evaluation and interpretation of banding
patterns and (6) Assessing PCR products for polymorphism (Mishra et al., 2014).

In natural plant populations, microsatellites have great potential for helping to understand
what determines patterns of genetic variation, particularly when used in concert with
chloroplast DNA (cpDNA) markers. Their utility has been demonstrated in studies of
genetic diversity, cultivar identification, mating systems, pollination biology and seedling
establishment (Ahmed, 2012). It is reported that among different classes of molecular
markers, SSR markers are useful for a variety of applications in plant molecular biology
(Mishra et al., 2014), this includes integrating the genetic, physical and sequence-based
physical maps in plant species, and simultaneously have provided breeders and geneticists
with an efficient tool to link phenotypic and genotypic variation (Mishra et al., 2014).

SSRs have several advantages over other molecular markers. For example, (1)
microsatellites allow the identification of many alleles at a single locus, (2) they are evenly
distributed all over the genome and they are co-dominant, (3) little DNA is required and the
analysis can be semi-automated and performed without the need of radioactivity

28
(Tharachand et al., 2012), (4) microsatellites can offer more detailed population genetic
insight than maternally inherited mitochondrial DNA (mtDNA) because of the high
mutation rate and bi-parental inheritance (5) highly polymorphic and specific (Ahmed,
2012), (6) very repeatable, (7) so cheap and easy to run (8) need a small amount of medium
quality DNA and with the advance of DNA isolation technology, it was possible to identify
loci in highly degraded ancient DNA (aDNA), where traditional enrichment procedures
have been unsuccessful, (9) with the development of high-throughput sequencing platforms,
such as the GS-FLX (Roche, Branford, CT, USA) SSR has recently become fast and
efficient (Ahmed, 2012). With the above mentioned features microsatellites have become
the genetic markers of choice in many plant systems (Trachand et al., 2012). On the other
hand these markers have several disadvantages: expensive, laborious and time-consuming.
The low frequency of SSRs in plants also hinders the large scale isolation of SSRs (Firas
and Abdulkareem, 2015).

2.8.3.2.4 Inter Simple Sequence Repeat (ISSR)

Soon after the discovery of the polymerase chain reaction (PCR) in 1983, new PCR-based
DNA marker systems were continuously being developed. In the early 1990’s, the
development of what would become today’s ‘inter-simple sequence repeat (ISSR)’ markers
was independently reported by several research groups (Ahmed, 2012). Inter Simple
Sequence Repeat (ISSR) markers have been proposed as a new source of genetic markers
that are inherited in Mendelian fashion and are scored as dominant markers. ISSR analysis
offers breeders and geneticists with competent means to link phenotypic and genotypic
variations and is rapidly being used by the research community in various fields of crop
improvement (Rao et al., 2007). ISSRs are first reported by Zietkiewicz et al., 1994 (Praneet
and Pankaj, 2015). ISSRs are DNA fragments about 100 - 3000 bp in length which is
located between flanking microsatellite regions (Figure 3) (Omondi et al., 2016). ISSR
marker technique is a PCR based method, which involves amplification of DNA present at
an amplifiable distance in between two identical microsatellite repeats oriented in opposite
direction. The technique uses microsatellites, usually 16-25 bp long as primers in a single
primer PCR reaction targeting multiple genomic loci to amplify the inter simple repeat
sequences of different sizes. The microsatellite repeats used as primers for ISSRs can be di,

29
tri, tetra or penta-nucleotides (Mishra et al., 2014). ISSRs use longer primers (15–30 mers)
as compared to RAPD primers (10 mers), which permit the subsequent use of high
annealing temperature leading to higher stringency. The annealing temperature depends on
the GC content of the primer used and ranges from 45 to 65ºC. The amplified products are
usually 100–3000 bp long and amenable to detection by both agarose and polyacrylamide
gel electrophoresis (Mishra et al., 2014). By single microsatellite primers ISSRs of different
sizes are amplified in a PCR reaction targeting multiple genomic loci. Thus, fragments of
several loci are generated at once, separated by gel electrophoresis and scored for presence
or absence (Omondi et al., 2016). In contrast to other molecular markers, the target
sequences of the ISSR primers are abundant throughout the eukaryotic genome and evolve
quickly, which consequently helps reveal a much larger number of polymorphic loci than
other dominant markers such as RAPD. These characteristics make ISSR markers attractive,
especially when the discussion is about species that have not yet been studied from the
genetic point of view using molecular markers, such as A. hypogaea. (Firas and
Abdulkareem, 2015).

Generally, ISSR markers are unmapped but can be used to saturate RFLP and SSR linkage
maps, and generate species specific, gene specific and trait specific markers. The
evolutionary rate of change within microsatellites is considerably higher than most other
types of DNA based markers, so the likelihood of polymorphism in these sequences is
greater. The source of variability in the ISSRs can be attributed to template DNA, nature of
primer used and detection method (Tadele, 2014). ISSR Polymorphism may relate to
mutations at the priming site that prevent amplification giving a presence/absence pattern
while insertion/deletion events within the SSR region or the amplified region would result in
the absence of a product or, more rarely, in length polymorphism, depending on the
amplifiability of the resulting fragment size (Mishra et al., 2014).

