GENETIC DIVERSITY OF CHICKPEA (Cicer arietinum L.

) CULTIVARS
FROM ETHIOPIA BY USING INTER SIMPLE SEQUENCE REPEAT
MARKERS

M.Sc. Thesis

TADELE TEMESGEN

June, 2014
Haramaya University
GENETIC DIVERSITY OF CHICKPEA (Cicer arietinum L.) CULTIVARS
FROM ETHIOPIA BY USING INTER SIMPLE SEQUENCE REPEAT
MARKERS

A Thesis Submitted to the

Department of Biology, School of Graduate Studies
HARAMAYA UNIVERSITY

In Partial Fulfillment of the Requirements for the Degree of

MASTER OF SCIENCE IN GENETICS

By
Tadele Temesgen

June, 2014
Haramaya University

ii
 School of Graduate Studies

Haramaya University

As Thesis Research advisors, we hereby certify that we have read and evaluated this
Thesis prepared under our guidance, by Tadele Temesgen Gubae entitled: “Genetic Diversi
ty of Chick pea (Cicer arietinum L.) Cultivars from Ethiopia by Using Inter Simple Seq
uence Repeat markers”. We recommended that it can be submitted as fulfilling of the
Thesis requirement.

Endashaw Bekele (Prof.) _______________ ________________

Major Advisor Signature Date

Yohannes Petros (Ph. D) ________________ _______________

Co – advisor Signature Date

As member of the board of Examiners of the M.Sc. Thesis Open Defence Examination, we
certify that we have read, evaluated the Thesis prepared by Tadele Temesgen Gubae and
examined the candidate. We recommended that the Thesis is accepted as fulfilling the
Thesis requirement for the degree of Master of Science in Genetics.

_________________ ________________ _________________

Chairperson Signature Date
__________________ _________________ ________________
Internal Examiner Signature Date
________________ ________________ _________________
External Examiner Signature Date

ii
 DEDICATION

This manuscript is dedicated to my beloved mother W/ro Alemetsehaye Zewdie who

has sown an academic interest in my mind, shaping me from early school age and
paving the way for today's effortful success even though she passed with out seeing
my success.

iii
 STATEMENT OF THE AUTHOR

First, I declare that this is my own work and that all sources of materials used for the
manuscript have been duly acknowledged. This thesis has been submitted in partial
fulfillment of the requirement for M.Sc. degree in genetics at Haramaya University and is
deposited at the University Library to be made available to borrowers under the rules and
regulations of the Library. I solemnly declare that this thesis is not submitted to any other
institution anywhere for the award of any academic degree, diploma, or certificate.

Brief quotations from this thesis are allowable without special permission provided that
accurate acknowledgement of source is made. Requests for permission of extended
quotation from or reproduction of this manuscript in whole or in part may be granted by the
head of the major department 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.

Name: Tadele Temesgen Signature: ……………………

Place: Haramaya University, Harmaya
Date of Submission: ……………………

iv
 BIOGRAPHICAL SKETCH

The author was born on April 23, 1989 in Amhara region, East Gojjam zone, Amber town
from his father Temesgen Gubae and mother Alemetsehaye Zewdie. He attended his
elementary school at Yegiwora Elementary School (1-6), junior school at Korck Elementary
- Junior School (7-8) and secondary-preparatory at Ginbot-20 Secondary and Preparatory
School (9-12). Then he joined Haramaya University in January 2009 and graduated with a
B.Sc. degree in Applied Biology in July 2011. After graduation, the author has been
sponsored by Minstery of Education and joined Haramaya University in September 2012 to
pursue his M.Sc. degree studies in Genetics.

v
 ACKNOWLEDGEMENTS

I would like to express my deepest thanks and sincere gratitude to my major advisor, Prof.
Endashaw Bekele, who accepted me to work in his genetic laboratory and made possible my
dreams of working M. Sc. thesis in the area of molecular genetics. I learnt, from his
developed personality and energetic nature, not only professionalism but also professional
ethics and seriousness. I really appreciate his generous thought, excellent guidance,
unreserved support (project related laboratory expenses, material assistance) and insightful
criticism in commenting on the manuscript that lead to the realization of this work.

I would like to extend my special thanks to my co-advisor, Dr. Yohannes Petros, for his
meticulous guidance, keen interest, encouragement, assistance, advice and valuable
suggestions starting from the proposal development to execution of thesis research work.
Again, I owe great thank to my friend, Dagimawit Chombe for introducing me to the
laboratory techniques, Tadesse Abate and Edossa fikiru for open discussion on statistical
procedures and Dr. Kassahune Tesfaye for technical advices during data analysis.

I am indebted to the School of Graduate Studies of the Haramaya University, Department of

Biology and to Ministry of Education for accepting me to pursue post graduate program and
provide financial support for the study. All administrative and research staffs of Ethiopian
Institute of Agricultural Research Centers were also very collaborative and deserve thanks
with appreciation. I would like to acknowledge Dr. Million Eshete in DZARC, Dr.
Gemechu Keneni in HARC and Ato Setu Bazie in SRARC with their Pulse Research
Program staff members who helped me during chickpea seeds collection. DZARC provided
me soil with sample and constructive information about the conditions (requirements) for
seeds germination in green house.

Finally, I am grateful to extend my deepest and heartfelt gratitude to my parents Ato

Temesgen Gubae and W/ro Banchiamilake Melikamu, my brothers, sisters, close relatives
and colleagues, for their care, support, encouragement, experience sharing and for all what
they did for me. Their all rounded and unconditional moral support enabled me to realize
my educational goal.

vi
 LIST OF ABBREVIATIONS

AFLP Amplified Fragment Length Polymorphism

AMOVA Analysis of molecular variance
ARARI Amhara Regional Agricultural Research Institute
Bp Base pair
dNTP Deoxy Nucleoside Tri Phosphate
EIARC Ethiopian Institute of Agricultural Research Center
ESTs Expressed Sequence Tags
FAOSTAT Food and Agricultural Organization Statistical Databases
FMs Functional markers
GMM Genic Molecular Markers
ICARDA International Center for Agricultural Research in the Dry Area

ICRISAT International Crops Research Institute for the Semi Arid Tropic
ISSR Inter Simple Sequence Repeats
Mha Million hectares
NJ Neighbor Joining
PCO Principal Coordinate Analysis
PCR Polymerase Chain Reaction
RAPD Random Amplified Polymorphic DNA
RFLP Restriction Fragment Length Polymorphism
SCAR Sequence Characterized Amplified Regions
SNP Single Nucleotide Polymorphism
SSLP Simple Sequence Length Polymorphism
SSR Simple Sequence Repeats
SSTR Simple Sequence Tandem Repeats
STS Sequences Tagged Sites
UBC University of British Colombia
UPGMA Un-weighted Pair Group Method with Arithmetic mean
UV Ultraviolet

vii
 TABLE OF CONTENTS

STATEMENT OF THE AUTHOR iv

BIOGRAPHICAL SKETCH v
ACKNOWLEDGEMENTS vi
LIST OF ABBREVIATIONS vii
LIST OF TABLES x
LIST OF FIGURES xi
LIST OF TABLES IN THE APPENDIX xii
LIST OF FIGURES IN THE APPENDIX xiii
ABSTRACT xiv
1. INTRODUCTION 1
2. LITRATURE REVIEW 4
2.1. Chickpea (Cicer arietinum L.) Description 4
2.1.1. Origin, taxonomy and distribution of chickpea 6
2.1.2. Production and importance of chickpea 8
2.1.3. Production constraints and tolerance to stress 9
2.2. Genetic Resource and Genetic Diversity 10
2.3. Genetic Erosion and Genetic Conservation 11
2.4. Genetic Markers and their Applications 13
2.4.1. Morphological markers 14
2.4.2. Biochemical markers (Isozymes) 15
2.4.3. Molecular markers 16
2.4.3.1. Restriction fragment length polymorphism (RFLP) 18
2.4.3.2. Random amplified polymorphic DNA (RAPD) 18
2.4.3.3. Amplified fragment length polymorphism (AFLP) 19
2.4.3.4. Simple sequence repeat (SSR) 20
2.4.3.5. Inter simple sequence repeats (ISSR) 21

viii
 TABLE OF CONTENTS (Continued)

3. MATERIALS AND METHODS 23

3.1. Plant Material and Sampling Strategy 23
3.2. DNA Extraction 25
3.3. Quality of Extracted DNA 25
3.4. Primer Selection and Optimization 26
3.5. PCR Amplification and Electrophoresis 26
3.6. Data Scoring and Statistical Analysis 27
4. RESULTS AND DISCUSSION 29
4.1. ISSR Markers and their Banding Patterns 29
4.2. Genetic Diversity as Revealed by Percent Polymorphism, Shannon Weaver and Gene
Diversity Values 31
4.3. Analysis of Molecular Variance (AMOVA) 32
4.4. Clustering Analysis 33
4.5. Principal Coordinate (PCO) Analysis 36
5. SUMMARY, CONCLUSION AND RECOMMENDATION 39
5.1. Summary 39
5.2. Conclusion 40
5.3. Recommendation 41
6. REFERENCES 42
7. APPENDIX 50

ix
 LIST OF TABLES

Table page

1. Description of chickpea varieties used in the experiment. 23

2. List of primers and their annealing temperature with respective sequences used for the
analysis. 26
3. Selected ISSR primers with their repeat motifs, amplification quality and scorable bands. 29
4. Number of polymorphic loci (NPL), percent polymorphism (PP), genetic diversity (H)
and Shannon Index information (I) of 27 chickpea cultivars based on each primer used. 32
5. Number of polymorphic loci (NPL), percent polymorphism (PP), genetic diversity (H)
and Shanon Index information (I) of 27 chickpea cultivars based on all 4 primers used. 32
6. Analysis of Molecular Variance (AMOVA) of chickpea cultivars in Ethiopia based on
their type as desi and kabuli. 33

x
 LIST OF FIGURES

Figure Page

1. Geographical distribution of different Cicer species. Source: Van derMaesen (1987). 7

2. Pictures of test gel of genomic DNA for the individual chickpea cultivars. 25
3. Molecular weight of amplified band-patterns for chickpea DNA samples amplified by
primer UBC – 842. 30
4. ISSR fingerprint generated from 21 representative chickpea cultivars using primer UBC–
849. 30
5. UPGMA dendrogram depicting clustering patterns for 27 Chickpea cultivars based on
Jaccard’s similarity coefficients. 35
6. Neighbor-joining analysis of 27 individuals based on Jaccard’s similarity coefficient. 36
7. Two dimensional representation of principal coordinate analysis for 27 Chickpea cultivars
based on Jaccard’s similarity coefficient using ISSR data. 37
8. Three dimensional representation of principal coordinate analysis of genetic relationships
among 27 cultivars of Cicer arietinum L. inferred from similarity matrix using the
Jaccard`s index. 38

xi
 LIST OF TABLES IN THE APPENDIX

Appendix Table Page

1. Advantages and Disadvantages of Molecular Markers. (Hikmet et al., 2004). 51

2. Commen features of commonly used molecular marker systems in cereals (FAO, 2003). 52

xii
 LIST OF FIGURES IN THE APPENDIX

Appendix Figure Page

1. Principles of inter simple sequence repeat marker. 53

2. The diagrammatic illustration of PCR process for single complete cycle. 53
3. The diagrammatic illustration of PCR process that used to amplify even very small
samples of DNA in molecular marker analysis. 54
4. Two types of chickpea seeds samples. 54
5. Banding pattern of chickpea DNA samples by primer UBC - 830 (A), UBC - 840 (B),
UBC - 842 (C) and UBC - 849 (D). 56
6. The author`s work: A &B) Cultivars in green house of Arat-killo campus of A.A.U C)
Preparing agarose gel D) DNA extraction by Invisorb® spin plant mini kit E&F) Loading
the DNA samples and amplicons on the gel. 56

xiii
GENETIC DIVERSITY OF CHICKPEA (Cicer arietinum L.) CULTIVA
RS FROM ETHIOPIA BY USING INTER SIMPLE SEQUENCE
REPEAT MARKERS

