SCHOOL OF APPLIED NATURAL SCIENCE DEPARTMENT OF

BIOLOGY GRADUATE PROGRAM

GENETIC DIVERSITY OF GROUNDNUT

(Arachis hypogaea L.) IN ETHIOPIA USING INTER SIMPLE
SEQUENCE REPEAT MARKER

BY
MOHAMMED ABDELLA

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

Requirements for the Degree of Master of Science in Biotechnology
 ADAMA SCIENCE AND TECHNOLOGY UNIVERSITY
SCHOOL OF APPLIED NATURAL SCIENCE DEPARTMENT OF
APPLIED BIOLOGY GRADUATE PROGRAM

ASSESSMENT OF GENETIC DIVERSITY OF GROUNDNUT

September- 2018

(Arachis hypogaea L.) IN ETHIOPIA USING INTER SIMPLE

Adama, Ethiopia
SEQUENCE REPEAT MARKER

BY
MOHAMMED ABDELLA
GSR/0196/09

Advisor: Dr. Mulugeta Kebede

Co-Advisor: Dr. Tileye Feyissa

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

the
Requirements for the Degree of Master of Science in Biotechnology

September- 2018

Adama, Ethiopia

ii
 SCHOOL OF GRADUATE STUDIES
ADAMA SCIENCE AND TECHNOLOGY UNIVERSITY

As thesis advisor, we here by certify that we have read and evaluated this thesis, 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. We
recommend that it be submitted as fulfilling this thesis requirement

Dr. Mulugeta Kebede _______________ _______________

Advisor signature Date

Dr. Tileye Feyissa _______________ _______________

Co-advisor signature Date

As the member of the board of the MSc thesis open defense examination, we certify that we
have read and evaluated the thesis prepared by Mohammed Abdella and examined the
candidate. We recommended that the thesis be accepted as fulfilling the thesis requirement for
the degree of Master of science 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 thesis. All scholarly matter that is included in the thesis
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 thesis. This thesis 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 thesis.

Name: Mohammed Abdella Signature: _____________________

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

Date of submission: ____________________

II
 DEDICATION

I dedicate this thesis to my family and all Ethiopian people who are in need to peace and
development. GOD BLESS 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. Tileye Feyissa (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.

I thank ASTU for offering me this precious free scholarship opportunity for “MSc degree”.
Next, I 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 am also thankful to Laboratory Assistants, other staff members of Institute of Biotechnology,

Addis Ababa University for their helping hands during carrying out this thesis research.

Special thanks to Ethiopian 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

CTAB - Cetyl Trimethyl Ammonium Bromide

EBI - Ethiopian Biodiversity Institute

ICRISAT - International Crops Research Institute for the Semi-Arid Tropic

ISSR - Inter Simple Sequence Repeat

NJ - Neighbor Joining

PCoA - Principal Coordinate Analysis

RAPD - Random Amplified Length Polymorphism

RLFP - Restriction Fragment Length Polymorphism

SSR - Simple Sequence Repeats

UPGMA - Unweighted Pair group Method with Arithmetic Averages

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...................................................................................................................2
1.3 Objectives..........................................................................................................................................4
1.3.1 General Objective.......................................................................................................................4
1.3.2 Specific Objectives.....................................................................................................................4
2. LITRATURE REVIEW..........................................................................................................................5
2.1 The Groundnut (Arachis hypogaea L.) Crop Origin and Distribution...............................................5
2.2 Taxonomy and Botanical Description................................................................................................6
2.3 Importance of Arachis hypogaea L...................................................................................................8
2.4 Major Constraints in Arachis hypogaea L. Production....................................................................9
2.5 Arachis hypogaea L. Distribution and Production Areas in Ethiopia..............................................10
2.6 Genetic Resources of Arachis hypogaea L. Gene Pool....................................................................11
2.7 Germplasm Collections of Arachis hypogaea L..............................................................................12
2.8 Types of Markers and Their Application in Genetic Diversity Studies............................................14
2.8.1. Morphological Markers...........................................................................................................14
2.8.2 Protein (biochemical) Markers..................................................................................................15
2.8.3 Molecular Markers....................................................................................................................16
3. MATERIALS AND METHODS...........................................................................................................27
3.1 Plant Materials.................................................................................................................................27
3.2 DNA Extraction, Quantification and Purity Checking.....................................................................29
3.3 Primer Selection and Optimization..................................................................................................30
3.4 PCR Amplification and Gel Electrophoresis...................................................................................30
VI
 3.5 Data Scoring and Statistical Analysis..............................................................................................31
4. RESULTS AND DISCUSSION............................................................................................................33
4.1 DNA Isolation, Purification and Quantification...............................................................................33
4.2 ISSR Polymorphism........................................................................................................................33
4.3 Polymorphism Information Content (PIC).......................................................................................36
4.4 Genetic Diversity.............................................................................................................................37
4.5 Genetic Relationship........................................................................................................................40
4.6 Cluster Analysis...............................................................................................................................42
4.7 Principal Coordinate Analysis.........................................................................................................47
5. SUMMERY AND CONCLUSION.......................................................................................................50
6. RECOMMENDATIONS...................................................................................................................52
7. REFFERENCES................................................................................................................................53
8. APPENDIX.......................................................................................................................................58

VII
 LIST OF TABLES

Table Page

Table 1. Regions and sites of Ethiopia from where the samples were collected...........................28

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

bands..............................................................................................................................................30

Table 3. PCR cycle parameter.......................................................................................................31

Table 4. DNA Amplification Profile and Polymorphism Generated in A. hypogaea L................34

Table 5. DNA Amplification Profile and Polymorphism Information Content Generated in A.

hypogaea L....................................................................................................................................37

Table 6. Overall genetic variability across all studied groundnut accessions...............................39

Table 7. Overall genetic variability across all studied ISSR primers............................................40

VIII
 LIST OF FIGURES

Figure Page

Figure 1. A stylized A. hypogaea plant. …………………………………… ……………..…..8

Figure 2. Groundnut producing areas in Ethiopia based on the data from CSA agricultural

sample survey………………………………………………………………………………....11

Figure 3. ISSR marker amplification region on the genome………………………………….25

Figure 4. Locations and sites of Ethiopia from where the samples were collected…………...27

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

857 and 881……………………………………………………………………….…………..36

Figure 6. UPGMA based dendrogram obtained for 43 accessins using four ISSR primers.…46

Figure 7. Neighbor joining analysis of accessions based on 56 PCR bands……………….....47

Figure 8. PCoA scatter plot diagram showing relationships among groundnut accessions......49