30
Figure 3. ISSR marker amplification region on the genome (Omondi et al., 2016).

The ISSR method has several benefits over other techniques: first, it is known to be able to
discriminate between closely related genotypes and second, it can detect polymorphisms
without any previous knowledge of the crop's DNA sequence (Ahmed, 2012). In addition, it
does not require genome sequence information; it leads to multilocus, highly polymorphous
patterns and produces dominant markers (Mishra et al., 2014). ISSR PCR is a fast,
inexpensive genotyping technique based on variation in the regions between microsatellites
(Ahmed, 2012). ISSRs segregate mostly as dominant markers following simple Mendelian
inheritance. ISSR analyses offer breeders and geneticists with competent means to link
phenotypic and genotypic variations in various fields of plant improvement (Tadele, 2014).
The technique was subsequently applied to numerous plant genetics studies: identification of
cultivars, genetic mapping/fingerprinting, assessment and characterization of genetic
diversity, bio geographical studies ,gene tagging, marker assisted selection, determining
SSR motif frequency and studies on natural populations/ speciation, marker assisted
selection, detection of soma clonal variation and molecular systematic. Despite these
advantages, ISSRs can have problems in reproducibility, and their dominant inheritance and
homology of co-migrating amplification products result in similar problems like for RAPDs
(Omondi et al., 2016).

31
 3. MATERIALS AND METHODS
3.1 Plant Materials
Seeds of 43 A. hypogea accessions were obtained from Ethiopian Biodiversity Institute
(EBI), Addis Ababa, Ethiopia which was originally collected from different geographical
locations of Ethiopia (Gursum 19, Babile 21, Jigjiga 1, Amhara 1, Bale 1) (Figure 4, Table
1). Seeds of all the 43 A. hypogaea accessions were grown in a greenhouse and fresh leaves
from 4 week old plants were used for genomic DNA extraction. Five young leaves were
collected from five randomly selected individual plants of each accession just before
flowering and equal proportions of leaves were grinded for genomic DNA extractions.
Genomic DNA was extracted from leaf samples following (Wang et al., 1994).

Figure 4. Locations and sites of Ethiopia from where the samples were collected

Table 1. Regions and sites of Ethiopia from where the samples were collected

32
Number Gene bank Code Collection region locality
accession number
1 19739 GOB 1 Oromia/Misrak/Babile Berkele/s 1
2 19740 GOB 2 Oromia/Misrak/Babile Berkele/s 2
3 19741 GOB 3 Oromia/Misrak/Babile Babile/ Shek Abdi
4 19742 GOB 4 Oromia/Misrak/Babile Awsherit 1
5 19743 GOB 5 Oromia/Misrak/Babile Awsherit 2
6 19744 GOB 6 Oromia/Misrak/Babile Lecole
7 19745 GOB 7 Oromia/Misrak/Babile Ifa Gende 1
8 19746 GOB 8 Oromia/Misrak/Babile Ifa Gende 2
9 19747 GOB 9 Oromia/Misrak/Babile Ifa Gende 3
10 19748 GOB 10 Oromia/Misrak/Babile Medigana 1
11 19749 GOB 11 Oromia/Misrak/Babile Medigana 2
12 19750 GOB 12 Oromia/Misrak/Babile Dendaro
13 19751 GOB 13 Oromia/Misrak/Babile Tofic 1
14 19752 GOB 14 Oromia/Misrak/Babile Tofic 2
15 19753 GOB 15 Oromia/Misrak/Babile Berkele
16 19754 GOB 16 Oromia/Misrak/Babile Gende
17 19755 GOB 17 Oromia/Misrak/Babile Gemechu
18 19756 GOB 18 Oromia/Misrak/Babile Tula
19 19757 GOB 19 Oromia/Misrak/Babile Tula About
20 19758 GOB 20 Oromia/Misrak/Babile Abdul 1
21 19759 GOB 21 Oromia/Misrak/Babile Abdul 2
22 19760 GOG 1 Oromia/Misrak/Gursum Llalemi 1
23 19761 GOG 2 Oromia/Misrak/Gursum Llalemi 2
24 19762 GOG 3 Oromia/Misrak/Gursum Awdal
25 19763 GOG 4 Oromia/Misrak/Gursum Oda 1
26 19764 GOG 5 Oromia/Misrak/Gursum Oda 2
27 19765 GOG 6 Oromia/Misrak/Gursum Oda 3
28 19766 GOG 7 Oromia/Misrak/Gursum Oda 4
29 19767 GOG 8 Oromia/Misrak/Gursum Oda 5
30 19768 GOG 9 Oromia/Misrak/Gursum Nur Selam 1
31 19769 GOG 10 Oromia/Misrak/Gursum Nur Selam 2
32 19770 GOG 11 Oromia/Misrak/Gursum Odaa 1
33 19771 GOG 12 Oromia/Misrak/Gursum Odaa 2
34 19772 GOG 13 Oromia/Misrak/Gursum Abader
35 19773 GOG 14 Oromia/Misrak/Gursum Harobata 1
36 19774 GOG 15 Oromia/Misrak/Gursum Harobata 2
37 19775 GOG 16 Oromia/Misrak/Gursum Harobata 3
38 19776 GOG 17 Oromia/Misrak/Gursum Harobata 4
39 19777 GOG 18 Oromia/Misrak/Gursum Awdal 1
40 19778 GOG 19 Oromia/Misrak/Gursum Awdal 2
41 19779 GSJ -1 Somali/Jigjiga Beledka
42 24208 GAW -1 Amhara/Adoawe Wangua
43 28662 GOBG-1 Oromia/Bale Ginir