ABSTRACT

Genetic diversity of 27 chickpea (Cicer arietinum L.) cultivars (12 kabuli type and 15 desi
type) were studied using Inter Simple Sequence Repeat (ISSR) markers to assess their
genetic diversity. The ISSR diversity analysis was conducted at Genetics Laboratory of
Addis Ababa University, Ethiopia. Four di-nucleotide repeat primers amplified a total of 24
clear and reproducible bands of which 22 were polymorphic. Both UPGMA phenograms
and neighbor joining (NJ) trees were constructed for the individual cultivars using
Jaccard’s similarity coefficient. The dendrogram clearly indicated three distinct clusters
and sub-clusters within each main cluster based on the type of cultivars. The 3D principal
coordinates (PCO) analysis also recovered the UPGMA and NJ tree groups although some
chickpea cultivars were intermixed with each other. The genetic diversity among chickpea
cultivars considered in the present study indicated that desi type chickpea exhibited a
genetic diversity value of 0.30 and Shannon diversity index of 0.47, while the kabuli type
chickpea had 0.33 and 0.50 for genetic diversity and Shannon diversity index values
respectively. Generally kabuli type chickpea cultivars show higher genetic diversity
(91.67%) than desi type (75%). Further more Analysis of molecular variance (AMOVA)
demonstrated highly significant (p = 0.00) genetic diversity within cultivars (97.71%) than
among cultivars (2.29%). Generally on the bases of bands generated by all primers, ISSR
was able to reveal moderate to high levels of genetic diversity within and among cultivars of
Ethiopian chickpea. Further study of ISSR using more number of primers or co-dominant
marker is recommended to give confirmative result.

Key Words: Chickpea cultivars, Ethiopia, Genetic diversity, ISSR markers.

xiv
 1. INTRODUCTION

Chickpea (Cicer arietinum L.) belongs to the genus Cicer that consists of 43 species (Van der
Maesen, 1987). It is a cool season leguminous crop that originated in south-eastern Turkey and
commonly grown in tropical, subtropical and temperate regions of the world. The crop has
several names in different parts of the world like Bengal gram (India), Chana (Urdu), Chickpea
or gram (English), Garbanzo (Latin America), Kichar or Chicher (German), Hommas, Hamaz
(Arab world), Nohud, Lablabi (Turkey), Poice chiche (French) and Shimbra (Ethiopia) (Van der
Maesen, 1987).

The primary centre of diversity is in the Fertile Crescent where the crop was first domesticated or
cultivated in an area of south-eastern Turkey and adjoining Syria. Chickpea is now cultivated
throughout the semi-arid regions of the world (Knights et al., 2007). Geographic spread of
chickpea secondary centers of diversity started to develop, about 2000 years ago in
Mediterranean, Europe, Indian sub-continent and north-east Africa, and some more recently in
Mexico and Chile with post-Colombus introduction (Redden and Berger, 2007).

Cicer arietinum, the only cultivated species within the genus Cicer, is a diploid plant with 2n =
2x = 16 (Arumuganathan and Earle, 1991). It has a genome size of approximately 931 Mbp
which is slightly less than the well-characterized tomato genome of 950 Mbp. It ranks third
among the cool season food legumes in terms of production after common bean (Phaseolus
vulgaris) and field pea (Pisum sativum) at global level and second in area coverage among the
pulses grown in Ethiopia preceded only by faba bean (Vicia faba).

There are two main types of chickpea, distinguished by their seed size, shape and color: one
produces relatively small seeds with an angular shape, light tan to dark color called desi, and the
other produces large, rounded white to pale cream color seeds, called kabuli. Kabuli chickpea
seeds are grown in temperate regions, whereas the desi type is grown in the semi-arid tropics
(Naser Maheri-Sis et al., 2008).

The world area under chickpea is about 11.08 million hectares (Mha), with a total production of
9.77 million ton, and an average productivity of 882 kg/ha. It has been cultivated mainly in the
Indian sub-continent, West Asia, and North Africa, but recently large acreages have been
introduced in the Americas and Australia. The yield potential of present-day chickpea varieties
exceeds 4 t ha-1; however, actual yield is less than 0.8 t ha-1 due to biotic and abiotic stresses on
its production (Madrid et al., 2008).

Chickpea is being valued for its high dietary protein content, its ability to fix atmospheric
nitrogen and the absence of specific major anti-nutritional factors. This make it an important
component of the cropping system and considered as nutritious and healthy food (Mohammed et
al., 2011). Chickpea seed contains 29% protein, 59% carbohydrate, 3% fiber, 5% oil and 4% ash.
Its protein is rich in lysine and arginine but is deficient in sulfur-containing amino acids
methionine and cysteine. It is also a good source of absorbable Ca, P, Mg, Fe and K
(Christodoulou et al., 2005).

Analysis of genetic diversity is important to examine differences between individuals of the

same species and also used to compare the genetic composition of members of different species
over a wider taxonomic range. During the past few decades, numerous attempts have been made
to examine the diversity and phylogeny of chickpea using karyotypic studies (Ohri and Pal,
1991), and seed storage proteins electrophoresis (isozyme analysis). However, these studies had
shown only low levels of genetic polymorphism for cultivated chickpea and were unable to
estimate genetic diversity at the accession level. Analysis of genetic diversity can be carried out
by using morphological characters, biochemical and modern molecular methods.

A genetic marker can be defined in one of the following ways: (a) a chromosomal landmark or
allele that allows the tracing of a specific region of DNA; (b) a specific piece of DNA with a
known position on the genome or (c) a gene whose phenotypic expression is usually easily
discerned, used to identify an individual or a cell that carries it, or as a probe to mark
chromosomes, or loci (King and Stansfield, 1990). Genetic markers fall into one of the three
broad classes: those based on visually assessable traits (morphological or agronomic traits), those
based on gene product (biochemical markers), and those relying on a DNA assay (molecular
markers).

2
Molecular markers are used to determine and analyze similarities between closely related species
and genotypes. Moreover, these molecular markers, which are based on PCR application, are
extremely important to increase agricultural yield and resistance for stress conditions, to
determine gene sequences, to improve different genotypes and to solve genetic related problems
(Zietkiewicz et al., 1994). The main reasons supporting the utilization of molecular markers in
national breeding programs are the heritability of the markers (100 %), and their cost, which is
potentially lower than the conventional selection (Winter and Kahl, 1995).

The major PCR-based marker systems that are currently available for genetic
study include Amplified Fragment Length Polymorphism (AFLP), Random Amplified
Polymorphic DNA (RAPD), Microsatellite (SSR) and Inter Simple Sequence Repeat (ISSR)
which are generally described as dominant markers (Weising et al., 2005). ISSR-PCR is a
technique, which involves the use of microsatellite sequences as primers in a polymerase chain
reaction to generate multi-locus markers. It is a simple and quick method that combines most of
the advantages of AFLP and RAPD (Zietkiewicz et al., 1994). These sequences are abundantly
dispersed throughout the genome and highly polymorphic in comparison with other molecular
markers (Morgante and Olivieri, 1993; Wang et al., 1994). Because of their long primer repeat
length(16-18bp SSR repeat) and highly polymorphic interaction at individual loci, ISSR markers
were used in the present study with the following objectives:

General Objective:

 To assess the pattern of genetic diversity and relationships of Ethiopian chickpea cultivars,
using ISSR markers.

Specific Objectives:

- To evaluate the potential informativeness of ISSR markers for identifying chickpea cultivars.
- To determine the amount of genetic diversity within and among two chickpea cultivars type.
- To assess generation of information in chickpea cultivars for breeding programs and
conservation.

3
 2. LITRATURE REVIEW

2.1. Chickpea (Cicer arietinum L.) Description

The name Cicer is of Latin origin, derived from the Greek word 'kikus' meaning force or
strength. Duschak (1871) traced the origin of the word to the Hebrew 'kirkes', where 'kikar'
means round. The word arietinum is also Latin, translated from the Greek 'krios', another name
for both ram and chickpea, an allusion to the shape of the seed which resembles the head of a
ram. It is currently cultivated in over 50 countries of the world and grown on 12 million hectares
producing around 10.9 million tons of seed in 2010 growing season (FAOSTAT, 2012).

Chickpea is one of the dominant annual pulse crop grown in several region of Ethiopia where the
annual rainfall is 700-2000mm, PH is 6.4-7.9 and moderate temperature. It is planted on an
elevation between 1400-2300 meters above sea level of heavy black vertisol at the end of rainy
season. These soils have high evapo-transpiration and dry rapidly, subjecting the plant to
moisture stress. It can also be grown on poor soil and can`t tolerate heavy rain or water stress.
Therefore, it fails to grow over wet tropical areas where it can be damaged by frost, hailstorm
and excessive rain. Chickpea can be cultivated as a sole crop, or in rotation with barley, grass
pea, linseed, mustard, peas, coffee, safflower, potato, sweet potato, sorghum and wheat but it
often follows wheat, barley and tef (Van derMaesen, 1987).

The stems of chickpea plants are branched, glandular pubescent, erect or spreading with 0.2-1m
tall. Its leaves are glandular pubescent with 3-8 pairs of leaflets and it has deep tap root (up to
2m, known as hardy crop) which enables chickpea to withstand drought conditions by extracting
water from deeper layer of the soil (Oplinger et al., 1990). Flowers are born in groups of two or
three and come in purple, white, pink or blue colors depending upon varieties. Each flower
produces a short, pubescent pod that appears to be inflated and carry a maximum of three seeds
each. The seeds come with either rough or smooth surfaces and can be cream, yellow, brown,
black or green in color (Van derMaesen, 1987).