IX
 ABSTRACT
Groundnut (Arachis hypogaea L.) belongs to the family Leguminoseae and genus Arachis. 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
been documented but molecular variations was not studied for this valuable crop. The main
objective of this study is to determine the genetic diversity of A. hypogaea accessions from
Ethiopia using ISSR marker. Using four ISSR primers, 54 reproducible bands were generated
of which 32 were polymorphic. The band size ranged from 100 bp to 1100 bp. The number of
amplified bands varied from 10 in primer 841 to 18 in primer 881. The maximum number of
polymorphic bands (100 %) was obtained using primer 857 whereas the minimum number of
polymorphic bands (27.8 %) was obtained in Primer 881. Average number of bands and
polymorphic bands per primer were 13.5 and 8 respectively. The polymorphic information
content (PIC) value ranged between 0.29 and 0.76 with an average of 0.49. The mean Nei’s
gene diversity and Shannon’s information index were 0.249 ± 0.153 and 0.336 ± 0.259
respectively. Genetic relationship between A. hypogaea accessions 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 with average value of 0.635. The lowest genetic similarity value was found
between accession GOBG-1 (Bale/Ginir) and GOB-14 (Babile/Tofic-2) 44%, followed by
GOG-1 (Gursum/Llalemi-1) and GAW-1 (Amhara/Wangua) and GOB-17 (Babile/Gemechu)
and GOBG-1 (Bale/ginir) at a similarity value of 46%, while highest similarity coefficient
was recorded between the accession GOB-10 (Babile/Medigana-1) and GOG-6 (Gursum/oda-
3) and GOB-7 (Babile/Ifa-gendi-1) and GOB-16 (Babile/Gende) 83% followed by that
between GOG-12 (Gursum/odaa-2) and GOB-9 (Babile/Ifa-gendi-3) at a similarity value of
82%, indicating that they belong to similar genetic background. The dendrogram based on
cluster analysis grouped the forty three A. hypogaea genotypes into five distinct clusters at
0.635 or 63.5% similarity coefficient, and the principal coordinate analysis revealed similar
grouping. PCoA analyses clustered the genotypes into individual groups where most of the
accessions 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 (Bale/Ginir) and GOB-14 (Babile/Tofic-2), GOG-1 (Gursum/Llalemi-1)
and GAW-1 (Amhara/Wangua) and GOB-17 (Babile/Gemechu) and GOBG-1 (Bale/ginir)
genotypes are the important ones to improve A. hypogaea through breeding programs to
which they could have specific trait of interest for the breeding program. Results of this study
would be promising as a genetic marker for the identification of A. hypogaea accessions and
an important source of knowledge for subsequent A. hypogaea 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
 1. INTRODUCTION
1.1 Background of the Study
Groundnut or peanut (Arachis hypogaea L.) belongs to the family Leguminoseae and
genus Arachis. Cultivated groundnut (Arachis hypogea L.) is a highly self-pollinated,
allotetraploid annual legume with 2n=4x=40 with a basic chromosome number of x=10
(Stalker, 1997). A. hypogaea 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 (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. A. hypogaea 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, this plant
improves soil by fixing nitrogen biologically without consuming non-renewable energies
and without disturbing agro-ecological balance (Jiaramraja and Fantahun, 2014).

A. hypogaea 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,
A. hypogaea is grown 26.4 million ha worldwide with a total production of 37.1 million
metric ton and an average productivity of 1.4 metric t/ha (Hamakareem et al., 2016). Major
A. hypogaea growing countries include China, India, the United States and Nigeria (Taru
et al., 2010). A. hypogaea is one of the four economically important oilseed crops with
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
currency earnings through export (Jiaramraja and Fantahun, 2014).

In Ethiopia A. hypogaea 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 A.
hypogaea production in Ethiopia are; Eastern Hararghe in Oromia and Metekel in
Benishangul-Gumuz (Addisu and Ermias, 2017; Fredu et al., 2015). Despite its
importance, the improvement of A. hypogaea productivity is stagnant in the country.
Research result showed that yields of more than 2.0 tons/ha can be obtained but the
national average yield produced by the farmers in Ethiopia is considerably low, 1.3

1
tons/ha, indicating the need of maximum effort to improve productivity (Gebbreselassie et
al., 2014).

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 (Raina et al., 2001).

Genetic diversity in A. hypogaea, based on morphological, biochemical and molecular

markers, has been reported by many researchers worldwide (peng et al., 2016; Raina et al.,
2001; Dwhani et al., 2017; Cuc et al., 2008; Roomi et al., 2014; Kochert et al., 1991;
Mace et al., 2007; Herselman 2003; Dwhani et al.,2017; Yusuf et al., 2017). However,
there is a gap in published works on genetic diversity of the A. hypogaea 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 Oromo Potato (Ijara, 2015), Tef, Coffee, Lentils, Rice and Sesame.
Therefore, the aim of this study is to determine the extent of genetic diversity in A.
hypogaea accessions found in Ethiopia using ISSR markers and identify highly diverse
genotypes for the purposes of broadening the genetic base of A. hypogaea 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

2
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 A. hypogaea were made

(Yusuf et al., 2017), but the molecular genetic diversity of has not been studied. 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. 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.

Molecular characterization using PCR-based ISSR markers provides a suitable method,

which can be used for varietal identification in A. hypogaea 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 (Raina et al, 2001). This research
generated basic information that could be useful for A. hypogaea breeding programs.
Better understanding on the diversity of this crop is important for improvement and
exploitation of A. hypogaea 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.

1.3 Objectives

1.3.1 General Objective

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

3
1.3.2 Specific Objectives
- To evaluate the potential informativeness of ISSR markers for identifying A.
hypogaea 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.

2. LITRATURE REVIEW

2.1 The Groundnut (Arachis 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

4
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 A. hypogaea 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 (Singh and Nigam, 2016).

Seven primary centers of diversity have been described for A. hypogaea: (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; and (7) Ecuadorian region for var. aequatoriana. Africa has been
described as a secondary center of diversity for cultivated groundnut (Krapovickas and
Gregory, 1994).

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). A.
hypogaea spread was; through Portuguese in the late 15 th century, carried two -seeded
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 var. hirsuta) 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, A. hypogaea became an important food crop in West Africa, Southeast and South

5
Asia, and USA. More than 100 countries now grows A. hypogaea (Singh and Nigam,
2016).

2.2 Taxonomy and Botanical Description

Arachis hypogaea is a member of the family Leguminoseae, subfamily Fabaceae, tribe
Aeschynomeneae, sub tribe Stylosanthenae, genus Arachis and species hypogaea (Susana,
2003). The genus Arachis has more than 70 wild species, of which only A. hypogaea 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 1) (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).

A. hypogaea is an allotetraploid with genome AABB, 2n = 4x = 40 (Krapovickas and

Gregory, 1994). Based on differences in branching pattern and in the presence of
reproductive nodes on the main stream, A. hypogaea is subdivided into two subspecies:
subsp. hypogaea and subsp. fastigiata. Subspecies 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 subspecies is subdivided
into botanical varieties hypogaea (Virginia and Runner) and hirsute (Peruvian humpback
or Chinese dragon type). Subspecies fastigiata has a sequential branching pattern,
reproductive nodes on the main stem; erect growth habit, earlier maturity 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 (Spanish type).
Krapovickas and Gregory (1994) later revised the classification of cultivated A. hypogaea
to include the two botanical var. peruviana (Valencia type) and aequatoriana (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.

6
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 A. hypogaea is generally self-pollinating although little out-crossing does occur
with the assistance of bees, which pollinate the flowers. The geocarpic habit of A.
hypogaea is a unique characteristic and could be responsible for dispersal and thus
population structure. Much of the dispersal is by water and therefore the species
distribution matches to a great extent 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 A.
hypogaea 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 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 subsp, hypogaea (alternating
pairs of vegetative: reproductive nodes) and subsp, fastigiata (sequential patterns of
reproductive nodes). However, intermediate types are commonly observed (Stalker, 1997).

7
Figure 1. A stylized A. hypogaea plant (Nigam, 2015).

2.3 Importance of Arachis hypogaea L.

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 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
which is composed of 26 inorganic constituents of which phosphorus, potassium,
magnesium and sulfur are high and virtually unaffected by heat (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, A. hypogaea are a rich source of 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) .

8
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% P 2O5 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 (sweet) product. The cake can be used for manufacturing artificial fiber. The
haulms (plant stalks) 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 A. hypogaea plant make it
an excellent cash crop for domestic markets as well as for foreign trade in several
developing and developed countries (Mattes, 2003). A. hypogaea is a legume crop;
therefore it is good in nitrogen fixation. A. hypogaea 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 A. hypogaea
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 Arachis hypogaea L. 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

9
(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 during early-season, mid-season, end-of-
season, and intermittent. Drought also predisposes A. hypogaea 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).