33
3.2 DNA Extraction, Quantification and Purity Checking
Genomic DNA extraction was done based on the modified CTAB method described in
(Wang et al., 1994) which involves minor modification for individuals of each accession to
yield optimal amounts of DNA. About 50mg Silica-gel dried leaves for each accession were
ground with mix and miller machine at Genetics Research Laboratory, Department of
Biology Addis Ababa University (AAU). For optimum amount of DNA for ISSR-PCR
reaction the first extraction was used, taken for polymerase chain reaction (PCR). The
samples were stored at 4°C until electrophoresis and subsequent dilution and PCR reaction.
The yield of DNA isolated was measured using a Nano Drop ND-8000 UV
spectrophotometer. Moreover, the purity and concentration of DNA was determined by
agarose gel electrophoresis by running the samples on 1 % TBE agarose gel.

3.3 Primer Selection and Optimization

A total of 9 ISSR primers that were available at the genetics laboratory were used for the
initial testing of variability and reproducibility. For optimization and screening of primers,
one individual representing each accession with 1:5 dilutions and concentration was
adjusted after running test gel using 1 % TBE agarose gel. All pre-selected 9 primers were
used for reproducibility and polymorphism test and evaluation. Finally, three dinucleotide
primers (primer 810, 841 and 857), and one penta-nucleotide primer (primer 881) (Table 2)
were selected based on polymorphism and reproducibility.

Table 2. List of ISSR primers tested for polymorphism and the reproducibility of the
amplified bands.

Number Primers Annealing Sequence of Amplification

To nucleotides (5’- 3’) pattern

1 UBC 810 45 GAGAGAGAGAGAGAGAT Polymorphic,

Reproducible
2 UBC 812 45 GAGAGAGAGAGAGAGAA X
3 UBC 824 48 TCTCTCTCTCTCTCTCG X
4 UBC 840 45 GAGAGAGAGAGAGAGAYT X
5 UBC 841 48 GAGAGAGAGAGAGAGAYC Polymorphic,
Reproducible
6 UBC 842 45 GAGAGAGAGAGAGAGAYG X

34
7 UBC 848 45 CACACACACACACACARC X
8 UBC 857 48 ACACACACACACACACYG Polymorphic,
Reproducible
9 UBC 881 48 GGGGTGGGGTGGGGTG Polymorphic,
Reproducible
Source: Primer kit 900 (UBC 900); Y = Pyrimidines (C or T), R = purines (A or G).

3.4 PCR Amplification and Gel Electrophoresis

The polymerase chain reaction was done using Biometra 2000 T3 Thermo cycler. PCR
amplification was carried out in a 25 μl total reaction mixture containing 1 μl template
DNA, 18 μl ddH20, 0.5 μl dNTP (1.25 mM), 2.5 μl PCR buffer (10xThermopol reaction
buffer), 2.5 μl MgCl2 (2 mM), 0.5 μl primer (20 pmol/μl) and 0.5 μl Taq Polymerase (5
U/μl). The amplification program was 4 min preheating and initial denaturation at 94°C,
then 39 x 30 s at 94°C, 1 min primer annealing at (45/48°C) based on primers used, 1.30
min extension at 72°C. The final extension for 7 min at 72°C followed (Table 3). The PCR
products were also stored at 4°C until loaded on gel for electrophoresis.

Table 3. PCR cycle parameter.

Step Temperature Duration

1. Initial Denaturation 94⁰C 4 mins

2. Denaturation 94⁰C 30 sec

3. Annealing 45⁰C-48⁰C 1 min

4. Extension 72⁰C 1.30 mins

5. Final Extension 72⁰C 7 mins

Total Cycles 39 cycles

The amplification products were checked first on test gel using 1% agarose gel using 1 X
TBE for the presence of ISSR-PCR products. The amplified products were run on to ISSR
gel using 1.67% agarose, with 1 X TBE using gel electrophoresis chamber. The ISSR gel
(1.67 % TBE) were prepared using 300 ml TBE mixed with 5.01 gram agarose using 500 ml

35
Erlenmeyer flask and then boiled in micro oven for 3 minutes. After it was cooled for about
20 minutes at room temperature, 12µl Ethidium Bromide (10mg/ml) added and
the gel poured on casting system and allowed to solidify for more than 2 hrs. Eight micro
litter ISSR amplification products and 2µl (6X) loading dye were mixed thoroughly and
loaded on the gel. A 100 base-pair ladder were used to estimate the molecular size of the
DNA fragments. The electrophoresis was run for 2 hours with at constant voltage of 100 V.
The ISSR profiles then visualized and photographed under UV light using Biometra Biodoc
analyzer.