In Ethiopia, chickpea seeds are generally used as snacks (in roasted and salted form), side dishes,
and breakfast or infant foods. They are consumed as green (Eshet), boiled (germinated) and

4
roasted (Kolo). The dried seeds are mixed with wheat or barley ground to powder to make
‘‘Kiyit Injera’’ and unleavened bread. Split chickpea seeds and removing the husk (Kik) and
powdered seeds (Shiro) are also used to make ‘wot’ a type of sauce eaten with injera. The
vegetative biomass is highly valued as a fodder in dry areas where grazing vegetation is scarce. It
is a good source of protein for feeds, except that the amino acids methionine and cystine are
deficient (Oplinger et al., 1990).

Generally there are two distinct types of cultivated chickpea based on 23 morphological
characters mainly seed size, shape and color, plant growth habit, pigmentation of vegetative
floral part (Gaur et al., 2010) as shown in appendix figure 4. (a) Desi chickpea (microsperma):
Chickpeas with colored and thick seed coat. The common seed colors include various shades and
combinations of brown, yellow, green and black. The seeds are generally small and angular with
a rough surface and high percentage of fibre. (b) Kabuli chickpea (macrosperma): are
characterized by white or beige-colored seed with ram’s head shape, low percentage of fiber, thin
seed coat, smooth seed surface, white flowers, and lack of anthocyanin pigmentation on the stem.
The kabuli types generally have larger seeds, smoother seed coat, and lighter color and receive
higher market price than desi types.

A third sub-type, intermediate chickpea, is characterized by medium to small seed sized, pea-
shaped and creamy colored seeds which may be as a result of crossing desi and kabuli types.
Hair like structures on its stem, leaves and pods secrete acid that provide the first line of defense
against pests, and reduce the need for chemical sprays (Yadav et al., 2007). It is commonly
accepted that kabuli chickpea originated from the desi type in the Mediterranean basin through
natural mutation and selection (Hawtin and Singh, 1981). The Kabuli cultivars are cultivated
principally in the Mediterranean basin, the Near East and America where the entire seeds are
used for human consumption after soaking and boiling. The large, smooth-coated and rapid
cooking Kabuli chickpea seed is usually preferred for human consumption. The desi type is
widely cultivated in Indian subcontinent, East Africa and Ethiopia. In Ethiopia approximately,
more than 85% of the area is covered with desi type where as the rest 15% is kabuli type (Singh
and Malhotra, 1984).

5
2.1.1. Origin, taxonomy and distribution of chickpea

Chickpea is one of the first domesticated, cool season autogamous grain legume grown in more
than 50 countries (Gaur et al., 2010). Harlan (1992) stated that chickpea has one definable center
of origin; Turkey around 5450BC and more than one secondary centers of diversity. India and
Ethiopia have been proposed as secondary centers of diversity for cultivated chickpea. There are
five major centers of diversity for chickpea that includes the Mediterranean basin, central and
West Asia, the Indian sub-continent and Ethiopia. Indeed, the archaeological record suggests that
cultivated chickpea was one of the first grain legumes to be domesticated in the Old World (Van
derMaesen, 1987).

The genus Cicer belongs to the family Leguminosae, subfamily Papilionoideae, tribe Cicereae
and comprises 43 species, out of which nine are annuals while the rest are perennials
(Muehlbaver, 1993). Among the nine annual species, Cicer arietinum is the only cultivated
species and eight other annuals are wild species. These include: C. reticulatum,
C. echinospermum, C. pinnatifidum, C. judaicum, C. bijugum, C. cuneatum, C. chorassanicum
and C.yamashitae (Rakesh et al., 2008). Van derMaesen (1987) classified the Cicer species into
four sections based on their morphological characteristics, life cycle and geographical
distribution. (1) Eight annual species namely C. arietinum, C. reticulatum, C. echinospermum,
C.pinnatifidum, C. bijugum, C. judaicum, C. yamashitae and C. cuneatum were placed in
Section Monocicer, (2) C. chorassanicum and C. incisum (perennial species) in section
Chamaecicer, (3) Twenty-threee perennial species in section Polycicer and (4) seven woody
perennial species in section Acanthocicer.

6
 C. anatolicum
C. pinnatifidum
C. bijugam

C. echinospermum

C. judiacum C. chorassanicum

C. yamashitae

C. cuneatum

Figure 1: Geographical distribution of different Cicer species Source: Van derMaesen (1987).

Chickpea is one of the world's most important annual crops cultivated over an area of 10 × 106
ha across North and Central America, the Mediterranean basin, East Africa, the Middle East,
Asia and Oceania. It has spread with human migration toward the West and South via the Silk
Route. The Cultivated species is found in West Asia and North Africa covering Turkey in the
north to Ethiopia in the south, and Pakistan in the east to Morocco in the west. While the wild
species are most abundant in Turkey, Iran, Afghanistan and central Asia, but the wild species
Cicer cuneatum was also collected from Ethiopia (Figure 1).

7
2.1.2. Production and importance of chickpea

The major chickpea growing countries in the world are India, Pakistan, Turkey, Ethiopia, U.S.A.,
Mexico and Australia (FAO, 1994). Like most pulses, chickpea production has increased only
marginally in the last three decades because of the decline in the area of production. However,
the productivity of the crop has improved by about 37% in the corresponding period due to the
development of high yielding cultivars and improved technologies (Singh and Saxena, 1999).

Chickpea is Cultivated on a large scale in arid and semi-arid environments, and has considerable
importance as food, forages and green manure among the poor and subsistence farmers. It is
valued for herbal medicine, cosmetics production and livestock industry as an alternative source
of protein from its threshed or dried vines and energy feedstuff (Christodoulou et al., 2005).
Chickpea is not only an important source of protein in human diets, but it also plays a significant
role in maintaining soil fertility particularly in dry areas, through biological nitrogen fixation. In
Ethiopia chickpea is important as part of cropping system since it mitigate the decline of soil
fertility by avoiding excessive use of chemical fertilizers to decrease environmental
contamination and soil acidification through crop rotation with cereals.

The seeds of chickpea contain protein of 16.7% to 30.6% for desi type and 12.6% to 29.0% for
kabuli type, commonly 2–3 times higher than that of cereal grains; carbohydrate of 51– 65% in
desi type and 54 – 71% in kabuli type (Wood and Grusak, 2007); lipid of 2.9% to 7.4% for desi
and 3.4% to 8.8% for kabuli types, and high percentage of different minerals nutrients such as
calcium, magnesium, potassium, phosphorus, iron, zinc and manganese (Ibrikci et al., 2003).
Chickpea has hypochlorestermic and hypolipidemic effect, and is effective in reducing
cholesterol levels (Geervani, 1991). In addition to lowering cholesterol, the high fiber content
prevents blood sugar levels from rising too rapidly after a meal, making chickpea a good choice
for individuals with diabetes, insulin resistance or hypoglycemia (McIntosh and Miller, 2001).

Chickpea does not contain any anti-nutritional factors except the raffinose type oligosaccharides
which cause flatulence. However the oligosaccharides can be neutralized by boiling or mere
soaking in water (Queiroz et al., 2002). Therefore, it is a staple food in many countries and plays
an enhancing role in vegetarians’ diet around the world, as a good source of energy, protein,

8
minerals, vitamins, fiber, and potentially health-beneficial phytochemicals. The leaves are said to
yield an indigo like dye. Its extract is rich in malic and oxalic acid which sometimes used for
medicinal purpose or as vinegar to prepare an adhesive (Geervani, 1991). Although it is not
water resistant, it is suitable for plywood and textile sizing due to the starch content. Generally,
it`s medical application includes using for the treatment of aphrodisiac, bronchitis, cutamenia,
cholera, constipation, diarrhea, dyspepsia, snakebite, sunstroke and warts (Duke, 1981).

2.1.3. Production constraints and tolerance to stress

High and more stable yields are the major goals of plant breeding programs. Breeding progress
depends on the magnitude of genetic variability among the genetic materials under consideration,
heritability of a given trait in a given environment and the level of selection intensity applied
(Singh et al., 2002). However, the maximum yield of crop cultivars, determined by their genetic
potential, is achieved rarely due to lack of improved seeds and several biotic or abiotic
production destabiling factors. These includes insufficient water (nutrients), plant diseases, insect
pests, poor traditional agronomic practices, low price, lack of sustainable marketing policy and
poor extension services. Therefore, identifying major production constraints and setting cultivar
improving technologies through genetics of resistance are the first systematic approaches to
solve these problems in plant breeding (Singh et al., 1994). Moreover, wild species hold promise
and deserve attention in resistance breeding, as they appear to be valuable sources of resistant
genes.

Several factors including the length of growing period, mean air temperature, soil drainage, soil
reaction, soil depth, occurrence of soil borne diseases etc. also determine the adaptation and
performance of chickpea production (Anbessa, 2003). Ascochyta blight, Rhizoctonia root rot,
Pythium rot, Fusarium wilt, White mold, and bacterial blight are diseases problems in chickpea
field production. These typical diseases also affect other legume crops when favored by periods
of high rainfall, humidity and high temperatures. There are also viruses isolated from chickpea
that include alfalfa mosaic, pea leaf roll, pea streak and bean yellow mosaic (Duke, 1981).

These biotic constraints are best controlled by using good quality seed, proper crop rotation,
proper tillage practices, burying diseased residue and using diseases resistant varities (Oplinger

9
et al., 1990). Thus screening, selecting and crossing the resistant varieties have been generating
higher heterosis from genetically diverse parents. Water logging, drought, heat and salinity are
the major abiotic stresses of chickpea production. Water is a major factor determining the kind
and amount of natural vegetation as well as crop yield in different regions. Low nutrient stress
preceded by drought may also be among the most important abiotic stresses with worldwide
distribution (Singh, 2002), especially nitrogen and phosphorus deficiency in tropic and sub tropic
areas.

2.2. Genetic Resource and Genetic Diversity

Crop genetic resource is the total genetic diversity of cultivated species and their wild relatives,
which include commercial varieties, landraces, special genetic stokes (mutants, breeders line
etc.) and weedy relatives having potential value to human. Genetic resource utilization is the
exploitation of natural resource to satisfy human need in addition to maintaining the environment
that initiates an issue of agricultural sustainability. It is nothing but meeting the need of human
population without compromising the ability of future generation to get their own needs
(Worede, 1988).