2.5 Arachis hypogaea L. 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 Ethiopia A. hypogaea
is grown and covered nearly 80,000 hectares (Fredu et al., 2015) of arable land per annum.
In terms of area distribution and production in Ethiopia, A. hypogaea is produced in
Oromia around (52, 921.26 ha) (East and West Welega, Illubabor, East and West
Hararghe), Benishangul-Gumuz (Metekel, Assosa, Kemashi, Pawe, Mao Komo)
(18,592.72 ha), Harari (2874.09 ha), Amhara (Awi, Oromia special zone) (2,380.15 ha),
and SNNP (South Omo, Gamo Gofa) (376.66 ha). 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 production. Next to Oromia, A. hypogaea is widely
cultivated in Benishangul Gumuz (CSA, 2015; Fredu et al., 2015). Though the crop is
grown different regions in Ethiopia, Oromia and Benishangul Gumuz regions account for
nearly 90% of the production in the country (Figure 2). Within these regions, Eastern
Hararghe zone in Oromia region and Metekel zone in Benishangul-Gumuz region are the
main centers for A. hypogaea production (Fredu et al., 2015).

10
 Figure 2. A. hypogaea producing areas in Ethiopia based on the data from CSA
agricultural sample survey (CSA, 2015).

2.6 Genetic Resources of Arachis hypogaea L. Gene Pool.

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 into a usable form and hybridization using pre- or post-fertilization
manipulations to establish hybrids (Nigam, 2015).

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

or parasexual means of genetic transformation. This approach has been used in A.

11
hypogaea classifying the genetic diversity into four gene pools: (1) The primary gene pool
consists of landraces and traditional cultivars of A. hypogaea 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 Germplasm Collections of Arachis hypogaea L.

Exploration for collecting seeds and living plants of cultivated A. hypogaea 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).

12
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,
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
Rhizomatoseae (Singh et al., 2004), which rarely produce seed and if produce, 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
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 and M, College Station, TX (Nigam, 2015). One of the most significant outcomes
of this broad collection effort was the publication of a new monograph of the genus
Arachis. The monograph represents a major milestone for the study and conservation of A.
hypogaea genetic resources, and provides a sound scientific foundation upon which future
work can be based (Singh and Nigam, 2016).

13
2.8 Types of 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, pure lines, 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
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).

14
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 homozygosity or heterozygosity, 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 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).

15
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 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

16
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 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).

17
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
marker 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).

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).

18
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).

After the invention of 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

19
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 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

20
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 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 of 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).

21
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 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
includeing: (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 consists 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

22
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 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

23
(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). 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, 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

24
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 (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).

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

25
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, 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).

26
 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 (Llalemi 1, Llalemi 2, Awdal, Oda 1, Oda 2, Oda 3, Oda
4, Oda 5, Nur Selam 1, Nur Selam 2, Odaa 1, Odaa 2, Abader, Harobata 1, Harobata 2,
Harobata 3, Harobata 4, Awdal 1, Awdal 2), Babile 21 (Berkele/s 1, Berkele/s 2, Babile/
Shek A., Awsherit 1, Awsherit 2, Lecole, Ifa Gende 1, Ifa Gende 2, Ifa Gende 3, Medigana
1, Medigana 2, Dendaro, Tofic 1, Tofic 2, Berkele, Gende, Gemechu, Tula, Tula About,
Abdul 1, Abdul 2), Somalia 1 (Jigjiga/Beledka), Amhara 1 (Adoawe/Wangua), Bale 1
(Ginir) (Figure 4; Table 1). Seeds of all the 43 A. hypogaea accessions were grown in a
greenhouse and fresh leaves from four 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.

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

27
Table 1. Regions and sites of Ethiopia from where the samples were collected

NO Gene bank Code Collection region locality Geographical location

accession Latitude Longitude
no (N) (E)
1 19739 GOB 1 Oromia/ Berkele/s 1 9012’25.33 42021’26.
Misrak/Babile ” 31”
2 19740 GOB 2 Oromia/ Berkele/s 2 9012’25.35 42021’27.
Misrak/Babile ” 34”
3 19741 GOB 3 Oromia/ Babile/Shek 9011’54.45 42021’41.
Misrak/Babile A. ” 22”
4 19742 GOB 4 Oromia/ Awsherit 1 9009’21.23 42022’21.
Misrak/Babile ” 34”
5 19743 GOB 5 Oromia/ Awsherit 2 9009’21.29 42022’21.
Misrak/Babile ” 28”
6 19744 GOB 6 Oromia/ Lecole 9007’36.32 42021’02.
Misrak/Babile ” 33”
7 19745 GOB 7 Oromia/ Ifa Gende 1 9 13’42.11 42018’22.
0

Misrak/Babile ” 18”
8 19746 GOB 8 Oromia/ Ifa Gende 2 9 13’41.11 42018’22.
0

Misrak/Babile ” 28”
9 19747 GOB 9 Oromia/ Ifa Gende 3 9 15’30.21 42018’21.
0

Misrak/Babile ” 12”
10 19748 GOB 10 Oromia/ Medigana 1 9 16’06.13 42018’12.
0

Misrak/Babile ” 43”
11 19749 GOB 11 Oromia/ Medigana 2 9 16’06.21 42018’24.
0

Misrak/Babile ” 28”
12 19750 GOB 12 Oromia/ Dendaro 9 17’25.21 42017’25.
0

Misrak/Babile ” 14”
13 19751 GOB 13 Oromia/ Tofic 1 9 16’06.36 42017’36.
0

Misrak/Babile ” 25”
14 19752 GOB 14 Oromia/ Tofic 2 9 16’06.41 42017’36.
0

Misrak/Babile ” 32”
15 19753 GOB 15 Oromia/ Berkele 9 10’48.27 42018’23.
0

Misrak/Babile ” 12”
16 19754 GOB 16 Oromia/ Gende 9 09’37.42 42018’50.
0

Misrak/Babile ” 10”
17 19755 GOB 17 Oromia/ Gemechu 9 07’25.34 42018’54.
0

Misrak/Babile ” 03”
18 19756 GOB 18 Oromia/ Tula 9 13’05.11 42019’28.
0

Misrak/Babile ” 32”
19 19757 GOB 19 Oromia/ Tula About 9 12’17.23 42019’38.
0

Misrak/Babile ” 42”
20 19758 GOB 20 Oromia/ Abdul 1 9 11’49.22 42019’43.
0

Misrak/Babile ” 37”
21 19759 GOB 21 Oromia/ Abdul 2 9 11’49.28 42019’58.
0

Misrak/Babile ” 23”
22 19760 GOG 1 Oromia/ Llalemi 1 9 19’33.14 42026’05.
0

Misrak/Gursum ” 26”