3.5 Data Scoring and Statistical Analysis of Diversity

Data Scoring and Statistical Analysis of Diversity ISSR bands were scored as present (1),
absent (0) and missing data (?) and entered into a binary matrix representing the ISSR
profile of each sample. The 0/1 matrix were used to calculate the Jaccard coefficient for all
possible pairs of samples using Free Tree 0.9.1.50 (Pavlicek et al., 1999) and NTSYS-pc
version 2.02 Rohlf (2000) software. Jaccard‟s similarity coefficient which is calculated as:
a
s ij=
a+ b+c
Where, a is the total number of bands shared between individuals i and j, b is the total
number of bands present in individual i but not in individual j and c is the total numbers of
bands present in individual j but not in individual i. The resulting similarity matrices were
employed to construct Neighbor Joining (NJ), which is used to compare individual
genotypes and evaluate patterns of genotype clustering and UPGMA-based phenogram, to
analyze and compare the individual genotypes (Dhwani et al., 2017). The major difference
between the two algorithms is that UPGMA assumes equal rates of evolution (molecular
clock assumption) along all branches, whereas neighbor joining assume variations in the rate
of change (Dhwani et al., 2017). Nie’s gene diversity and Shannon’s Weaver diversity index
(I) were computed with POPGENE software 1.32 (Yeh et al., 1997). The matrix of genetic
similarity will be also used in a principal coordinate analysis (PCoA) to resolve the patterns
of clustering among the genotypes based on Jaccard’s coefficient. The calculation of
Jaccard’s coefficient were made with PAST software version 1.18 (Hammer et al., 2001).

36
 4. RESULT

4.1 DNA Isolation, Purification and Quantification

The amount of DNA isolated from various genotypes of A. hypogaea Ranged from
529 to 2904 mg/pl (Data not shown). The genotype GOG-12 yielded the highest amount of
DNA (2904 ng/pl), whereas the lowest amount of DNA (529 ng/pl) was obtained from
genotype GOB-18. The ratio of absorbance (A260/A280) ranging from 1.70 to 1.96 revealed
that the DNA obtained was free from contaminants.

4.2 ISSR Polymorphism

Out of the 9 primers tested initially, four of them with clear and reproducible banding
pattern were selected and used in this investigation (Table 2). The molecular weight of the
bands amplified using the four primers were in the range of 100 to 1100bp (Figure 5). Four
ISSR primers produced PCR fragment that ranged from 10 to 19 which were total generated
56 bands across the 43 accessions, of which 41 were polymorphic (Table 4). The
polymorphic bands of the primers ranged from 10 (primer 841) to 19 (primer 881) across the
genotypes. Average number of bands and polymorphic bands per primer were 14 and 10.25
respectively. The percentage of polymorphism for primers ranged from 60 to 100, with an
average polymorphism percent of 73.2 % (Table 4). In this investigation, the di-nucleotide
primers, namely 810, 841 and 857 were observed to have fourteen (64.29 percent), ten (60
percent) and thirteen (100 percent) polymorphic loci, respectively. The penta-nucleotides
primer 881 was observed to have 13 (68.42 percent) polymorphic loci, respectively.

37
Table 4: DNA Amplification Profile and Polymorphism Generated in A. hypogaea L. Using
4 ISSR Primers

Primer Sequence Ta oC Total number Polymorphic % of

of bands bands polymorphism
810 (GA)7T 45 14 9 64.29%

841 (GA)7YC 48 10 6 60%

857 (AC)7G 48 13 13 100%

881 (GGGGT)3G 48 19 13 68.42%

Total 56 41 73.2%

Our data indicated that ISSR technology can detect considerable polymorphisms (73.2%) in
our genotypes, suggesting that it will be useful in A. hypogaea germplasm characterization
and fingerprinting purposes. This study provides fundamental evidence that ISSR marker is
a simple, informative, reproducible and suitable approach to evaluation of molecular
diversity in groundnut. A representation of the ISSR band profile obtained with primer
UBC810, 841, 857 and UBC 881 are shown in (Fig. 5).

38
Figure 5. ISSR fingerprint generated from accessions of A. hypogaea from primers:
810,841, 857 and 881.

4.3 Polymorphism information content (PIC)

Polymorphism information content is the probability of detection of polymorphism by a
primer/primer combination between two randomly drawn genotypes and depends on the
number of detectable alleles and the distribution of their frequency. The PIC value, as a
relative measure level of polymorphism varied from 0.36 (UBC 841) to 0.76 (UBC 857)
with an average value of 0.53 (Table 5).

Table 5: DNA Amplification Profile and Polymorphism Generated in A. hypogaea Using 4

ISSR Primers.

Primer Total Polymorphic % of polymorphism PIC

number of bands
bands

810 14 9 64.29% 0.43

841 10 6 60% 0.36

857 13 13 100% 0.76

881 19 13 68.42% 0.58

Total 56 41 73.2% 0.53

Fifty percent of the primers (2 primers) had PIC values above 0.53 indicating that these
primers are highly informative for determining A. hypogaea accessions polymorphism.
Comparison of the number of polymorphic bands with the PIC values revealed that a greater
number of polymorphic bands were associated with higher values of PIC. The higher PIC
value indicated the informativeness of the primers. The most informative primer in the
present study was UBC 857 with PIC value of 0.76.

39
4.4 Genetic Diversity
The mean observed number of alleles (Na) was 1.6512 ± 0.4822. A value of the mean
effective number of alleles (Ne) was 1.5187 ± 0.4121. The Nei’s gene diversity (H) ranged
from 0.303 ± 0.216 in ‘GOB 17 and GOG 13’ to 0.4997 ± 0.209 in ‘GSJ-1 and GOB 19’
with an average value of 0.401 ± 0.213 (Table 3). The Shannon’s indices (I) ranged from
0.4804 ± 0.3073 in ‘GOB 17’ to 0.6929 ± 0.4102 in ‘GSJ-1’ with an average value of 0.586
± 0.359. Total gene diversity (Ht) and gene diversity among accessions (Hs) were 0.3066 ±
0.042 and 0.131 ± 0.065, respectively, indicating the existence of moderate genetic diversity
among accessions. The coefficient of gene differentiation (Gst) among accessions was
0.5836. Based on the Gst value, the mean estimated number of gene flow (Nm) between
accessions was found to be 0.3567 (Table 6).