Genetic diversity refers to the variation of genes within or among populations/species due to
evolution, domestication and plant breeding. It makes possible to develop new breeds of
plants or domestic animals and allow wild species to adapt the changing conditions (Hunter,
1996). Here, natural evolution build up genetic diversity and domestication causes further
differentiation in small part of wild species to become adapted for human requirement.
Therefore, genetic diversity is the foundation of genetic variation among individual varieties for
adaptation, domestication, survival and evolution of species. Genetic diversity of plant
populations is largely influenced by factors such as reproduction system, genetic drift,
evolutionary history and life history. In broad-spectrum, out crossing species have higher levels
of genetic diversity than selfing and clonal plants (Gezahegne Girma, 2007).

Knowledge of genetic diversity and population structure relatedness among germplasm is the key
for successful breeding program, effective plant conservation or for elucidating the taxonomy,
evolution and origin of crop species (Staub et al., 1997). Joint analyses of molecular and

10
phenotypic diversity attempt to predict the breeding value for different phenotypic traits
depending on the molecular marker diversity or parental genotype. The most important use of
germplasm collection has been their exploitation as sources of resistance and tolerance to biotic
and abiotic stresses, and to improve the nutritional quality of the crop, both for human and
animal consumption (Robertson et al., 1997).

Ethiopia is one of the world`s richest country in crop diversity and its genetic resources are of
critical value. The Ethiopian region is often referred to as a major vavilovian center of diversity
for several domesticated crops and their wild relatives. It is a center of origin for various crops
such as tef (Eragrostis tef), coffee (Coffee arabica), noug (Guizotia abyssinica), enset (Ensete
ventricosum) and mustard (Brassica carinata). It is also a center of diversity for a number of
crop species that have been introduced centuries ago and developed in to wide genetic diversity.
These include pulse crops such as faba bean, chickpea, lentil, cowpea, grass pea and major cereal
crops such as durum wheat, finger millet and some oil crops like linseed, caster bean, sesame and
safflower (Feven, 2002).

2.3. Genetic Erosion and Genetic Conservation

Crop productivity can be improved by genetic modification of cultivars or by altering the

growing environment. Study of genetic diversity is the process by which variation among
individuals or groups of individuals is analyzed by a specific method or a combination of
methods. It is required for populations to evolve and cope up with environmental change, new
disease and pest epidemics. Today there is a great concern over the disappearance of the genetic
resources, partly because of the substitution of a diverse set of genetically variable crop land
races with few genetically uniform improved varieties (Harlan, 1992).

As a single gene or few major genes control most of the desired traits, it is obvious that growing
uniform cultivars over vast areas is potentially dangerous since the products are frequently
vulnerable to changing conditions such as unexpected drought or virulent viruses because of
their narrow genetic base. Loss of genetic diversity is often associated with reduced reproductive
fitness. This means Species with greater genetic diversity are more likely to be able to evolve in
response to a changing environment than those with low diversity. However, species that lack

11
genetic diversity may experience low fertility among individuals, even in the environments that
are not changing (Hunter, 1996). These genetic erosions are severe problems in conservation of
biodiversity.

Genetic characterization and evaluation of available germplasm does not only unveil the
magnitude and pattern of genetic diversity of germplasm for conservation but also enables one to
determine useful genes in genotype progress through future breeding activities (Arumuganathan
and Earle, 1991). There are two strategies of germplasm conservation, ex-situ and in-situ. Both
systems are being followed in Ethiopia`s crop genetic resource conservation. But in-situ, a
community-based conservation of farmers` varieties in different agro-ecological regions, is not
yet rigorously undertaken as that of ex-situ. Genetic resource depletion resulting from several
forces like promotion of mono cropping should also be tackled by devising appropriate mode of
conserving the genetic resources both at in situ and gene bank levels.

Analysis of chickpea by biochemical and DNA based markers sometimes revealed low levels of
intra-specific diversity and at other times showed high levels (Hameed et al., 2009). These have
mainly been explained because of the self-pollinating nature of chickpea and mutations,
respectively. Assessment of the extent of genetic diversity within chickpea is fundamental for
chickpea breeding and conservation of genetic resource. Criteria for estimation of the genetic
diversity can be through morphological (Upadhaya et al., 2007) or molecular studies.

The importance and need of chickpea cultivars at global level requires evaluation of germplasm
to assist the future breeding programs. Seetharam et al. (2009) stated that genetic diversity
analysis in plants has been traditionally assessed using morphological traits. He argued that this
assessment of phenotypic variation may not be a reliable measure of genetic differences as gene
expressions are under environmental influence and called for the use of DNA based markers.
The assessment of genetic diversity is important not only for crop improvement but also for
competent management and conservation of germplasm.

12
2.4. Genetic Markers and their Applications

A genetic marker can be defined as a specific gene that produces a recognizable trait and can be
used in family or population studies. There are different genetic markers with different properties
in terms of degree of information provided for evaluating genetic variation: morphological (agro-
morphological), biochemical (isozymes) and DNA based markers (molecular markers) (Staub et
al., 1997). They are measurable inherited genetic variations that can be widely used by breeders
and conservationists to study genetic diversity or to understand genetic components. Traits that
serve as genetic markers are polymorphic; the more polymorphic the trait, the greater its
potential value to germplasm management (Seetharam et al., 2009).

Molecular markers developed directly from the parts of genes are referred as “genic” molecular
markers (GMM) and used for detection of nucleotide diversity in genes controlling agronomic
traits in plant populations. GMMs developed from coding sequences like ESTs or fully
characterized genes frequently have been assigned known functions. Based on the site of
polymorphism and latter’s effect on phenotypic variation, GMMs have been classified into two
groups (Anderson and Luebberstedt, 2003):
i) Gene-targeted markers (GTMs): derived from polymorphisms within genes, however not
necessarily involved in phenotypic trait variation, e.g. untranslated regions (UTRs) of EST
sequences (Aggarwal et al., 2007).
(ii) Functional markers (FMs): derived from polymorphic sequences or sites within genes and,
thus, more likely to be causally involved in phenotypic trait variation. The FMs, depending on
the involvement in the phenotypic trait variation, are further classified into two subgroups: (a)
indirect functional markers (IFMs), for which the role for phenotypic trait variation is indirectly
known, and (b) direct functional markers (DFMs), for which the role for the phenotypic trait
variation is well proven. They have some advantages over random markers that are generated
from anonymous region of the genome, because FMs are linked to the desired trait allele. Such
markers are derived from the gene responsible for the trait of interest and target the functional
polymorphism in the gene. A FM allows breeders to track specific alleles within pedigrees of
population and to minimize linkage drag flanking the gene of interest (Anderson and
Luebberstedt, 2003).

13
Markers are entities that are heritable as simple Mendelian traits and easy to score (Schulman et
al., 2004). The major drawbacks of morphological and biochemical markers are, variability, age
dependent and could be influenced by environmental factors. DNA based markers on the other
hand are not influenced by environment and do not exhibit epistatic interactions. The limitless
availability of DNA based markers permits construction of linkage maps with hundreds or even
thousands of markers using a single mapping population (Nayak et al., 2010).

2.4.1. Morphological markers

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. Phenotypic traits have the advantage that they
may be directly related to the fitness of the populations and usefulness for plant breeding.
Genetic characterization by using morpho-agronomic character was undertaken on chickpea and
most of the results indicate the existence of adequate genetic diversity at the phenotypic level
(Upadhaya et al., 2008). But the quantitative characters may not necessarily reveal the diversity
at gene level because some changes to genes can not be observed at a morphological or
physiological level. That means structural differences in the gene product may not alter its
biological activity sufficiently to result in altered phenotype.

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. They have great advantages since morphological characters are simple
to score and inexpensive too. Some of them are very plastic in nature which could be easily
affected by environmental change and affect the exact relationships of plant species, (Vithanage
et al., 1995). These quantitative traits are influenced by environmental factors, implying that they
show continuous variation.

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

14
not mean that any of the biochemical or molecular or both replace morphological markers, rather
they are used in combination. Morphological studies indicated that the three annual species C.
reticulatum, C. echinospermum and C. bijugum are closest to C. arietinum (Robertson et al.,
1997).

2.4.2. Biochemical markers (Isozymes)

Biochemical markers 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.
Therefore, depending up on number of loci, state of homo-zygosity or hetero-zygosity, and
enzyme configuration, from one to several bands are visualized. The positions of these bands can
be polymorphic and considered as informative loci. Isozymes have provided a valuable tool for
the study of genetic variation in crop populations and to investigate species relationships
(Schulman et al., 2004).

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 crop plants such as, chickpea, field pea, lentil, wheat and rapeseed. Seed proteins are
considered as practical and reliable methods because seed storage proteins and nucleotide
sequences are largely independent of environmental fluctuations. Moreover, some enzymatic
assays are possible only at certain growth stage and in specific tissues. Since most isozyme loci
are mono-morphic in cultivated chickpea, the paucity of isozyme polymorphism restricts their
application in germplasm characterization and tagging of genes of interest, but they were
effectively exploited in the case of inter-specific crosses (Kusmenoglu et al., 1992).

The accessions of cultivated species of Cicer exhibited similar seed storage protein patterns
(Ahmad and Slinkard, 1992) which show a very low genetic diversity in cultivated chickpea. The
total isozymes that can be readily analyzed in plant tissue are usually less than 50% . Isoenzyme
electrophoresis of chickpea had revealed insufficient polymorphism particularly in cultivated
chickpea as a result of a narrow genetic variability and low allelic number (Feven, 2002).
However, Kazan et al. (1993) showed that there is already developed map based on the

15
morphological and isoenzyme markers for crosses between C.arietinum, C. reticulum and C.
echinospermum chickpea species.

In general, biochemical markers include various physico-chemical variations of bio-molecules

such as seed storage proteins, blood proteins, carbohydrates, lipids and allozymes. Such bio-
chemical markers are intermediates between morphological and DNA based markers since they
are often the intermediate result of gene expressions. 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 loci in the genome, highly biased
genomic sampling, requirement of fresh tissue, and sometimes limited variation (Weising et al.,
2005). Therefore, their major shortcomings are lower polymorphism, lower genome coverage,
technical difficulties and the need for specific developmental stage for certain enzymes
(Schulman et al., 2004).

2.4.3. Molecular markers

According to Vithanage et al. (1995) molecular markers are "land marks" which can be
identified on the genome and can be applied in the identification of cultivars and clones. They
are also used for genetic mapping, diversity analysis, marker assisted selection, hybrid
prediction, germplasm management, population genetics, screening duplicate accessions in gene
bank, selection of recurrent parental genome in back cross and molecular systematics (Weising et
al., 2005). Molecular markers are based on naturally occurring polymorphisms in DNA
sequences; base pair deletion, substitutions, or additions. They are superior to both
morphological and biochemical markers because they are relatively abundant through the
genome even in highly inbreed cultivars, completely independent of environmental conditions.
Some of them exhibit co-dominant mode of inheritance and can be detected at any stage of plant
development (O’ Neill et al., 2003).