28
 23 19761 GOG 2 Oromia/ Llalemi 2 9019’33.38 42026’05.
Misrak/Gursum ” 38”
24 19762 GOG 3 Oromia/ Awdal 9018’19.23 42026’11.
Misrak/Gursum ” 36”
25 19763 GOG 4 Oromia/ Oda 1 9019’14.11 42027’06.
Misrak/Gursum ” 18”
26 19764 GOG 5 Oromia/ Oda 2 9018’37.42 42028’38.
Misrak/Gursum ” 21”
27 19765 GOG 6 Oromia/ Oda 3 9018’40.13 42028’38.
Misrak/Gursum ” 16”
28 19766 GOG 7 Oromia/ Oda 4 9018’30.32 42029’41.
Misrak/Gursum ” 38”
29 19767 GOG 8 Oromia/ Oda 5 9018’30.28 42029’41.
Misrak/Gursum ” 25”
30 19768 GOG 9 Oromia/ Nur Selam 1 9019’30.26 42028’38.
Misrak/Gursum 32”
31 19769 GOG 10 Oromia/ Nur Selam 2 9019’30.53 42028’38.
Misrak/Gursum ” 45”
32 19770 GOG 11 Oromia/ Odaa 1 9021’54.13 42029’50.
Misrak/Gursum ” 34”
33 19771 GOG 12 Oromia/ Odaa 2 9021’54.24 42029’50.
Misrak/Gursum ” 46”
34 19772 GOG 13 Oromia/ Abader 9017’51.28 42024’14.
Misrak/Gursum ” 33”
35 19773 GOG 14 Oromia/ Harobata 1 9017’12.43 42023’43.
Misrak/Gursum ” 24”
36 19774 GOG 15 Oromia/ Harobata 2 9017’12.38 42023’43.
Misrak/Gursum ” 28”
37 19775 GOG 16 Oromia/ Harobata 3 9016’12.18 42023’18.
Misrak/Gursum ” 21”
38 19776 GOG 17 Oromia/ Harobata 4 9016’22.20 42023’35.
Misrak/Gursum ” 12”
39 19777 GOG 18 Oromia/ Awdal 1 9017’20.56 42026’26.
Misrak/Gursum ” 65”
40 19778 GOG 19 Oromia/ Awdal 2 9017’20.48 42026’26.
Misrak/Gursum ” 51”
41 19779 GSJ -1 Somali/Jigjiga Beledka 9017’51.23 42039’08.
” 34”
42 24208 GAW -1 Amhara/Adoawe Wangua 10048’45.4 36025’35.
3” 12”
43 28662 GOBG1 Oromia/Bale Ginir 7011’86.21 40037’46.
21

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 plant molecular genetics Research

29
Laboratory, Department of Microbial, Cellular and Molecular Biology Department, Addis
Ababa University. 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 % agarose gel.

3.3 Primer Selection and Optimization

A total of 9 ISSR primers 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 % agarose gel.
All pre-selected 9 primers were screened for reproducibility and polymorphism. Finally,
three dinucleotide primers (primer 810, 841 and 857), and one penta-nucleotide primer
(primer 881) were selected based on polymorphism and reproducibility (Table 2).

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

NO Primers Annealing Sequence of Amplification pattern

To ( oC) nucleotides (5’- 3’)

1 810 45 GAGAGAGAGAGAGAGAT Polymorphic, Reproducible

2 812 45 GAGAGAGAGAGAGAGAA Not amplified
3 824 48 TCTCTCTCTCTCTCTCG Not reproducible
4 840 45 GAGAGAGAGAGAGAGAYT Not amplified
5 841 48 GAGAGAGAGAGAGAGAYC Polymorphic, Reproducible
6 842 45 GAGAGAGAGAGAGAGAYG Not amplified
7 848 45 CACACACACACACACARC Not reproducible
8 857 48 ACACACACACACACACYG Polymorphic, Reproducible
9 881 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 1.0 μl template
DNA, 17.5 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,

30
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 with a final extension of 7 min at 72°C (Table 3). The PCR
products were also stored at 4°C until loaded on gel for electrophoresis.

Table 3. PCR cycle parameter.

Step Temperature oC 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 g agarose using 500 ml
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 gel casting tray 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 at constant
voltage of 100 V. The ISSR band patterns were visualized and photographed under UV
light using Biometra Biodoc analyzer.

3.5 Data Scoring and Statistical Analysis

Data Scoring and Statistical Analysis of ISSR bands were scored as present (1) and absent
(0) 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:

31
 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) and UPGMA-based phenogram. The
unweighted pair group method with arithmetic average (UPGMA) was used in order to
determine the genetic relationship among accessions and generates phenogram using
NTSYS- pc version 2.02 (Rohlf, 2000). The neighbor joining (NJ) method was used to
compare individual accessions and evaluate patterns of accession clustering using Free
Tree 0.9.1.50 Software (Pavlicek et al., 1999). 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 was 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).

32
 4. RESULTS AND DISCUSSION

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 ratio of absorbance (A260/A280) ranging from
1.70 to 1.96 revealed that the DNA obtained was relatively of high in quality.

4.2 ISSR Polymorphism

Out of the 9 primers screened initially, four of them with clear and reproducible banding
pattern were selected and used in this investigation (Table 2). The size of the bands
amplified using the four primers were in the range of 100 to 1100 bp (Figure 5). The four
ISSR primers produced PCR fragments that ranged from 10 to 18 which generated 54
bands across the accessions, of which 32 were polymorphic (Table 4). The polymorphic
bands of the primers ranged from 10 (primer 841) to 18 (primer 881) across the genotypes.
Average number of bands and polymorphic bands per primer were 13.5 and 8 respectively.
The percentage of polymorphism for primers ranged from 27.8% in primer 881 to 100% in
primer 857, with an average polymorphism percent of 59.8 % (Table 4). In this
investigation, the di-nucleotide primers, namely 810, 841 and 857 were observed to have
61.5 %, 50 % and 100 % of polymorphism, respectively. The penta-nucleotides primer 881
was observed to have 27.8 % polymorphism. The main reasons behind these variable
results of molecular markers might be due to the different genetic sources of A. hypogaea
collections, primer variation in quality and quantity and distribution or frequency of the
primer sequence throughout the genome of A. hypogaea. In this study, the di-nucleotide
ISSR primers 810 and 841 with GA repeat, 857 with CA and 881 with GGGGT motives,
detected genetic diversity among accessions. Generally primers with (GA) and (AC)
repeats show higher polymorphism in plants than primers with other penta-nucleotides
(Tadele et al., 2014; Tshilenge-Lukanda et al., 2014). Moreover, the choice of appropriate
primer motives in ISSR fingerprint is critical to detect high polymorphism and reveal
relationship between genotypes. Similar result was reported by

The molecular analysis results using four ISSR primers revealed an average level of 59.8
% of polymorphism among accessions. This is relatively high for a self-pollinated species
such as A. hypogaea. The sort of genetic variability in this crop we found is of the same
type as reported by different previous researchers. For example, Raina et al. (2001) who

33
reported 54% of polymorphism among 13 A. hypogaea accessions using ISSR markers.
This level of genetic variation is relatively low when compared with other studies, Dwhani
et al., (2017) have reported a higher level of polymorphism 87% in A. hypogaea using
ISSR markers. Similarly, Suvendu et al. (2009) who reported 74.7% polymorphism using
ISSR markers among 20 A. hypogaea accessions.

Various molecular markers showed different efficiency for evaluating DNA fingerprinting
in A. hypogaea. For example, Herselman (2003) found a very low level of polymorphism
(on average 2.78%) among 21 closely related cultivated South African A. hypogaea
genotypes using AFLP marker, and Dwivedi et al. (2001) reported a low genetic diversity
(18%) among 26 accessions of cultivated A. hypogaea, including interspecific derivatives,
landraces, and released cultivars using RAPD markers. Kochert et al. (1991) found a very
low level of RFLP variability among 8 U.S A. hypogaea cultivars and 14 wild Arachis
species. (Mace et al., 2007) observed a high level of polymorphism within germplasm of
A. hypogaea which are resistant to bacterial wilt. He reported that 99.4% of the genotypes
showed polymorphism when screened with thirty two SSR primers.

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

Using 4 ISSR Primers

Primer Sequence 3’- Annealing To Total number Polymorphic % of

5’ (oC) of bands bands polymorphism
810 (GA)7T 45 13 9 61.5 %
841 (GA)7YC 48 10 5 50 %

857 (AC)7G 48 13 13 100 %

881 (GGGGT)3G 48 18 5 27.8 %

Total 54 32 59.8 %
Source: Primer kit 900 (UBC 900); Y = Pyrimidines (C or T), R = purines (A or G).