Table 6. Overall genetic variability across all studied A. hypogaea accessions.

Sample Na ± Ne ± H± I± Ht ± Hs ± Gst Nm
size (SD) (SD) (SD) (SD) (SD) (SD)

43 1.6512 1.5187 0.401 0.586 0.3066 0.131 0.5836 0.3567

± ± ± ± ± ±

0.4822 0.4121 0.213 0.359 0.042 0.065

Na = Observed number of alleles. Ne = Effective number of alleles. H = Nei's (1973) gene diversity. I =
Shannon's Information index. Ht = Total genetic diversity. HS = Gene diversity among accesssions. Gst =
Coefficient of genetic differentiation among accessions. Nm = Estimate of gene flow from Gst or Gcs. e.g.,
Nm = 0.5(1 - Gst)/Gst.

In terms of primers, the highest Observed number of alleles (0.980 ± 0.640) and effective
number of alleles (1.85 ± 0.12) was shown by primer 857 and the least Observed number of
alleles (1.600 ± 0.520) and effective number of alleles value (1.42 ± 0.41) was shown by
primer 841. The highest gene diversity (0.46 ± 0.036) and Shannon index (0.657 ± 0.037)
was shown by primer 857 and followed by primer 810, with gene diversity and Shannon
index value of (0.46 ± 0.036 and 0.413 ± 0.322), respectively (Table 7) and the least value
of genetic diversity and Shannon index was recorded in primer 841 with 0.25 ± 0.22 and

40
(0.364 ± 0.326) value respectively. Both gene diversity measurements and Shannon’s
indices, showed moderate polymorphism among Ethiopian groundnuts.

Table 7. Overall genetic variability across all studied ISSR primers

Primer Na ± (SD) Ne ± (SD) H ± (SD) I ± (SD)

810 1.643 ± 0.500 1.54 ± 0.42 0.31 ± 0.23 0.413 ± 0.322

841 1.600 ± 0.520 1.42 ± 0.41 0.25 ± 0.22 0.364 ± 0.326

857 1.980 ± 0.640 1.85 ± 0.12 0.46 ± 0.036 0.657 ± 0.037

881 1.680 ± 0.480 1.52 ± 0.39 0.29 ± 0.21 0.419 ± 0.291

Average 1.726 ± 0.535 1.58 ± 0.32 0.32 ± 0.17 0.463 ± 0.244

Na = Observed number of alleles. Ne = Effective number of alleles. H = Nei's (1973) gene diversity. I =
Shannon's Information index.

4.5 Genetic relationship

Knowledge about genetic constitute differences of genotypes is highly important in breeding
programs. The importance is because of the need to identify parents with high genetic
distances to produce progenies with higher heterosis. In this study, generated similarity
matrix by ISSR based on the Jaccard’s similarity coefficient matrices showed similarity
ranged from 44% to 83% i.e. 0.44 to 0.83 or 56% to 17% with an average value 36.5% of
genetic diversity or dissimilarity (Data not shown). The average similarity across the 43
genotypes was found to be 63.5% or indicating a moderate level of genetic similarity
amongst them. The lowest genetic similarity value (i.e. maximum diversity) was found
between genotypes GOBG-1 and GOB-14 (44%) followed by that between (GOG-1 and
GAW-1) and (GOB-17 and GOBG-1) at a similarity value of (46%), while highest
similarity coefficient (i.e. minimum diversity) was found between the genotypes (GOB-10
and GOG-6) and (GOB-7 and GOB-16) (83%) followed by that between (GOG-12 and
GOB-9) at a similarity value of (82%). High genetic similarity values (Sij) of 0.82 to 0.83
were obtained for the genotypes, indicating limited genetic diversity.

41
This high level of similarity could be due to having common ancestors and or selecting
similar traits during breeding programs in Gursum or Babile landraces. However, the
distances between the accession of the landraces were much lower indicating the genetic
relatedness of the various geographic accessions which may be caused by the high rates of
gene flow due to exchange of germplasm among farmers and groundnut trades across the
regions. Generally, from the above result of Jaccard’s similarity value collection of
groundnut accessions, GOBG-1 from (Bale/Ginir) and GOB-14 (Oromia/Babile) followed
by that between GOG-1 (Oromia/Gursum) and GAW-1 from (Amhara/Wangua), GOB-17
(Oromia/Babile) and GOBG-1 (Bale/Ginir) were the most divergent and therefore might
have a larger possibility of heterosis in a breeding programs aimed to improve productivity
or agronomical traits in groundnut.