The choice of marker system is largely dependent on the intended application, ease of use, costs
involved in development and genotyping. Molecular markers and linkage maps are the
prerequisites for undertaking molecular breeding activities. However, the progress towards
development of a reasonable number of molecular markers has been very slow in cultivated

16
varieties of chickpea. One of the main reasons for this may be attributed to the low level of
genetic diversity present in the cultivated gene pools of the species, at least with the detection
tools that are currently available (Sharma et al., 1995).

DNA fingerprinting, describes any multi locus approach of visualizing DNA polymorphisms
either by hybridization or polymerase chain reaction (PCR). It is an important tool for
characterization of germplasm and establishment of varieties identity /hybrids/parental sources in
plant breeding and germplasm management. Molecular studies have also clearly indicated a
close relationship between C. arietinum, C. reticulatum and C. echinospermum, which strongly
support the idea of C. reticulatum as the possible wild progenitor of cultivated chickpea (King
and Burke 1999).

DNA based markers such as RAPD (Sudupak et al., 2002), RFLP (Udupa et al., 1993), AFLP
(Nguyen et al., 2004), SSR (Keneni et al., 2012) and ISSR (Weising et al., 2005) were used to
study genetic diversity and relationships in chickpea. Most of these studies reported abundant
diversity in wild Cicer but narrow genetic diversity in cultivated chickpea. Molecular markers
are also used in different areas of genome analysis like genetic diversity (intra and inter-
specific), cultivar identification (Upadhyaya et al., 2008 and Sefera et al., 2011), construction of
genetic linkage maps (Nayak et al., 2010), identification of QTLs (Cobos et al., 2005), and
marker assisted selection (Gupta and Varshney, 2000).

In recent years, different marker systems such as RFLP, RAPD, ISSR, SCAR, STS, AFLP, SSR,
SNPs, and others have been developed and applied to a range of crops including cereals (FAO,
2003). Their common features are summarized below in appendix table 2. The co-dominant
markers such as isozymes, RFLP, SSR and SNP markers have distinct advantages over dominant
markers since (a) they allow plant breeders to identify individuals homozygous for the desired
alleles in the f2 generation of a cross without the need of f3 progeny testing (Yu et al., 1991) and
(b) their co-dominant nature permits easy transfer of markers between genetic maps of different
crosses (Thomas and Scott, 1993).

17
2.4.3.1. Restriction fragment length polymorphism (RFLP)

RFLPs have been used 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 (Mohita, 1998).

RFLP, the first widely used non-PCR based molecular markers, have been used to study the
genetic relationships in the nine annual chickpea species and showed little genetic diversity
(Udupa et a1 1993). Thus, highest genetic variation was observed in C. pinnatifidum and the
lowest in C. macracanthum. However, as compared with PCR based techniques RFLP analyses
is labor intensive, time-consuming, expensive and require relatively pure and intact DNA
(Weising et al., 2005). Later, with the development of PCR-based random amplified
polymorphic DNA (RAPD) and simple sequence repeats (SSRs), most of the problems
associated with RFLP were overcome.

2.4.3.2. Random amplified polymorphic DNA (RAPD)

RAPD, the first PCR based molecular marker technique, identification can be used at any stage
of plant development and is not affected by environmental factors (Lisek et al., 2006). It
is simple, fast, relatively cheap and highly polymorphic technique which does not require
sequence information for primer design and no need of probe development. As a result, it is
useful in studies on chickpea`s genetic diversity, phylogeny, gene tagging and evolutionary
biology (Ratnaparkhe et al., 1998). But the technique has an inherent limitation of being
sensitive to laboratory changes, less reproducible and dominant marker, which can not
distinguish between the homozygote and heterozygote dominants.

RAPD was used in diversity analysis of different plants such as coffee (Aga et al., 2000), cereals
and horticulture (Birmata et al., 2004). It was also used in study the relationships among annual
Cicer species and revealed existence of genetic relationships among all species. Melese Dadi
(2005) employed RAPD marker to assess genetic diversity among 17 elite Ethiopian chickpea
genotypes. Out of six RAPD primers tested, only one primer generated polymorphic patterns
between the genotypes, which indicate narrow genetic base of the cultivated species.
18
This low degree of genetic diversity detected by RFLP and RAPD markers in cultivated chickpea
limit mapping of large number of these markers in a given cross. Sudupak et al. (2002) also used
RAPD markers to investigate genetic relationships among the annual Cicer species. The RAPD
analysis placed C.arietinum, C. reticulatum and C. echinospermum in a single cluster, C.
yamashitae and C. chorassanicum in a second cluster, C. pinnatifidum, C. bijugum and C.
judaicum in a third cluster and C. cuneatum in the fourth cluster. The cultivated species C.
arietinum was closest to the wild species C. reticulatum, its most probable progenitor (Ngyen et
al., 2004).

2.4.3.3. Amplified fragment length polymorphism (AFLP)

AFLP is a molecular technique that combines restriction based RFLP marker and PCR based
RAPD marker. The technique first uses the restriction enzymes to digest genomic DNA,
followed by ligation of double-stranded adaptors to the sticky ends of the restriction fragment to
generate template DNA (Vos et al., 1995). 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.

The AFLP technique seems to be more reliable than other PCR-based molecular markers (Reiter
2001). In addition, this technique has an option for high throughput by means of multiplexing
primer pairs, automated fragment detection and scoring. Polymorphisms arise from sequence
changes in the restriction sites or the insertions/deletions of selective bases in the fragments.
Bands are generally detected as presence or absence, making AFLPs a dominant marker
system. Advantages of AFLP markers are it generate higher polymorphism and no primer
sequence requirement. Therefore, AFLP has been used in chickpea for the development of
genetic linkage map and to assess genetic diversity within and among different chickpea species
(Nguyen et al., 2004). But it is technically demanding and requires high quality DNA, which
limits the application of the technique (Gedil et al., 2001).

19
2.4.3.4. Simple sequence repeat (SSR)

Microsatellite is a simple DNA sequence of short tandem repeated segments (1-6 base pairs) at a
unique physical location in the genome. They are highly polymorphic DNA based markers with
discrete loci and co-dominant alleles. Polymorphism among individual occurs due to variation in
the number of repeats. Microsatellite markers are also known as simple sequence repeats
(SSR’s), simple sequence tandem repeats (SSTR) and simple sequence length polymorphism
(SSLP) markers. They were first developed for use in human genome and were later found to be
abundant in plants (Morgante and Olivieri, 1993). Initial identification and screening of
microsatellite loci are time consuming. The sequences flanking microsatellite sites are generally
conserved within species and often in closely related species. The flanking sequences of
microsatellite loci of cultivated chickpea were found to be conserved in related annual species
(Gupta and Varshney, 2000).

Among various molecular markers currently available SSR markers have been proven and
recommended as markers of choice for a variety of applications in plant breeding because of
their multi-allelic nature, co-dominance inheritance and relative abundance or extensive genome
coverage (Gupta and Varshney, 2000). RFLP is not readily adapted to high sample throughput
and RAPD assays are not sufficiently reproducible, while both SSRs and AFLPs are efficient in
identifying polymorphisms, SSRs are more readily automated. As a result several hundred SSR
markers have been developed in chickpea.

Keneni et al. (2012) use 33 miccrosatellite markers to study the genetic diversity and population
structure of 155 Ethiopian chickpea (Cicer arietinum L.) germplasm accessions from different
geographical origins. Here, specific markers on the gel revealed a total of 111 bands with a range
of 2-5 bands per marker and the existence of more polymorphism in the introduced genotypes
than in the landraces. This showed the existence of high genetic diversity in Ethiopian chickpea
germplasm accessions, which can be useful for more systematic germplasm management and
utilization in breeding programs.

Sethy et al. (2006) also used 25 polymorphic SSR markers to analyse intra-specific genetic
diversity within 36 geographically diverse chickpea accessions. Based on cloning and

20
sequencing of size variant alleles at two microsatellite loci revealed that the variable numbers of
AG repeats in different alleles were the major source of polymorphism. Further Udupa et al.
(2004) studied dynamics of microsatellite evolution in chickpea and select di ant tri nucleotide
repeat (TA) n and (TAA) n, respectively. Based on polymorphism they observed that the two loci
do not evolve in complete independence.

2.4.3.5. Inter simple sequence repeats (ISSR)

The Inter Simple Sequence Repeats (ISSR) is microsatellite based multi locus marker technique,
which is simple and useful for estimating genetic diversity in several crop plants. It has the
advantage of RAPD and shows higher level of polymorphism, reproducibility and cost
effectiveness per polymorphism. ISSRs segregate mostly as dominant markers following simple
Mendelian inheritance. However, they have also been shown to segregate as co dominant marker
s in some cases thus enabling distinction between homo-zygotes and hetero-zygotes (Wang et
al., 1992). The microsatellite repeats used as primers in ISSR markers can be di-nucleotide, tri-
nucleotide, tetra-nucleotide or penta-nucleotide with degenerate bases extended into the flanking
sequences (Zietkiewicz et al., 1994) as shown in appendix figure 1.

ISSR assays can be undertaken for any species that contains a sufficient number and distribution
of SSR motifs and have the advantage that genomic sequence data are not required. They are
now being applied for cultivar identification, phylogenetic relationships and assessment of
genetic diversity in various plant species (Hou et al., 2005). ISSR analyses offer breeders and
geneticists with competent means to link phenotypic and genotypic variations in various fields of
plant improvement. So ISSR fingerprinting has been successfully applied to determine genetic
diversity and relationships in a number of crop species (Godwin et al., 1997). Recently, a large
scale genetic diversity study in chickpea revealed detection of 1683 alleles in 2915 accessions
from composite collections using 48 ISSR markers (Upadhyaya et al., 2008).

ISSR markers are observed to be highly variable within the species and reveal many more
polymorphisms since they use longer primers that allow more stringent annealing temperatures
(Hillis et al., 1996). Applications of ISSR technique in gene tagging and marker assisted
selections are becoming more popular. Thus the advantage of ISSR markers lies in their linkage

21
to SSR loci and likely to mark gene rich regions (Kojima et al., 1998). ISSR markers are as well
practical to study hybrid seed genotyping and varietal identification. Ranade et al. (2000) studied
the hybrid seed genotyping and plant varietal identification using ISSR markers in cotton for
GOT (Grow out Test) for genetic purity. Moreover, this marker observed to be very useful in
detecting genetic diversity and population structure of Coffee (Tesfaye, 2006), Lentil (Edossa et
al., 2010), Rice (Gezahagne, 2007) and White lupine (Oumer, 2011) collected from all over
Ethiopia.