Though a limited number of ISSR markers were used in the study, the results confirm that
ISSR markers are efficient in detecting polymorphism between accessions of A. hypogaea
from different geographic location. Until the present day information available on the
reproductive biology of A. hypogaea, suggested that it is a predominantly self-pollinating
plant. However, the result of this study might be attributed to two reasons: one against and
the other in favor of the self-pollinating nature of the A. hypogaea plant. In the former
case, the result obtained could be accounted to mixed type of mating, typical of plant

34
species, in which there is a gene flow, and thus there might be moderate gene flow among
the local populations by effectors such as wind, insect, human (seedling movement) and
birds. The other is that they might have preferential or diverse adaptive genes that are not
fixed through self-pollination nature of this plant. However, this study used a small sample
size, geographic range and limited primers. Therefore, to find clear patterns of diversity for
the whole country and reach a sound conclusion, further studies should be conducted with
large sample sizes and geographic range using many ISSR primers.

The ISSR analysis used in the present study involved amplification of regions between
adjacent, inversely oriented microsatellites using a SSR motif containing primers anchored
at the 3’ or 5’ end by two or four arbitrary, often degenerate nucleotides (Zietkiewicz et
al., 1994). The ISSR method provides an alternative choice to obtain highly reproducible
markers without any necessity for prior sequence information for various genetic analyses.
It takes advantage of the ubiquitously distributed SSRs in the eukaryotic genomes.
Because of these abundant and rapidly evolving SSR regions, ISSR amplification has the
potential of revealing much larger numbers of polymorphic fragments per primer that any
other marker system used such as RAPD or microsatellite (Praneet and Pankaj, 2015).

The present study shows, however, that there is a great level of variability among ISSR
primers for generating polymorphic loci, suggesting that it will be useful in A. hypogaea
accessions characterization and fingerprinting purposes. ISSRs are regions that lie within
the microsatellite repeats and offer great potential to determine intra-genomic and inter-
genomic diversity compared to other arbitrary primers, since they reveal variation within
unique regions of the genome at several loci simultaneously. ISSR marker accesses
variation in the numerous microsatellite regions dispersed throughout the various genomes
(particularly the nuclear genome) and circumvents the challenge of characterizing
individual loci that other molecular approaches require. Compared to RAPD, ISSR assays
are highly reproducible (Zietkiewicz et al., 1994). A representation of the ISSR band
profile obtained with primer 810, 841, 857 and UBC 881 are shown in (Fig. 5).

35
 Figure 5. ISSR band patterns generated from accessions of A. hypogaea L. 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 0.29 (primer 881) to 0.76 (primer
857) with an average value of 0.49 (Table 5). The study of (Cuc et al., 2008) in which
an average of 0.46 PIC was calculated, are in support of our findings. Similarly (Roomi et
al., 2014) observe an average of 0.548 PIC value for SSR markers among A. hypogaea
accessions.

36
Table 5: DNA Amplification Profile and Polymorphism Generated in A. hypogaea L.
Using 4 ISSR Primers.

Primer Total number Polymorphic % of polymorphism PIC

of bands bands

810 13 9 61.5 % 0.49

841 10 5 50 % 0.42

857 13 13 100 % 0.76

881 18 5 27.8 % 0.29

Total 54 32 59.8 % 0.49

Markers with high PIC values are described as highly polymorphic and thus detect higher
level of genetic variation in an organism. Hildebrand et al. (1992) asserted that PIC value
is an important primary data which determines informativeness of a genetic marker and
that a PIC value of 0.70 and above is highly informative whiles a value of around 0.44 is
moderately informative. PIC is a statistic that measures the usefulness of a genetic marker
for linkage analysis. Therefore, a greater proportion (75%) of the markers used in this
study have moderate to high informativeness for linkage analysis in A. hypogaea,
indicating that these primers are informative for determining A. hypogaea accessions.
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.
However, the present study reveals the most informative primer in the present study was
primer 857 with PIC value of 0.76 and the least informative primer was 881 with PIC
value of 0.29. The observed variation in PIC values in this study could be attributed to the
type of primers and genotypic differences in the A. hypogaea material used.

4.4 Genetic Diversity

The binary data matrix generated by the amplified fragments of the 43 A. hypogaea
accessions in the ISSR-PCR analyses was used for the computations of Nei’s genetic
distances and Shanon’s indices for every pairwise comparison of the accessions for the
analysis of ISSR data. 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 0.183 ± 0.116 between GOG-6 (Gursum/Oda-3) and GOB-10

37
(Babile/Medigana-1) to 0.3154 ± 0.209 between GOBG-1 (Bale/Ginir) and GOB-14
(Babile/ Tofic-2) with an average value of 0.249 ± 0.153 (Table 3). The lower Nei’s gene
diversity observed (0.183 ± 0.116) for the accessions collected from different parts of
Ethiopia, this could be associated with usage of small sample size-representing different
parts of A. hypogaea growing regions. The Shannon’s indices (I) ranged from 0.2804 ±
0.1873 in GOB-7 (Babile/Ifa-gendi-1) and GOB-16 (Babile/Gende) to 0.3929 ± 0.2102 in
GOBG-1 (Bale/Ginir) and GOB-14 (Babile/Tofic-2) with an average value of 0.336 ±
0.259. Total gene diversity (Ht) and gene diversity among accessions (Hs) were 0.3066 ±
0.042 and 0.231 ± 0.065, respectively, indicating the existence of moderate genetic
diversity among accessions. In the case of di allelic loci (binary data) the maximum value
of the Ht and Hs indices equals 0·5, revealing maximum genetic diversity (Nei, 1978).

The different levels of genetic diversity for A. hypogaea may be explained, in part by the
use of different molecular markers, and in part by the different sampling strategies, such as
the geographic distance between sampled accessions. The results revealed that GOG-6
(Gursum/Oda-3) and GOB-10 (Babile/Medigana-1) accessions were closely related,
having the lowest genetic distance of 0.183 ± 0.116, which could be attributed to the levels
of management implemented and focus on limited accessions disseminated by different
stakeholders. The highest genetic distance 0.3154 ± 0.209 values belonged to GOBG-1
(Bale/Ginir) and GOB-14 (Babile/Tofic-2) accessions, which were the most distant
accessions. These values can be employed in a breeding program such that the accessions
with the lowest genetic similarities could be selected as parents to improve the A.
hypogaea accessions.

The coefficient of gene differentiation (Gst) among accessions was 0.38. Based on the Gst
value, the mean estimated number of gene flow (Nm) between accessions was found to be
0.56 (Table 6). The genetic differentiation of a species reflects the interactions of various
evolutionary processes including long-term evolutionary history, such as shifts in
distribution, habitat fragmentation and population isolation, mutation, genetic drift, mating
system, gene flow and natural selection (Schall et al., 1998). The coefficient of genetic
differentiation (Gst) was 0.38, suggesting an average genetic differentiation between
accessions. The average value of differentiation reflects the interactions of various factors
including: a reduced geographic range in most of the accessions, inter and intra-regional
climates, their breeding system, and genetic drift or genetic isolation of samples.

38
The estimated number of migrants per generation (Nm) was 0.56. In angiosperms, the
level of Nm is divided into three grades: high, Nm equal to or larger than 1.0; moderate,
Nm ranging from 0.250 to 0.99; and low, Nm ranging from 0.00 to 0.249 (Slatkin, 1985).
Gene flow is generally considered as the main factor that could homogenize the genetic
structure of populations in their distribution area. According to Slatkin (1987), Nm¼ 1 is
sufficient to overcome the effects of genetic drift. Also, species with low gene flow have
higher genetic differentiation than species with high gene flow. However, our results
indicated that virtually moderate gene flow occurred between the accessions of A.
hypogaea.

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

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

size (SD) (SD) (SD) (SD) (SD)

43 1.6512 1.5187 0.249 0.336 0.3066 0.231 0.38 0.56

± ± ± ± ± ±

0.4822 0.4121 0.153 0.259 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.