4.6 Cluster analysis

Evaluation similarity coefficients or dendrogram helps to select superior individuals to
obtain hybrids with greater segregation and heterotic effects during recombination. Based on
Jaccard’s coefficients UPGMA and neighbor joining (NJ) analysis were used to construct
dendrogram for 56 ISSR-PCR fragments scored from 43 individuals. The above values on
Jaccard’s similarity coefficient were used to construct the UPGMA (unweighted pair group
method with arithmetic mean) dendrogram employing NTSYSpc. Clustering analysis put
the genotypes into one cluster at 59.2% similarity. They were however clustered in to five
clusters based on the cut-off point of 63.5% similarity equivalent to the mean genetic
similarity of all the accessions Figure 7. Cophenetic correlation created from comparing
similarity and output matrix of the dendrogram was 87% or 0.87, which indicated that the
cluster generated by Jaccard’s similarity was a good fit to the matrix. .

Cluster I being the major cluster included twenty one genotypes at a similarity of 64%,
which further divided into two sub clusters. In sub cluster I eight genotypes were included
viz. GOBG -1, GOB -1, GOB -2, GOB -6, GOB -15, GOB -21 GOG -11 and GOG-13 at a
similarity value of 67%. These genotypes showed relatively high similarity with one other
compared to other clusters containing different genotypes. Genotypes GOG-13 and GOB -
15 showed maximum similarity followed by GOG -11 and GOB -1, the genotype GOBG -1

42
is the most divergent genotype of this group. In sub cluster II thirteen genotypes were
included viz. GOG -2, GOG -4, GOG -5, GOG -6, GOG -10, GOG -14, GOG -16 and GOG
-19, GOB -3, GOB -7, GOB -8, GOB -10, and GOB -16 at a similarity value of 66%.
Genotypes GOG -6 and GOB -10 showed maximum similarity followed by GOB -7 and
GOB -16, genotype GOG -2 is the divergent genotype of this group. cluster II included four
genotypes viz. GOB -12, GOB -18, GOG -9 and GSJ-1 at a similarity value of 65%.
Genotypes GOB -18 and GOG -9 showed maximum similarity and the genotype GSJ-1 is
the most divergent genotype of this group. Cluster III included six genotypes viz. GOB -5,
GOB -13, GOB -19, GOG -3, GOG -7 and GOG -18 at a similarity value of 64.8%.
Genotypes and GOB -5 and GOG -18 showed maximum similarity followed by GOB -19
and GOG -7, genotype GOG -3 is the most divergent genotype of this group. Cluster IV
included nine genotypes viz. GOB -4, GOB -9, GOB -11, GOB -20, GOG -8, GOG -12,
GOG -15, GOG -17 and GAW -1. These genotypes showed relatively low similarity with
one other compared to other clusters containing different genotypes. Genotypes GOB -9 and
GOG -12 showed maximum similarity and the genotype GOG -15 is the most divergent
genotype of this group. Cluster V included three genotypes viz. GOB -14, GOB -17 and
GOG -1 at a similarity value of 65%. Genotypes GOB -14 and GOB -17 showed maximum
similarity and the genotype GOG -1 is the most divergent genotype of this group.

The dendrogram of individuals (Figure 6) did not divide the individuals into distinct groups
resembling the geographically-defined accessions. The accession clustering follows
geographical proximity which shows the presence of extensive gene flow among
neighboring regions or zones. The UPGMA analysis of the individuals in each accession
revealed that almost all individuals were distributed and inter-mixed with individuals of
another locality. While comparing the clusters or subclusters within different groups of
germplasm, no specific/significant trend of grouping of genotypes was observed. However,
a loose clustering of genotypes as per their geographical origin was observed. For example,
germplasm from Babile (62.5%) and Gursum (61.5%) were grouped in cluster I sub cluster I
and II, respectively (Figure 6). Generally, individuals were evenly distributed along the
dendrogram, revealing moderate level of genetic diversity.

43
Figure 6. UPGMA based dendrogram obtained for 43 accessions using 4 ISSR primers.
Key: GOG – Oromia/Gursum, GOB – Oromia/babile, GOBG – Bale/Ginir, GSJ –
Somalia/jigjiga, GAW – Amhara/Wangua

Based on genotyping data for 56 ISSR-PCR fragments, a NJ tree was constructed using
DARwin5. Like that of the UPGMA analysis of the individual accession the dendrogram
derived from neighbor-joining analysis of the whole ISSR data were not showing a clear
grouping (Figure 7). No accessions were tended to form their own cluster all over the tree
i.e. all individuals were inter-mixed. Generally, the dendrogram analysis using A. hypogaea
individual plant form inter-mixed cluster between accessions since average levels of genetic

44
variation is detected in almost all accessions investigated. As visible in the dendrogram, the
genotypes that were closer were more similar than those that were lying apart.

Figure 7. Neighbor joining analysis of accessions based on 56 PCR bands amplified by four
ISSR primers.
Key: GOG – Oromia/Gursum, GOB – Oromia/babile, GOBG – Bale/Ginir, GSJ –
Somalia/jigjiga, GAW – Amhara/Wangua

45
4.7 Principal coordinate analysis
Principal coordinate analysis (PCoA), as a complementary technique for cluster analysis, is
one of the important multivariate statistical approaches to group based on similarity
coefficients. The dependability of the dendrogram and the principal coordinate analysis
intensely supports the reliability of the marker system. All the data obtained using 4 ISSR
primers were used for PCO analysis using Jaccard’s coefficients of similarity. The first four
components of the coordinates of the PCO having eigen values of 1.61, 1.44, 1.24 and 1.11
with variance of 10.2%, 9.1%, 7.9 and 7.0%, respectively, used to show the grouping of
individuals using two co-ordinates (Figure 8) . The result indicated that the first two
principal coordinates, PCoA1 and PCoA2 explained 34.3% and 25.6% of the variation
respectively and together explained 59.9% of the total variation. Similar to the UPGMA
clustering pattern, the 43 germplasm accessions of groundnut were grouped into five groups
(clusters) based on the principal co-ordinate analysis.