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. The potential for integrating ISSR-PCR into programs of plant improvement is
enormous. The major areas of ISSR-PCR application in different crops include: genetic diversity
and phylogenetic analysis, genomic fingerprinting, genome mapping, gene tagging, marker
assisted selection, determining SSR motif frequency and studies on natural populations/
speciation (Pradeep Reddy et al., 2002).

22
 3. MATERIALS AND METHODS

3.1. Plant Material and Sampling Strategy

A total of 29 cultivated chickpea cultivars were used for this study. This includes two separate
sets of experimental materials, 14 kabuli type and 15 desi type varieties. The materials were
collected from the High Land Pulse Crop Improvement Programmes of three Agricultural
Research Centers, namely, Debre Ziet, Holeta and Sirinka in EIARC. Among these Holeta ARC
varities were breeding lines (elite chickpea varieties). The descriptions of varieties used in the
experiment are summarized in table 1.

Table 1: Description of chickpea varieties used in the experiment

SN Kabuli Varieties Year of release Breeder/maintainer Source Seed color

1 DZ-10-4 1974 DZARC/EIARC Ethiopia White cream
2 Arerti 1999 DZARC/EIARC ICARDA White cream
3 Shasho 1999 DZARC/EIARC ICRISAT White cream
4 Chefe 2004 DZARC/EIARC ICRISAT White cream
5 Habru 2004 DZARC/EIARC ICARDA White
6 Ejeri 2005 DZARC/EIARC ICARDA White
7 Teji 2005 DZARC/EIARC ICARDA White
8 Acos Dubie 2009 DZARC/EIARC Mexico White cream
9 Yelibie 2006 SRARC/ARARI ICRISAT Yellowish
10 Kobo 2010 SRARC/ARARI ICRISAT Yellowish
11 Kaseche 2010 SRARC/ARARI ICRISAT White
12 Akuri 2011 SRARC/ARARI ICRISAT Cream
13 ICC-4973 Breeding line HARC India White
14 ICC-19180 Breeding line HARC ICRISAT White

23
 Table 1. (Cont…)

SN Desi Varieties Year of release Breeder/maintainer Source Seed color

1 DZ-10-11 1974 DZARC/EIARC Ethiopia Brown
2 Dubie 1978 DZARC/EIARC Ethiopia Grey
3 Mariye 1985 DZARC/EIARC ICRISAT Brown
4 Worku 1994 DZARC/EIARC ICRISAT Golden
5 Akaki 1995 DZARC/EIARC ICRISAT Brown
6 Naatolii 2007 DZARC/EIARC ICRISAT Light Golden
7 Minjar 2010 DZARC/EIARC ICRISAT Golden
8 Mastewal 2006 DZARC/ EIARC ICRISAT Golden
9 Fetenech 2006 SRARC/ ARARI ICRISAT Reddish
10 Kutaye 2005 SRARC/ ARARI ICRISAT Red
11 ICC- 4948 Breeding line HARC India Dark Brown
12 ICC- 15996 Breeding line HARC ICRISAT Reddish
13 ICC- 5003 Breeding line HARC India Dark Brown
14 1CC- 4918 Breeding line HARC India Brown
15 Pm- 233 Breeding line HARC ICARDA Light Brown
DZ - Debre Ziet, SR – Sirinka, H – Holeta and ARC - Agricultural Research Centers.

Ten chickpea seeds of each cultivar were sown in 20-25cm diameter plastic pots containing
black vertisol soil with organic manure. The soil is ideal for chickpea production and maintained
in Addis Ababa University glass house (Arat-killo). Watering was done once a day regularly but
unfortunately two of the kabuli type cultivars (Akuri and ICC-4973) died in the green house
because of powdery mildew and were excluded from the analysis. Thus only 27 cultivars were
included in this study and after three weeks of germination, five individual plants per pot were
selected randomly for evaluation. Healthy young leaf samples were harvested from each selected
plant and dried in silica gel for genomic DNA extraction.

24
3.2. DNA Extraction

®
Genomic DNA extraction was done based on the Invisorb spin plant mini kit extraction

protocol (Robert-Rossle, 2009). Which allows rapid and efficient isolation of high quality
genomic DNA from a wide variety of plant materials (upto 100mg of fresh, frozen or dried
plants part) with some modifications on concentration as well as incubation and centrifugation
time. It combines the lysis of starting material with very efficient binding of sufficient and
pure genomic DNA onto a spin filter surface without cahaotropic ions. DNA extractions were
carried out from silica gel dried leaf samples by grinding ten leaf lets per cultivar using pestle
and mortar at genetic research laboratory, Department of Biology, Addis Ababa University
(AAU).

3.3. Quality of Extracted DNA

The quality of extracted genomic DNA was tested using agarose gel electrophoresis for which
5μl of the DNA sample was mixed with 3μl loading dye (Bromophenol blue) and loaded to
0.83℅ gel and electrophoresed in 100ml, 1x TBE running buffer at constant voltage of 80V for
50 minutes. After electrophoreses, the gel was stained with 50μl ethidium bromide (10mg/ml) in
450 ml, distilled water for 25 minutes, and de-stained in distilled water for a further 25 minutes.
Gel picture, was taken under UV trans-illuminator by BioDocAnalyze apparatus with digital
canon camera that was connected to desk top computer (Figure 2), then genomic DNA samples
with high band intensity and less smear were selected for PCR in further analysis.
Icc- 19180
Aco-Dube
DZ-10-11
DZ-10-14

Icc- 5003
Icc -4918

Icc- 1596
Icc -4948

Matewal

Pm- 233
Naatolii

Fetnech

kaseche
Mariye

Kutaye
Minjar
Worku
Shasho

Yelibie
Habru
Arerti
Akaki
Dubie

Chefe
Kobo
Ejeri

Teji

Figure 2: Pictures of test gel of genomic DNA for the individual chickpea cultivars.
25
3.4. Primer Selection and Optimization

In the present study, ISSR was used for the first time to assess genetic variation of cultivated
chickpea cultivars released from Ethiopia. A total of twenty-one Inter simple sequence repeat
primers obtained from the Genetic Research Laboratory (Primer kit UBC) were used for the
initial testing of polymorphism and reproducibility. From these stocks only four di-nucleotide
repeat ISSR primers (Table 2) were selected for this study based on the degree of polymorphism
and distinctness of generated bands. They were used to detect polymorphism in chickpea
cultivars while the other seventeen primers (UBC – 813, 816, 829, 832, 834, 835, 836, 837, 841,
843, 847, 850, 853, 857, 863, 872, and 890) didn`t amplify the genomic DNA.

Table 2: List of primers and their annealing temperature with respective sequences used for the
analysis.

No. Primer code Primer sequence(5’-3’) Annealing Temperature

1 UBC – 830 TGTGTGTGTGTGTGTGG 48oC
2 UBC – 840 GAGAGAGAGAGAGAGAYT 48oC
3 UBC – 842 GAGAGAGAGAGAGAGAYG 48oC
4 UBC – 849 GTGTGTGTGTGTGTGTYA 48oC
Y= Pyrimidines (C or T); All primers are di-nucleotid repeats

3.5. PCR Amplification and Electrophoresis

PCR amplification was carried out in Biometra 2000 T3 thermo-cycler with a 25μl reaction
mixture containing 1μl template DNA, 14.6μl ddH20, 2.8μl dNTP (1.25mM), 2.6μl Taq
buffer (10xThermopol reaction buffer), 2.5μl Mgcl2 (2mM), 1μl primer (20pmol/μl) and 0.5μl
Taq Polymerase (5u/μl). The amplification program was 4 minutes pre heating and initial de-
naturation at 94oC, followed by 40cycles of 15 seconds transition at 94oC, 1 minutes primer
annealing at 48oC, 1:30 minutes initial chain elongation at 72oC using the fastest possible
transition temperatures. The last cycle was followed by an additional extension at 720C for 7
minutes to ensure that the primer extension reaction was completed. The PCR products were
stored at 40C until loading on gel for electrophoresis.

26
An agarose gel (1.67gm agarose with 100 ml 1xTBE) was prepared and 9μl of the PCR products
of each sample mixed with 4μl loading dye (0.12% bromo-phenol blue and 30% glycerol), and
loaded on to the gel. A molecular ladder of 500 bp was loaded on to the gel wells to estimate
molecular weight of ISSR fragments. The electrophoresis was done for 2 hours at constant
voltage of 100V. The gel was stained with 50μl ethidiumbromide (10mg/ml) which was mixed
with 500 ml distilled water for 45 minutes and de-stained for 40 minutes. Finally the gel picture
was taken with UV- illumination Bio-Doc analyzer.

3.6. Data Scoring and Statistical Analysis

ISSR bands were scored visually and each fragment, amplified using ISSR primers was treated
as a unit character, and scoured as ‘1’ for presence, ‘0’ for absence and ‘?’ for ambiguous data.
Based on recorded bands POPGENE version 1.32 software (Yeh et al., 1999) was used to
calculate genetic diversity for varieties used as number of polymorphic loci, percent
polymorphism, Gene diversity (H) and Shannon diversity index (I). Analysis of molecular
variance (AMOVA) was used to calculate variation among and within cultivars using Areliquin
version 3.01 (Excoffier et al., 2006). NTSYS- pc version 2.02 (Rohlf, 2005) and Free Tree
0.9.1.50 (Pavlicek et al., 1999) software’s were used to calculate Jaccard’s similarity coefficient
by using the following formula:-

Sij= a
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 number of bands present in individual j but not in individual i.

The Unweighted Pair Group Method with Arithmetic mean (UPGMA) (Sneath and Sokal, 1973)
was used to analyze and compare the cultivars and generate phenogram using NTSYS- pc
version 2.02 (Rohlf, 2005). The Neighbor Joining (NJ) method (Saitou and Nei, 1987) was used
to compare individual varieties and evaluate patterns of cultivars clustering using Free Tree
0.9.1.50 Software (Pavlicek et al., 1999). The major difference between the two algorithms is
27
that UPGMA assumes equal rates of evolution (rigid molecular clock assumption) along all
branches, Whereas neighbor joining assume variations in the rate of change (Nei and Kumar
2000; Lan and Reeves 2002).

To further examine the patterns of variation among individual samples on 3D, and highlight the
resolving power of ordination (Seetharam et al., 2009), a principal coordinated analysis (PCO)
was performed based on Jaccard’s similarity coefficient (Jaccard, 1908). The calculation of
Jaccard’s coefficient was made with PAST software version 1.18 (Hammer et al., 2001). The
first three axes were latter used to plot the three dimensional PCO with STATISTICA version
6.0 software (Hammer et al., 2001).