In terms of primers, the highest observed number of alleles 1.980 ± 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 1.42 ± 0.41 was shown by primer
881. The highest gene diversity 0.46 ± 0.036 and Shannon index 0.497 ± 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, and the least value of genetic diversity
and Shannon index was recorded in primer 881 with 0.25 ± 0.22 and 0.364 ± 0.326,
respectively (Table 7).

39
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 1.680 ± 0.480 1.52 ± 0.39 0.29 ± 0.21 0.419 ± 0.291

857 1.980 ± 0.640 1.85 ± 0.12 0.46 ± 0.036 0.497 ± 0.037

881 1.600 ± 0.520 1.42 ± 0.41 0.25 ± 0.22 0.364 ± 0.326

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 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
of 36.5% of genetic diversity or dissimilarity. The average similarity across the 43
accessions was found to be 63.5% or indicating a moderate level of genetic similarity
amongst them. The level of genetic dissimilarity observed among accessions with the four
ISSR primers varied between 17 and 56% suggesting that the accessions analyzed were
different but genetically closely related.

The lowest genetic similarity value (i.e. maximum diversity) was found between
accessions GOBG-1 (Bale/Ginir) and GOB-14 (Babile/Tofic-2) (44%) followed by that
between GOG-1 (Gursum/ Llalemi-1) with GAW-1 (Amhara/ Wangua) and GOB-17
(Babile /Gemechu) with GOBG-1 (Bale/Ginir) at a similarity value of 46%, which might
be attributed to having long geographical distance, while highest similarity coefficient (i.e.
minimum diversity) was found between the genotypes GOG-6 (Gursum/Oda-3) with
GOB-10 (Babile/Medigana-1) and GOB-7 (Babile/Ifa-gendi-1) with GOB-16
(Babile/Gende) 83% followed by that between GOG-12 (Gursum/Odaa-2) with GOB-9
(Babile/Ifa-gendi-3) at a similarity value of 82%. High genetic similarity values (Sij) of
82% to 83% were obtained for the genotypes, indicating limited genetic diversity, which

40
might be attributed to having similar ecological environment and short geographical
distance.

Highest levels of divergence between the accession from GOBG-1 (Bale/Ginir) and GOB-
14 (Babile/Tofic-2) could be attributed to the fact that the accessions have been popularly
cultivated in the respective regions over time giving enough time for significant genetic
differentiation along geographical lines. At the same time, it could indicate genetic
evidence of A. hypogaea being a diverse taxon as reported by Tang et al. (2007). Tang et
al. (2007) reported that the genetic diversity in cultivated peanut was much richer at the
molecular level when compared with that in the morphological and biochemical levels.
This therefore would mean that the studied accessions from GOBG-1(Bale/Ginir) and
GOB-14 (Babile/Tofic-2) has the highest amount of diversity that can be used for A.
hypogaea improvement in Ethiopia. In the same manner, it can be argued that GOG-1
(Gursum/ Llalemi-1) with GAW-1 (Amhara/Wangua) and GOB-17 (Babile/Gemechu) and
GOBG-1 (Bale/Ginir) has diversity that can be used for improvement of A. hypogaea at
Babile, Gursum, Amhara/Wangua and Bale/Ginir. Generally, the higher level of genetic
variation found in this study may be due to the fact that geographically isolated
accessions in certain geographic locations could accumulate genetic differences and
evolve unique genotypes as they adapt to different environment

The comparison of the genetic distances between accessions from GOB-10 (Babile
/Medigana-1) with GOG-6 (Gursum/Oda-3) and GOB-7 (Babile/Ifa-gendi-1) with GOB-
16 (Babile/Gende) revealed a closer genetic relationship with each other than all other
accessions from the same region (Babile/Gursum). Additionally, this high level of
similarity could be due to having common region and or selecting similar traits during
breeding programs in Gursum or Babile accessions. However, the distances between the
accessions 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 A. hypogaea trades across the regions and limited time for
significant genetic differentiation along geographical lines. Similarly, GOG-12
(Gursum/Odaa-2) exhibited a closer relationship with GOB-9 (Babile/Ifa-gendi-3). The
low genetic distances observed between these accessions could possibly reflect the initial
bottleneck during domestication maintained by the inherent self-pollination mechanism of
A. hypogaea crop. Thus the enhancement of diverse populations by using diverse
germplasm resources is needed in the future for various purposes such as population

41
genetic structure, germplasm analysis, identification of cultivars, selection of parents and
phylogenetic relationships.

On the contrary, highest genetic distance observed between GOBG-1(Bale/Ginir) with

GOB-14 (Babile/Tofic-2) could possibly indicate that the diversity in these accessions
could be used for the improvement of A. hypogaea crop in these regions; it would be
expected due to their geographical isolation and therefore might have a larger possibility of
heterosis in a breeding programs aimed to improve productivity or agronomical valuable
traits in A. hypogaea. This study has also observed that there is high diversity between
GOG-1 (Gursum/Llalemi-1) with GAW-1 (Amhara/Wangua) and GOB-17
(Babile/Gemechu) and GOBG-1 (Bale/Ginir) which could be exploited to improve the A.
hypogaea accessions in Ethiopia. This study has proved that although the overall genetic
base of A. hypogaea is moderate, there exists some level of diversity between A. hypogaea
accessions from the collection areas that can be exploited for the improvement of A.
hypogaea crop as a measure for crop improvement and food security in Ethiopia.

4.6 Cluster Analysis

Evaluation of 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 54 ISSR-PCR fragments scored from 43 accessions. 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 6). Similar result was reported by
Dwhani et al., (2017) for similarity coefficient of clustering A. hypogaea genotype using
ISSR marker. 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, where r > 0.9 indicates a very good fit, r -
0.8 - 0.9 indicates good fit and r < 0.8 indicates a poor fit. Similar result was observed by
Yousefi et al., (2015), who reported 0.87 cophenetic correlation coefficient acquired for
the clustering method representing a good fitness between the dendrogram clusters and the
similarity matrice in Thymus collections using ISSR marker.

42
Cluster I being the major cluster included twenty one accessions at a similarity of 64%,
which further divided into two sub clusters. In sub cluster I eight accessions were included
viz. GOBG -1 (Bale/Ginir), GOB -1 (Babile/Berkele/s 1), GOB -2 (Babile/Berkele/s 2),
GOB-6 (Babile/Lecole), GOB-15 (Babile/Berkele), GOB-21 (Babile/Abdul 2), GOG-11
(Gursum/Odaa 1) and GOG-13 (Gursum/Abader) at a similarity value of 67 %. These
accessions showed relatively high similarity with one other compared to other clusters
containing different accessions. Accessions GOG-13 (Gursum/Abader) and GOB-15
(Babile /Berkele) showed maximum similarity followed by GOG-11 (Gursum/Odaa 1) and
GOB-1 (Babile/Berkele/s 1); the accession GOBG -1 is the most divergent accessions of
this group. In sub cluster II thirteen accessions were included viz. GOG-2
(Gursum/Llalemi 2), GOG-4 (Gursum/Oda 1), GOG-5 (Gursum/Oda 2), GOG-6
(Gursum/Oda 3), GOG-10 (Gursum/Nur Selam 2), GOG-14 (Gursum/Harobata 1), GOG-
16 (Gursum/Harobata 3), GOG-19 (Gursum /Awdal 2), GOB-3 (Babile/Shek A.), GOB-7
(Babile/Ifa Gende 1), GOB-8 (BabileIfa/Gende 2), GOB-10 (Babile/Medigana 1), and
GOB-16 (Babile/Gende) at a similarity value of 66%. Accession GOG-6 (Gursum/Oda 3)
and GOB-10 (Babile/ Medigana 1) showed maximum similarity followed by GOB-7
(Babile/Ifa Gende 1) and GOB-16 (Gursum/Harobata 3), accession GOG-2
(Gursum/Llalemi 2) is the divergent accessions of this group. Cluster II included four
accessions viz. GOB-12 (Babile/Dendaro), GOB-18 (Babile/Tula), GOG-9 (Gursum/Nur
Selam 1) and GSJ-1 (Somali/Jigjiga/Beledka) at a similarity value of 65 %. Accessions
GOB-18 (Babile/Tula) and GOG-9 (Gursum/Nur Selam 1) showed maximum similarity
and the accession GSJ-1 (Somali/Jigjiga/Beledka) is the most divergent accessions of this
group. Cluster III included six accessions viz. GOB-5 (Babile/Awsherit 2), GOB-13
(Babile/Tofic 1), GOB-19 (Babile/Tula About), GOG-3 (Gursum/Awdal), GOG-7
(Gursum/Oda 4) and GOG-18 (Gursum/Awdal 1) at a similarity value of 64.8 %.
Accessions and GOB-5 (Babile/Awsherit 2) and GOG-18 (Gursum/Awdal 1) showed
maximum similarity followed by GOB -19 (Babile/Tula About) and GOG -7 (Gursum/Oda
4), accession GOG-3 (Gursum/Awdal) is the most divergent accessions of this group.
Cluster IV included nine accessions viz. GOB-4 (Babile/Awsherit 1), GOB -9 (Babile/Ifa
Gende 3), GOB -11 (Babile/Medigana 2), GOB-20 (Babile/Abdul 1), GOG-8
(Gursum/Oda 5), GOG-12 (Gursum/Odaa 2), GOG-15 (Gursum/Harobata 2), GOG-17
(Gursum/Harobata 4) and GAW-1 (Amhara/Wangua) at a similarity value of 63.5 %.
These accessions showed relatively low similarity with one other compared to other
clusters containing different accessions. Accessions GOB-9 (Babile/Ifa Gende 3) and