The plots helped to visualize the genetic relationships among the accessions and supported
the results obtained from the phylogenetic tree analysis. Cluster I contained fourteen
genotypes i.e. GOG (3, 4, 5, 13, 14, 15, 16 and 19), GOB (2, 8, 12, 13 and 19) and GOBG-1.
Cluster II contained eight genotypes i.e. GOG (7, 8, 9 and 18), GOB (5, 11 and 18) and
GAW-1. Cluster III included eight genotypes i.e. GOG (2 and 11), GOB (1, 3, 7, 10 and 16)
and GSJ-1. Cluster IV included seven genotypes i.e. GOG (1, 6 and 10) and GOB (6, 14, 15
and 21). Cluster V contained six genotypes i.e. GOG (12 and 17) and GOB (4, 9, 17 and
20). As visible in the plot, the genotypes that were closer were more similar than those that
were lying apart. Generally, the PCoA result indicated that most of the accessions did not
group together with other genotypes from the same geographical region. One possible
reason for this could be the exchange of A. hypogaea germplasm among farmers across
regions. This result confirms the result obtained in all the above diversity indices. However,
most of the landraces that represent different geographical regions were found to form
distinct groups and spread all over the plot (Figure 8). The result of principal co-ordinate
analysis (PCoA) was partially in accordance with the cluster analysis.

46
Figure. 8. PCoA scatter plot diagram showing relationships among A. hypogaea accessions.

47
 5. CONCLUSION

Evaluation of genetic diversity based on morphological features has not proven to be

efficient as they are highly influenced by environments. To overcome these problems,
biochemical and molecular techniques have been found to yield better results. ISSR marker
technique is a PCR based method, which involves amplification of DNA present at an
amplifiable distance in between two identical microsatellite repeats oriented in opposite
directions. Such DNA markers are considered a best tool for determining genetic
relationship/diversity as they are abundant in number, highly polymorphic and is
independent of environmental interaction i.e. are highly heritable. The development of
precision molecular techniques for genetic analysis has almost replaced the morphological
and biochemical analysis to a great extent. The present study is the first report on genetic
diversity of A. hypogaea using ISSR marker. The molecular analysis results using four ISSR
primers revealed a level of 73.2% of polymorphic loci among accessions. This is relatively
high for a self-pollinated species such as A. hypogaea. This level of genetic variation is in
consistent with Rungnoi et al. 2012 who reported 72.4% of polymorphism among 363
accessions using ISSR markers and also similar with Suvendu et al. 2009 who reported
74.7% polymorphism using ISSR markers among 20 A. hypogaea accessions. This level of
genetic variation is relatively high when compared with Raina et al. (2001) who reported
54% of polymorphism among 13 A. hypogaea accessions using ISSR markers. Other studies
have reported a higher level of polymorphic loci 87% in ISSR markers (Dwhani et al.,
2017). This could suggest that the primers used may represent a key factor for detecting
molecular variability. To get an objective discrimination of genetic diversity of cultivated
peanut and make a correct classification of them, substantially more DNA markers and more
genotypes would be needed. The estimates of genetic relationships can be useful for
organizing germplasm for conservation of genetic resources, for the identification of
cultivars, for selection of parents for hybridization, for predicting favorable heterotic
combinations and for reducing the number of accessions needed to ensure sampling a broad
range of genetic variability in breeding programmes. Accessions with the most distinct DNA
profiles are likely to contain the greatest number of novel alleles.

48
The genetic diversity data generated by four ISSR primers revealed that moderate level of
genetic diversity exists in A hypogeae Based on Jaccard’s similarity coefficient, highest
genetic similarity was observed between GOB -10 with GOG -6 and GOB -7 with GOB -16
and the second highest genetic similarity was observed between GOG -12 with GOB -9.
These results could suggest that the homogeneity among the accessions (geographic region
based) could be due to genetic flux or that they may had a common origin, alternatives that
needs to be further explored. On the other hand, the least genetic similarity was detected
between GOBG -1 with GOB -14 followed by GOG -1 with GAW -1 and GOB -17 with
GOBG -1 or this genotypes showed a good amount of genetic divergence and would
therefore be useful in broadening genetic base of landraces of groundnut in Ethiopia. The
size and number of polymorphic fragments, percentage of polymorphic loci, together with
the overall gene diversity indices reported in the study indicated moderate level of genetic
diversity among the germplasm lines. The most diverse accessions identified in this study
should be given a due emphasis to include them in groundnut breeding program of the
country. Both PCoA and UPGMA cluster analysis approved the clustering of all 43
genotypes into five groups. Cluster analyses of the present study showed that most
genotypes of the landraces did not group together with other genotypes from the same
geographical region. The close relationship between genotypes from the different
geographical locations might be due to gene flow caused by the exchange of germplasm
through farmers and traders across regions of Ethiopia. In conclusion, this study reported a
successful fingerprinting of A. hypogaea accessions using ISSR and demonstrated the
usefulness of these markers in estimating the extent of genetic variation in A. hypogaea
germplasm. The use of ISSR markers in A. hypogaea must be further continued in order to
drive specific linkage between ISSR markers and genes controlling agronomically important
characters. These diagnostic molecular tools will greatly assist in the identification of new
and different sources of diversity which may help breeders to decide what genotypes to
cross for making new genetic combinations and to determine which genetic resources
should be retained in a collection in order to conserve maximum genetic diversity in the
gene bank.