28
 4. RESULTS AND DISCUSSION

4.1. ISSR Markers and their Banding Patterns

Out of the twenty-one primers tested initially, four of them gave relatively clear banding pattern
and they were selected to distinguish chickpea cultivars in this study (Table 3). But 17 of the 21
primers did not amplify DNA of any chickpea cultivars. These primers, which did not amplify
DNA of all the genotypes, may not have found complementary sequences on the genomic DNA.
Such non-amplifying primers are also reported in other crop plants (Tao et al., 1993). The size of
the fragments amplified by using four primers were in the range of 500 bp to 3500 bp (Figure 3).
The banding pattern of chickpea samples using primer UBC-849 is shown in figure 4 and others
are presented in appendix figure 5. The number of amplified fragment and polymorphic loci
produced per individual varied depending on the primer used. Here, the number of bands
produced by each primer ranged from three bands for primer 840 to eight for primer 849.

The di-nucleotide repeats (GA) 8 gave least amplification and (GT) 8 gave the most amplification
in the present study. In contrast (Pradeep Reddy et al., 2002) studied Inter simple sequence re-
peat (ISSR) polymorphism and its application in plant breeding, where he found that Primers
with (AG), (GA), (CT), (TC), (AC), (CA) repeats show higher polymorphism than primers with
other di-, tri- or tetra-nucleotide repeats.

Table 3: Selected ISSR primers with their repeat motifs, amplification quality and scorable
bands.

ISSR Primers Repeat Motif Amplification Quality Number of Scored Bands

UBC–830 (TG)8 G Very Good 7

UBC–840 (GA)8 YT Good 3

UBC–842 (GA)8 YG Good 6

UBC –849 (GT)8YA Excellent 8

Total 24

29
 MARKER
Shasho Shasho
Arerti
Akaki
Akaki
Minjar Dubie
Naatolii
Icc-4948

UBC–849.
Worku
DZ-10-14
Ejeri
Minjar

primer UBC – 842.

Kutaye
Fetenech
Chefe
Icc- 4948
Kobo Mariye
DZ–10-14
Icc-5003
Mastewal
Icc-918 Kutaye

30
Pm -233 Chefe
Kobo
Icc-996 Icc- 5003

DZ-10-11 Icc- 4918

Pm -233
kaseche
Icc-15996
Acos DZ –10-11
Kaseche
Yelibie
Acos
Habru Yelibie

Icc-19180 Habru
Icc-19180
Teji
Teji
Naatolii MARKER

Figure 4: ISSR fingerprint generated from 21 representative chickpea cultivars using primer
Figure 3: Molecular weight of amplified band - patterns for chickpea DNA samples amplified by

Dubie
500 bp
1000bp
4000 bp
3000 bp

1500 bp
2000 bp
4.2. Genetic Diversity as Revealed by Percent Polymorphism, Shannon Weaver and Gene
Diversity Values

Out of the total 24 loci scored, 22 were observed to be polymorphic. In all individuals the
number of polymorphic loci ranged from two for primer-840 to eight for primer-849. Primer 830
and 849 showed 100 percent polymorphism, while primer 840 and 842 showed 66.67 and 83.33
percent polymorphism respectively in the overall analysis. Considering the percent
polymorphism, least polymorphism was observed for primer-840 (Table 4).

Among the Cicer arietinum cultivars evaluated using ISSR markers with all primers desi type
chickpea exhibited a genetic diversity value of 0.30 and Shannon diversity index of 0.47, while
the kabuli type chickpea had 0.33 and 0.50 for genetic diversity and Shannon diversity index
values respectively. The average genetic diversity for the sample was 0.32 (Table 5). Primer 849
showed highest genetic diversity and Shannon diversity index values (0.46 and 0.65,
respectively) and primer 842 was the least (0.25 and 0.39, genetic diversity and Shannon
diversity index values, respectively).

Thus, kabuli chickpea cultivars were observed to show higher percent polymorphism and
Shannon’s diversity index than desi chickpea cultivars with an average genetic diversity of 0.32.
This may be due to possible gene flow between cultivars through effectores (human, insects,
birds) and can be further supported with presence of close relatives of desi cultivars. Because
kabuli chickpea cultivars are originated from the desi type in the Mediterranean basin through
natural mutation and selection (Hawtin and Singh, 1981).

31
Table 4: Number of polymorphic loci (NPL), percent polymorphism (PP), genetic diversity (H)
and Shannon Index information (I) of 27 chickpea cultivars based on each primer used.

With individual primers

Primers code NPL PP (%) H±SD I±SD
UBC–830 7 100 0.39±0.12 0.57±0.13
UBC–840 2 66.67 0.27±0.25 0.40±0.35
UBC–842 5 83.33 0.25±0.18 0.39±0.24
UBC–849 8 100 0.46±0.04 0.65±0.04
Average 5.5 87.5 0.34±0.15 0.50±0.19

Table 5: Number of polymorphic loci (NPL), percent polymorphism (PP), genetic diversity (H)
and Shanon Index information (I) of 27 chickpea cultivars based on all 4 primers used.

With all primers

Cultivars NPL PP (%) H±SD I±SD
Desi type 18 75 0.30±0.21 0.47±0.30
Kabuli type 22 91.67 0.33±0.15 0.50±0.20
Average 20 78.33 0.32±0.18 0.49±0.25

4.3. Analysis of Molecular Variance (AMOVA)

Analysis of molecular variance was carried out on the overall ISSR data score of chickpea
cultivars with grouping as desi and kabuli type. It was done by computation of the distance
between “haplotypes”, each individual`s data pattern as one “haplotypes” and computing
variance components for each level (Excoffier et al., 2006). As shown in table 6, partition of
genetic diversity by analysis of molecular variance revealed that higher percentage of variation
was attributed to within cultivars (97.71%) and the rest 2.29 percent variation was attributed to
among cultivars. This could be due to cross pollinating agents and seed intermixing during
harvesting. The Variation among groups and within groups was found to be highly significant at
(p=0.00).

32
In addition, the AMOVA result of Edossa et al., 2010 on lentil, Tesfaye K. 2006 on coffee,
Oumer Abdie 2011 on white lupine and many other earlier studies support the higher genetic
diversity within populations than among population. Edossa studied the morphological and
molecular diversity of Ethiopian lentil (Lens culinaris Medikus) using four ISSR primers and
found 56.28% diversity within populations than among populations (43.72%). Oumer Abdie
(2011) also studied genetic diversity of Ethiopian white lupine using four ISSR primers and
reported 74.62% of genetic diversity within populations and 25.38% among the populations of
white lupine species.

Since the plant is highly self-pollinated species, higher genetic diversity was expected among
cultivars than within cultivars. However, AMOVA result indicated larger genetic diversity
within the cultivars type than among cultivars and this might be accounted to two contrasting
reasons. The one like any other plant species, the genetic diversity of Cicer arietinum L. species
is affected by multiple evolutionary forces like mating types, gene flow, genetic drift,
evolutionary history, mode of reproduction and natural selection. There might be moderate gene
flow among the desi and kabuli type cultivars by insect, human and birds. On the other hand
preferential adaptive genes of chickpea cultivars are not fixed in self-pollination throughout
evolutionary history.

Table 6: Analysis of Molecular Variance (AMOVA) of chickpea cultivars in Ethiopia based on

their type as desi and kabuli.

Source of d.f SS Variance Percentage Fixation P

Variation components of variation Indices
Among Cultivars 1 1.293 0.02304 Va 2.29 0.02285 0.00

Within Cultivars 25 24.633 0.98533 Vb 97.71 0.00

Total 26 25.926 1.00838 100

SS- sum of square, df- degrees of freedom

33
4.4. Clustering Analysis

Cluster analysis of UPGMA (Unweighted Pair-Group Methods Using arithmetic Averages) and
NJ (Neighbor Joining) analysis dendrogram were computed for all 27 individuals of
Cicer arietinum L. cultivars based on 24 PCR bands amplified by four di-nucleotide ISSR
primers. The UPGMA dendrogram resulting from a SAHN clustering analysis and NJ analysis
on the basis of Jaccard’s coefficients of similarity was constructed. The Jaccard’s similarity
coefficient was obtained after pair-wise comparisons performed using binary character matrices
(of the presence and absence) that were produced from amplified fragments.

The dendrogram derived from the whole ISSR data showed three distinct clusters and sub-
clusters within each main cluster as indicated on figure 5. The first cluster consisted of almost all
the desi type chickpea cultivars except Kobo and Arerti cultivars. At about 76% similarity
coefficient, the first cluster forked into two sub-clusters, one consisting Akaki, ICC-15996, DZ –
10-11, Kutaye, Dubie, Naatolii, Pm-233 and the other consisting of ICC-4918, Kobo, ICC-4948,
Arerti, Mastewal, ICC-5003. Arerti and Kobo showed more than 66% and 79% similarity
respectively with other second sub cluster of desi type cultivars. This might be due to the fact
that kabuli cultivars are inter-specific hybrids of wild and cultivated desi type chickpea and
hence these cultivars might have close genetic similarity with desi chickpea.

The second cluster consisted of all the kabuli type chickpea cultivars and Minjar, Worku, Mariye
from desi type. Around 60% similarity, two sub-clusters were distinct in the second cluster. The
first sub-cluster consisted of Minjar, Shasho, Acos, Yelibie and Habru, while Worku, Ejeri,
Mariye, DZ–10-14, and Kaseche were grouped under the second sub-cluster. Worku and Mariye
show 82% and 70% closely relatedness with second sub-cluster of kabuli cultivars.

The third cluster consists of Fetenech, Chefe, ICC-19180 and Teji chickpea cultivars where the
clustering of Fetenech here negates the expectation that it would be grouped in the first cluster.
Fetenech might have had a tight similarity with Chefe, ICC-19180 and Teji as shown in NJ
analysis. From the dendrogram established by neighbor-joining method (Figure 6) it is clearly
noted that few cultivars lacked purity (Kobo, Mariye, minjar and ICC-4948) and intermixed in
clustering. Some of the individuals from each cultivar type appear to have longer extended

34
branchs from their respective group, which indicate that they are more variable. That means,
individuals escaped from the cultivars might have accumulated many diverse adaptive gene
complexes adapted to environmental changes than the other individuals of the same cultivar type.

Generally, the desi and kabuli cultivars clearly observed to have more or less distinctly separated
groups in both UPGMA and neighbor joining analysis. Both trees recovered almost the same tree
topology with similar clustering, although few individuals appeared to escape from groups in
case of neighbor-joining analysis.

Akaki
ICC-15996
DZ-10-11
Kutaye
Dubie
Natolli
Pm-233
ICC-4918 C-I
Kobo
ICC-4948
Arerti
Mastewal
ICC-5003
Minjar
Shasho
Acos-dubie
Yelbie
Hebru
C-II
Worku
Ejeri
Maryie
DZ-10-4
kaseche
Fetenech
Chefe C-III
ICC-19180
Tejie
0.44 0.56 0.69 0.82 0.94
Coefficient

Fiure 5: UPGMA dendrogram depicting clustering patterns for 27 Chickpea cultivars based on
Jaccard’s similarity coefficients.