43
GOG-12 (Gursum/Odaa 2) showed maximum similarity and the accession GOG-15
(Gursum /Harobata 2) is the most divergent accessions of this group. Cluster V included
three accessions viz. GOB-14 (Babile/Tofic 2), GOB-17 (Babile/Tula) and GOG-1
(Gursum /Llalemi 1) at a similarity value of 65 %. Accessions GOB-14 (Babile/Tofic 2)
and GOB-17 (Babile/Tula) showed maximum similarity and the accession GOG-1
(Gursum/Llalemi 1) is the most divergent accessions of this group.

The dendrogram for the accessions (Figure 6) did not divide the accessions into distinct
groups resembling the geographically-defined accessions. The accession clustering follows
geographical proximity which shows the presence of 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 sub clusters within different groups of germplasm, no
specific/significant trend of grouping of accessions was observed. However, a loose
clustering of accessions 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).

Additionally, in constructed dendrogram of A. hypogaea, accessions coming from the

same geographical location were distributed in various clusters. For instance, the
accessions from Gursum and Babile were distributed in all 5 distinct clusters. Although
such evidence has already been reported reported by (peng et al., 2016; Raina et al., 2001),
suggesting that there was no obvious differentiation in the population structure of A.
hypogaea from different geographic origins. Moreover, accessions from distant
geographical locations were clustered together with other accessions from different
regions. For example, ‘GOBG-1’ accession from Bale/Ginir was clustered with GOB-1
(Babile/Berkele/s 1), GOB-2 (Babile /Berkele/s 2), GOB-6 (Babile/Lecole), GOB-15
(Babile/Berkele), GOB-21 (Babile/Abdul 2), GOG -11 (Gursum/Odaa 1) and GOG-13
(Gursum/Abader).

Similarly, the accession from GAW-1 (Amhara/wangua) was clustered with GOB-4
(Babile/Awsherit 1), GOB -9 (Babile/Ifa Gende 3), GOB -11 (Babile/Medigana 2), GOB-
20 (Babile/Abdul 1), GOG-8 (Gursum/Oda 5), GOG-12 (Gursum/Odaa 2), GOG-15
(Gursum/Harobata 2), GOG-17 (Gursum/Harobata 4) accessions from Babile and Gursum
which is unexpected because they are geographically far distant locations. From the

44
evidence of the current study it would seem likely that the accession clustering is the result
of the traditional ways that A. hypogaea have been distributed across the region and also
by frequent exchange of seeds between farmers.

The study of genetic diversity would be important for designing appropriate improvement
program. Based on the Jaccard’s similarity UPGMA clustering, the present study showed
high genetic diversity in GOBG-1(Bale/Ginir) with GOB-14 (Babile/Tofic-2), (GOG-1
(Gursum/Llalemi-1) with GAW-1 (Amhara/Wangua) and GOB-17 (Babile/Gemechu) and
GOBG-1 (Bale/Ginir) accessions. Improvement programs should, therefore, target
selection of individual plants with desirable traits from these accessions, since the
accessions are more diverse. However, this does not mean that less diverse accessions do
not consist plants with important traits. But it only means that the probability of getting
individual plants with good traits from such accessions is very low as compared to those
that are more diverse. Therefore, a more detailed sort of selection should also be practiced
in such accessions. The genetic distance between populations, on the other hand, is a
valuable parameter for genetic improvement programs. Hybridization/crossing between
any distantly related accessions is expected to yield more heterotic and vigorous plants
constituting much of the different traits contained in the above distant accessions.
Therefore, hybridization or crossing between distantly related accessions of the present
study could be an appropriate strategy for improvement programs. Using both (genetic
diversity and distance) parameters simultaneously for improvement programs would be the
best approach. In this case, the best individuals selected and crossed with individuals
selected from accessions that is/are comparatively distant from the former accessions could
yield good results.

45
 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 54 ISSR-PCR fragments, a NJ tree was constructed. 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. This lack of association of the individual plants into their
respective location in case of NJ may indicate the vulnerability or exposure of the plants to
human intensive management, exchange of seeds via marketing and presence of gene flow
due to limited distance. Generally, the dendrogram analysis using A. hypogaea individual
plant form inter-mixed cluster between accessions since average levels of genetic variation
is detected in accessions investigated. The NJ tree topology also further confirmed the

46
UPGMA dendrogram, in which most of the accessions from the same collection areas
formed a separate cluster.

Figure 7. Neighbor joining analysis of accessions based on 56 PCR bands amplified by 4

ISSR primers.
Key: GOG – Oromia/Gursum, GOB – Oromia/Babile, GOBG – Bale/Ginir, GSJ –
Somalia/Bigjiga, GAW – Amhara/Wangua

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 four
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. The result indicated that the first two
principal coordinates, PCoA1 and PCoA2 explained 34.3 % and 2.6 % of the variation

47
respectively and together explained 36.9 % of the total variation. Similar to the UPGMA
clustering pattern, the 43 accessions of A. hypogaea were grouped into five groups
(clusters) based on the principal co-ordinate analysis (Figure 8).