49
 6. RECOMMENDATIONS

♦♦♦ The limitations of resources like Primers, dNTPs, Taq Polymerase and other reagents
due to their high cost on top of limited local availability, hindered the present study so that
only limited sample sizes and few primers were used. Hence, further study with more
sample size and geographic range would give more clear patterns of diversity for the whole
study underlying causes of the observed genetic differences noted in this study.

♦♦♦ Analysis with molecular markers, such as AFLP, SSR and other newly emerged
molecular markers, which can be easily used for genetic screening and complete
characterization of existing populations are recommended.

♦♦♦ Analysis of genetic diversity in plant species using more than one methods helps to
better understand the levels of genetic variation, the genetic structure of populations and
determine migration route. Therefore, researchers, policy makers and other stakeholders
need to come-up with sustainable use and conservation.

♦♦♦ This study is not exhaustive in terms of representative samples, and its size and area
coverage including outside the country. Hence, more survey and sample collection have to
be carried out to reveal more diversity of the species.

50
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 8. APPENDIX
REAGENTS AND SOLUTIONS:

A. 1.0 M Tris-HCl (pH 8.0)

Dissolve 12.11gm Tris HCl in sterile de-ionized water, adjust pH to 8.0 with conc. HCl, and
make up volume to 100 ml with de-ionized water and autoclave at 15 psi for 20 min.

B. 0.5 M EDTA ( pH 8.0)

EDTA (dissolved salt; Mw = 372.3) = 18.61gm

Dissolve, 18.61gm EDTA in sterile de-ionized water, adjust pH to 8.0 with 5N NaOH, make
up volume to 100 ml with de-ionized water and autoclaved at 15 psi for 20 min.

C. 5 M NaCl

NaCl = 29.2 gm

Dissolve 29.2gm NaCl in sterile de-ionized water, make up volume to 100 ml with de-
ionized water and autoclave at 15 psi for 20 min

D. Extraction buffer

1M Tris-HCl (pH 8.0) = 10 ml

0.5 M EDTA (pH 8.0) = 2 ml

3 M NaCl = 46.6 ml

2% CTAB (w/v) = 2 g

0.2% ß-Mercaptoethanol = 0.2ml

Dissolve the above and make up to 100 ml with de-ionized water and autoclave at 15 psi For
20 min.

56
E. 10% working C-TAB

10% CTAB = 10 g

5 M NaCl = 14 ml

Dissolve the above in water, make up to 100 ml and autoclave at 15 psi for 20 min.

F. 3M NaOAC (pH 4.8)

Sodium Acetate = 24.61 gm

Dissolve 24.61 gm NaOAC in sterile de-ionized water, adjust pH to with glacial acetic acid,
make up volume to 100 ml with de-ionized water and autoclave at 15 psi for 20 min.

G. Choloroform : Iso-amyl alcohol mixture (24:1) (100 ml)

Choloroform = 96 ml

Iso-amyl alcohol = 4 ml

H. 70% ethanol (100ml)

Absolute alcohol = 70 ml

Double distilled water = 30 ml

I. RNase stock

1 M Tris- HCL (pH 8.0) = 100 µl

5 M NaCl = 300 µl

RNase = 10 mg

Adjust volume to 1 ml with de-ionized water, boil for 15 min and allow to cool slowly and
stored at -20ºC

J. TE (10:1)

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1 M Tris-HCl (pH 8.0) = 1.0 ml

0.5 M EDTA (pH 8.0) = 0.2 ml

Dissolve the above and make up volume to 100 ml with de-ionized water and autoclave at
15 psi for 20 min.

K. 10 X TBE (pH 8.0) Tris base = 108.0 g Boric acid = 55.0 g EDTA (0.5M) = 20ml
Dissolve the above and make up volume to 1000 ml with double distilled water.

LIST OF INSTRUMENTS USED IN:

A. DNA extraction, purification, quantification.

(a) Electronic balance

(b) Micropipette (2µl,20µl,100µl,1000µl)

(c) Water bath

(d) Layophilizer

(e) Deep Freezer (-20°C)

(f) Refrigerator

(g) Fume hood

(h) Table top centrifuge

(i) Vortex

(j) Eppendorf tubes(0.5ml,2ml)

(k) Tips (200µl,1000µl)

(l) Quant Fluorimeter

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(m) Quartz Cuvette (n) pH meter

(o) Water purification system

(p) Hybridization oven

(q) Vertical autoclave

B. ISSR analysis

(a) Thin walled PCR tubes

(b) PCR

(c) Laminar air flow

(d) Micro centrifuge

(e) Pipett

(f) Pipette tips

C. Gel Electrophoresis

(a) Gel electrophoretic unit

(b) UV transilluminator

(c) Power pack (300)

(d) Microwave oven

(e) Laboratory shaker

(f) Gel Doc. System

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