35
Figure 6: Neighbor-joining analysis of 27 individuals based on Jaccard’s similarity coefficient.

4.5. Principal Coordinate (PCO) Analysis

All the data obtained using four ISSR primers were used in PCO analysis using Jaccard’s
similarity coefficients to display the genetic associations between the two cultivar types
diagramatically. The orientation of the axis was defined by coordinates, whose lengths are given
by eigen values, which provide information’s about the dimensionality of the data and how the
individuals of the cultivars are related to each other and the main axes. The first three
coordinates of the PCO showed an Eigen-value of 2.43, 1.93 and 1.31. Thus, Eigen vectors from
the first, second and third principal coordinate axes accounted in 13.23, 10.52 and 7.14 percent
of the total variation respectively.

36
These three coordinates were used to construct the three dimensional (3D) graph to show
individuals grouping (Figure 8). For comparison, the two dimensional representation (2D) of the
PCO analysis were also presented in figure 7. The 3D graph observed to have better grouping of
individuals in to their respective cultivars type except few intermixing than the 2D
representation. Because in 2D there was no clear distinction between cultivar types, instead of
being scattered all over the plot.

0.4
ICC-4918
0.3 Akaki
Kutaye
Yelbie
DZ-10-11
Natolli
0.2 Minjar
Kobo
Shasho ICC-4948 ICC-15996
0.1 Pm-233
Mastewal
Arerti
Hebru
Coordinate 2

0 Dubie

-0.1 Acos-dubie

-0.2 ICC-5003
kaseche
-0.3 Maryie DZ-10-4
Worku
Tejie
Fetenech Ejeri
-0.4 Chefe

ICC-19180
-0.4 -0.3 -0.2 -0.1 0 0.1 0.2 0.3 0.4 0.5
Coordinate 1

Figure 7: Two dimensional representation of principal coordinate analysis for 27 Chickpea

cultivars based on Jaccard’s similarity coefficient using ISSR data.

37
Figure 8: Three dimensional representation of principal coordinate analysis of genetic
relationships among 27 cultivars of Cicer arietinum L. inferred from similarity
matrix using the Jaccard`s index.

38
 5. SUMMARY, CONCLUSION AND RECOMMENDATION

5.1. Summary

Currently, a number of molecular markers have been widely used to study diversity in many
plants (Karp et al., 1996). Given the proliferation of molecular markers, a comparison between
markers seems highly inevitable on the basis of study objectives and the nature of the markers.
Among many desired quality of molecular markers, automation (PCR-based), polymorphisms
and reproducibility are the highly demanded features of the molecular techniques to be used in
the intraregional diversity analysis. Thus PCR-based markers may enhance the efficiency of
conventional breeding methods for the introgression of traits among different chickpea cultivar
types. ISSR markers are one of the molecular markers that have these characteristics to study
variability in different crops.

In this study, ISSR markers were observed to be appropriate molecular markers for generating
intera-specific genetic diversity to evaluate extent of genetic diversity within and among Cicer
arietinum L. cultivars. Out of the total 24 scorable bands produced by four di-nucleotid primers,
22 bands were polymorphic. This indicates that molecular markers revealed polymorphism at the
DNA level, make ISSR marker an ideal and a very powerful tool for characterization of genotype
and estimation of genetic diversity in chickpea cultivars. Despite the fact that the diversity
analysis indicated the existence of diversity among 2 chickpea cultivar types, the total genetic
diversity is generally narrower.

Among chickpea cultivars considered in the present study, kabuli type chickpea showed slightly
higher genetic diversity than desi type. Partitioning of genetic diversity, AMOVA, using
ungrouped cultivars revealed that out of the total genetic diversity, most of the ISSR diversity
was distributed within cultivars (97.71%) and the remaining diversity was distributed among
cultivars (2.29%). Thus results of AMOVA revealed to support the larger genetic diversity
found in several crops within cultivars rather than among cultivars.

Based on jaccard`s similarity coefficients of UPGMA, high genetic similarity was observed
between DZ-10-11 and Kutaye chickpea cultivars (0.942) followed by the value between Akaki

39
and ICC-15996 (0.90). The least similarity was observed between Akaki cultivar with Teje
cultivar (0.42). The Fetenech cultivar shares relatively smaller similarity values with all cultivars
from the desi type chickpea. The desi chickpea cultivar showed higher similarity values than the
kabuli cultivar.

Generally in this study, though all the diversity parameters quantified the availability of gene
diversity among the cultivated chickpea cultivars of Ethiopia, the total genetic diversity is
narrower. The low genetic diversity found among the Ethiopian cultivars evidence the narrow
genetic bases used in the national breeding program of Ethiopia and it could be attributed to
strong selection by breeders and local farmers for traits of their interest.

5.2. Conclusion

In this study, four di–nucleotide repeated ISSR primers were employed to assess the pattern of
genetic diversity and relationships among desi and kabuli type chickpea cultivars. They were
able to reveal the genetic diversity ranged from moderate to high levels and identified the highest
as well as the least diverse cultivars in Ethiopian chickpea. Here, high genetic diversity was
observed among kabuli type cultivars and moderate levels of variation were shown in desi type
cultivars. The Shannon’s diversity index also confirmed the existence of higher diversity in
kabuli cultivars. The genetic similarity based on Jaccard’s similarity coefficients was observed
high value between DZ-10-11 and Kutaye chickpea cultivars (0.942), while the least similarity
was observed between Akaki and Teje cultivars (0.42).

Generaly, dendrogram drawn based on jacard similarity coefficient revealed some level of
association between cultivars and individuals from the same type of cultivars were largely
grouped together. Until the present day, information available on the reproductive biology of
Cicer arietinum L., suggested that it is a predominantly self-pollinating plant. However, the
result of this study showed opposite to this due to the occurrence of higher genetic diversity
within cultivars than among cultivars.

40
5.3. Recommendation

Although the ISSR analysis indicated the existence of diversity among the 27 chickpea cultivars,
the total genetic diversity is narrower. This low genetic diversity among the Ethiopian chickpea
cultivars indicated that the cultivars are closely related. Therefore, the National Breeding
Program should take cautious action to broaden the genetic base of the chickpea cultivars in the
country to reduce its vulnerability to diseases and insect pest outbreak. Hence, the Ethiopian
chickpea Breeding Program should revise its chickpea breeding approach in a way that assists in
broadening the available narrower genetic diversity of the cultivars. This could be achieved
through gene transfer from wild relatives to cultivated species, implementing crossing programs,
enhancing variability through mutation breeding, utilize biotechnological tools in identifying,
characterizing and transfer of noble genes from distant relatives of the species.

Generally the patterns of genetic diversity obtained in this study suggested that:
► The study is not exhaustive in terms of sample size and marker systems. Hence, more sample
collection including wild relatives and morphological analysis has to be carried out.
► Rates of self- pollination and cross-pollination require in-depth investigation to account for
the speculated gene flow.
► Further study of ISSR marker by using more number of primers is recommended in order to
give confirmative results.
► In addition, studies using other molecular markers can give more information about chickpea
genotype to be used effectively for breeding and conservation program.

41
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APPENDICES

50
 7. APPENDIX

7.1. Appendex Ι

Appendix Table 1: Advantages and Disadvantages of Molecular Markers. (Hikmet et al., 2004).

Type of markers Advantages Disadvantages

Restriction Fragment -Co-dominant markers -Need large amount of good
Length Polymorphism -Highly reproducible quality DNA
(RFLP) -Can use filters many times -Laborious (compared to RAPD)
-Good genome coverage -Difficult to automate
-Can be used across species -Need radioactive labeling
-No sequence information -Cloning and characterization of
-Can be used in plants reliably probe are required
-Needed for map based cloning
Randomly Amplified -High genomic abundance -No probe or primer information
Polymorphic DNA -Good genome coverage -Dominant markers
(RAPD) -No sequence information -Not reproducible
-Ideal for automation -Can not be used across species
-Less amount of DNA -Not very well-tested
-No radioactive labeling
-Relatively faster
Simple Sequence Repeat -High genomic abundance -Can not be used across species
(SSR) -Highly reproducible -Need sequence information
-Fairly good genome coverage -Not well-tested
-High polymorphism
-No radioactive labeling
-Easy to automate
-Multiple alleles
Amplified Fragment -High genomic abundance -Very tricky due to changes in
Length Polymorphism -High polymorphism patterns with respect to materials
(AFLP) -No need for sequence information used
-Can be used across species -Cannot get consistent map (not
-Work with smaller RFLP fragments reproducible)
-Useful in preparing contig maps -Need to have very good primers

ISOZYMES -Useful for evolutionary studies -Laborious

-Isolation lot easier than that of DNA
-Can be used across species
-No radioactive labeling
-No need for sequence information
51
-Limited in polymorphism
-Expensive (a unique system )
-Have to know tissue location
-Not easily automated
Appendix Table 2: Commen features of commonly used molecular marker systems in cereals
(FAO, 2003).

Features RFLPs RAPDs AFLPs SSRs SNPs

DNA required (μg) 10 0.02 0.5-1.0 0.05 0.05

DNA quality High High Moderate Moderate High

PCR based No Yes Yes Yes Yes

Number of polymorphic 1.0-3.0 1.5-50 20-100 1.0-3.0 1.0

loci analyzed
Ease of use / Relative Not easy Easy Easy Easy Easy

Amenable to automation low moderate Moderate High High

Reproducibility / Relative high unreliable High High High

Development cost/Relative low Low Moderate High High

Cost per analysis/Relative high Low Moderate Low Low

Dominance/codominance codominant dominant Dominant Codominant codominant

52
7.2. Appendix ΙΙ

Appendix Figure 1: Principles of inter simple sequence repeat marker.

Appendix Figure 2: The diagrammatic illustration of PCR process for single complete cycle.

53
Appendix Figure 3: The diagrammatic illustration of PCR process that used to amplify even very
small samples of DNA in molecular marker analysis.

Kabuli Desi

Appendix Figure 4: Two types of chickpea seeds samples.

54
A

55
 D

Appendix Figure 5: Banding pattern of chickpea DNA samples by primer UBC - 830 (A), UBC -
840 (B), UBC - 842 (C) and UBC - 849 (D).

A B C

D E
F

Appendix Figure 6: The author`s work: A &B) Cultivars in green house of Arat-killo campus of
A.A.U. C) Preparing agarose gel. D) DNA extraction by Invisorb® spin
plant mini kit. E&F) Loading the DNA samples and amplicons on the gel.

56