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 (Gursum/Awdal), GOG-4 (Gursum/Oda 1), GOG-5 (Gursum/Oda
2), GOG-13 (Gursum/Abader), GOG-14 (Gursum/Harobata 1), GOG-15 (Gursum
/Harobata 2), GOG-16 (Gursum/Harobata 3), GOG-19 (Gursum/Awdal 2), GOB -2
(Babile/Berkele/s 2), GOB-8 (BabileIfa/Gende 2), GOB-12 (Babile/Dendaro), GOB-13
(Babile/Tofic 1), GOB-19 (Babile/Tula About) and GOBG -1 (Bale/Ginir). Cluster II
contained eight genotypes i.e. GOG-7 (Gursum/Oda 4), GOG-8 (Gursum/Oda 5), GOG-9
(Gursum/Nur Selam 1), GOB-5 (Babile/Awsherit 2) , GOB -11 (Babile/Medigana 2), GOB
18 (Babile/Tula) and GAW-1 (Amhara/Wangua). Cluster III included eight genotypes i.e.
GOG-2 (Gursum/Llalemi 2), GOG -11 (Gursum /Odaa 1), GOB -1 (Babile/Berkele/s 1),
GOB-3 (Babile/Shek A.), GOB-7 (Babile/Ifa Gende 1), GOB-10 (Babile/Medigana 1),
GOB-16 (Babile/Gende) and (Somali/Jigjiga/Beledka). Cluster IV included seven
genotypes i.e. GOG-1 (Gursum/Llalemi 1), GOG-6 (Gursum/Oda 3), GOG-10
(Gursum/Nur Selam 2), GOB -6 (Babile/Lecole), GOB-14 (Babile/Tofic 2), GOB -15
(Babile/Berkele) and GOB -21 (Babile/Abdul 2). Cluster V contained six genotypes i.e.
GOG-12 (Gursum/Odaa 2), GOG-17 (Gursum /Harobata 4), GOB-4 (Babile/Awsherit 1),
GOB -9 (Babile/Ifa Gende 3), GOB-17 (Babile /Tula) and GOB-20 (Babile/Abdul 1).

The PCoA result indicated that most of the accessions did not group together with other
genotypes from the same geographical region (Figure 8). One possible reason for this
could be the exchange of A. hypogaea accessions among farmers across regions. This
result confirms the result obtained in all the above diversity indices. However, most of the
accessions that represent similar geographical regions were found to form distinct groups
and spread all over the plot (Figure 8). This could have led to a generally low coefficient
of variation observed in groundnut accessions, an indication of high level of uniformity
and the presence of gene flow. This suggested that the source of these accessions could be
the same due to seed exchange among the farmers. The result of principal co-ordinate
analysis (PCoA) was partially in accordance with the cluster analysis.

48
Figure. 8. PCoA scatter plot diagram showing relationships among A. hypogaea
accessions.

49
 5. SUMMERY AND 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.

In Ethiopia, 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
59.8 % of polymorphic loci among accessions. To get an objective discrimination of
genetic diversity of cultivated A. hypogaea 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 accessions 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.

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 GOG-6 (Gursum/Oda-3) with GOB-10
(Babile/Medigana-1)and GOB-7 (Babile/Ifa-gendi-1) with GOB-16 (Babile/Gende) and
the second highest genetic similarity was observed between GOG-12 (Gursum/Odaa-2)
with GOB-9 (Babile/Ifa-gendi-3). These results could suggest that the homogeneity
among the accessions (geographic region based) could be due to gene flow 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 (Bale/Ginir) and GOB-14
(Babile/Tofic-2) followed by GOG-1 (Gursum/ Llalemi-1) with GAW-1 (Amhara/
Wangua) and GOB-17 (Babile /Gemechu) with GOBG-1 (Bale/Ginir) or this genotypes

50
showed a good amount of genetic divergence and would therefore be useful in broadening
genetic base of accessions of A. hypogaea 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 accessions. The most diverse accessions identified in this
study should be given a due emphasis to include them in A. hypogaea breeding program of
the country.

Both PCoA and UPGMA cluster analysis approved the clustering of all 43 accessions into
five clusters. Cluster analyses of the present study showed that most genotypes of the
accessions 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 accessions 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. These 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. The results also provide important milestone for future
conservation and improvement programs of A. hypogaea. Furthermore, results from gene
flow estimates suggest that there could be a possibility of sampling plants with the same
genetic constitution from different administrative regions. Therefore, representing a
population by as many collections as possible would be the best approach.

51
 6. RECOMMENDATIONS

 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.

 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.

 It is recommended that the present study be complemented with a parallel

investigation on adaptive traits (drought, pest and insect resistance) and other
economic traits (growth and form) before a definite strategy is adopted.

52
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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.

E. 10% working C-TAB

10% CTAB = 10 g

5 M NaCl = 14 ml

58
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)

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.

59
 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

(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

60
(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

DNA Extraction Procedure

1. Pour CTAB solution (700 μl per sample) in a 15ml-tube and add 0.2 vol % Mercapto-
ethanol (use fume hood!). Mercapto-ethanol is stored at 4°C. Incubate the CTAB at 65°C.

2. Weigh in 100 mg fresh leave material (50mg dry material) per sample. Pulverise
thoroughly using a clean mortar and pestle. For fresh material add liquid nitrogen or quartz
sand for dry material. First grind down slightly, then more powerful (cells have to be
crashed). Use safety goggles!

3. Transfer the powder into a sterile Eppendorf cap (use a new, sterile spatula for each
sample.

4. Add 700 μl of warm (65°C) CTAB solution to the powdered sample (open the caps
carefully), dissolve the powder and incubate the sample for 30 minutes at 65°C.

5. Centrifuge for 5 minutes at 15000 rpm, 20°C.

61
6. Transfer the supernatant (only clear liquid) in a new Eppendorf-cap. Use blue pipette
tips which are cut.

7. Add new CTAB solution (700 μl) to the tissue pellet and stir slightly with a new 1000 μl
pipette tip, incubate 30 min at 65°C. Step 6 and 7 are repeated. The same is carried out for
a third extraction. Each fraction proceeds with step 9 and is treated separately.

8. Add 600 μl Chloroform to the cap with supernatant. (This chloroform step should be
carried out immediately).

9. Shake the samples thoroughly by inverting the Eppendorf-caps for approximately 5

minutes.

10. Centrifuge for 5 min at 15000 rpm.

11. Transfer the supernatant (only clear liquid) in a new Eppendorf-cap. Use blue pipette
tips which are cut. Work carefully; do not transfer suspended matter (normally the
Chloroform is covered by a thin layer of fine sediment material). Chloroform has to be
disposed of in a special waste bottle.

12. Repeat the chloroform extraction (step 8-11) to make sure that all impurities are
removed and then proceed with step 13.

13. Add cooled Isopropanol (4°C), approximately 2/3 of the solution volume. Shake
carefully by inversing the Eppendorf cap. In most cases DNA becomes visible as white
threads. Freeze for more than 2 h at -20°C. (BREAK POSSIBLE).

14. Centrifuge 10 min at 15000 rpm in a cooled centrifuge.

15. Aspirate liquid using yellow tips (without touching pellet!). If pellet is solid enough the
larger part of the liquid may be poured out. (Alternatively add TE and proceed with
Qiagen kit).

16. Add 200 μl Ethanol 70 % to the pellet.

17. Centrifuge for 10 min at 15000 rpm in a cooled centrifuge.

18. Aspirate Ethanol using yellow tips. Dry the DNA-pellet at room temperature. (Usually
15 min are sufficient; after drying no liquid drops are to be seen)

62
19. Dissolve pellet in 100 μl TE (1x, p.a. grade) and store at 4°C. (BREAK POSSIBLE)

20. Add cooled 7.5 M NH4Ac-solution (4°C, half of the solution volume). Mix carefully.

21. Add cool Ethanol 100 % (double of the solution volume). Mix carefully. Freeze for
more than 2 h at -20°C. (BREAK POSSIBLE)

22. Centrifuge 30 min at 15000 rpm in a cooled centrifuge. Aspirate fluid carefully.

23. Add 200 μl Ethanol 70%.

24. Centrifuge 10 min. at 15000 rpm in a cooled centrifuge. Aspirate liquid and dry pellet
at room temperature. Dissolve the pellet in 100 μl TE (1x, p.a. grade)

25. Repeat steps 20 to 23 with 3 M NaAC-solution (instead of NH4Ac), then proceed with
step 26.

26. Centrifuge 10 min. at 15000 rpm in a cooled centrifuge. Aspirate liquid and dry pellet
at room temperature. Dissolve the pellet in 100 μl TE (1x, p.a. grade).

63
1