GENETIC DIVERSITY OF OROMO POTATO (Plectranthus edulis

(VATKE) AGNEW) AS REVEALED BY INTER SIMPLE SEQUENCE

REPEAT MARKERS (ISSR)

M.Sc. Thesis

IJARA SHIFERAW

JANUARY, 2015
HARAMAYA UNIVERSITY
 GENETIC DIVERSITY OF OROMO POTATO (Plectranthus edulis
(VATKE) AGNEW) AS REVEALED BY INTER SIMPLE SEQUENCE
REPEAT MARKERS (ISSR)

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 BIOTECHNOLOGY

By
Ijara Shiferaw Adugna

January, 2015
Haramaya University
 HARA MAYA UNIVERSITY

SCHOOL OF GRADUATE STUDIES

As thesis research advisors, here, we certify that we have read and evaluated this thesis prepar
ed under our guidance by Ijara Shiferaw entitled “Genetic Diversity of Oromo Potato
(Plectranthus edulis (Vatke) Agnew) as revealed by Inter Simple Sequence Repeat Markers”
We recommend that it be submitted as fulfilling the thesis requirement.

Tileye Feyissa (PhD) ________ ____________________________

Major Advisor Signature Date

Yohannes Petros (PhD) ________ ____________________________

Co-Advisor Signature Date

As member of Board of Examiners of the MSc Thesis Open Defense Examination, we certify
that we have read and evaluated the Thesis prepared by Ijara Shiferaw, and examined the
candidate. We recommend the thesis it be accepted as fulfilling the Thesis requirement for the
degree of Master of Science in Biotechnology.

_______________________ ___________ ______________________

Name of Chairman Signature Date

_______________________ ___________ ______________________

Name of Internal Examiner Signature Date

_______________________ ___________ ______________________

Name of External Examiner Signature Date

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

i
 DEDICATION

This piece of work is dedicated to my father Shiferaw Adugna, my mother Birhane Tolcha and
my brothers, Haile Shiferaw, Jima Shiferaw and Adamu Shiferaw, who have helped me both
morally and financially for this success.

ii
 STATEMENT OF THE AUTHOR

By my signature below, I declare that this thesis is my own work and that all other sources of
materials used for this thesis have been duly acknowledged. This thesis has been submitted in
partial fulfillment of the requirements for MSc degree at the Haramaya University and is
deposited at the University’s Library to be made available to borrowers under rules 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 an
accurate acknowledgement of the source is made. Request for permission for 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: Ijara Shiferaw Signature____________________________

Place: Haramaya University

Date of submission: ___________________

iii
 BIOGRAPHICAL SKETCH

The author was born on October 07, 1988, from his father, Shiferaw Adugna, and, his mother,
Birhane Tolcha, in Kuyyu Woreda, North Shewa (Selale) Zone, Oromiya Regional State. He
attended his primary and junior secondary education at Seyoum Demissew and Gerba
Gurracha No.2 Schools, respectively. He was enrolled at Gerba Gurracha Secondary School
and attended his preparatory education at Gerba Gurracha Preparatory School. After the
completion of his Preparatory School education, he joined Aksum University in September,
2007 and graduated with BSc. degree in Applied Biology in July 2007. Soon after his
graduation, he was employed by the Ministry of Education (MoE) at Jigjiga University and
served from September, 2010 to July, 2011 as a Graduate Assistant until he joined the School
of Graduate Studies at Haramaya University to pursue a study leading to M.Sc. degree in
Biotechnology.

iv
 ACKNOWLEDGEMENTS

I would like to express my special thanks and heartfelt appreciation to my major advisor, Dr.
Tileye Feyissa, who helped me to be engaged in this research program and accepted me to
work in his laboratory and made possible my dreams of working M.Sc. thesis in the area of
molecular genetics. I acknowledge and value his competent guidance and unlimited
encouragement throughout my study period. I would like to extend my special thanks to my
co-advisor, Dr. Yohannes Petros, for his meticulous and valuable guidance, keen interest, and
encouragement and constructive criticism starting from the proposal development to thesis
write up.

I would also like to extend my heartfelt thanks to Dr. Kassahun Tesfaye for providing the
necessary support during the course of my research work. I would like to express my
appreciation and thanks to Ministry of Education (MoE) for giving me the MSc training
opportunity and for its financial support for this thesis work. I gratefully thank Jigjiga
University for giving me an opportunity for post-graduate study, sponsorship of the research
work and payment of my salary during the study period and Haramaya University, the
Department of Biology for accepting me as post-graduate student. Of all, I owe a special debt
of gratitude to Ato Tilahun (who helped me by giving me GPS at Sodo) and Nitsuh Aschale
for her cooperation by collecting samples from Gojam. I am grateful to Fantahun Meheret and
Edossa Fikru for their limitless excellent guidance during laboratory work and molecular data
analysis. I would like to thank my friends, Getachew Tadesse, Fikadu Asfaw, Mulatu T., Fufa
Beyene, Birhanu Hurisa, Berhanu Kapital, Tadesse Abate, Hawi Nigussie, Lewuye Gete,
Dagmawit Tobiaw (PhD candidate) and Belachew Seraw, and all my classmates at Haramaya
University and my friends at Addis Ababa University especially Tiruye Taye and Muluken
Birara for their support and encouragement.

Finally, I would like to take this opportunity to express the profound gratitude from the
bottom my heart to my beloved father Shiferaw Adugna and my mother Birhane Tolcha for
their continuous support both spiritually, morally and for their love. I never forget my family
who went through hard times together, cheered me on, and celebrated each accomplishment:

v
All my brothers including Adamu Shiferaw and Jima Shiferaw and my uncle Mulu Tola to the
success of my study.

vi
 ACRONYMS AND ABBREVIATIONS

AFLP Amplified Fragment Length Polymorphism

AMOVA Analysis of Molecular Variance

CTAB Cetyltrimethyl Ammonium Bromide

dNTP Deoxy Nucleotide Tri Phosphate

EDTA Ethylene Diamine Tetra Acetic acid

FAO Food and Agricultural Organization

ISSR Inter Simple Sequence Repeat

MoE Ministry of Education

NJ Neighbor Joining

PCO Principal Coordinate Analysis

PPL Percent of Polymorphic Loci

RAPD Random Amplified Polymorphic DNA

RFLP Restriction Fragment Length Polymorphism

SSR Simple Sequence Repeats

UBC University of British Colombia

UPGMA Unweighted Pair Group Method using the Arithmetic Average

vii
 TABLE OF CONTENTS
PAGE
STATEMENT OF THE AUTHOR iii
BIOGRAPHICAL SKETCH iv
ACKNOWLEDGEMENTS v
ACRONYMS AND ABBREVIATIONS vii
TABLE OF CONTENTS viii
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. LITERATURE REVIEW 4
2.1. Origin, Taxonomy and Botanical Description of Plectranthus edulis 4
2.2. Importance of P. edulis 6
2.3. Genetic Diversity 8
2.4. Genetic Markers and Their Application in Genetic Diversity Analysis 9
2.4.1. Morphological markers 9
2.4.2. Biochemical markers 10
2.4.3. Molecular markers 11
2.4.3.1. Non-PCR based molecular markers 12
2.4.3.1.1. Restriction fragment length polymorphism (RFLP) markers 12
2.4.3.2. PCR based molecular markers 12
2.4.3.2.1. Random amplified polymorphic DNA (RAPD) markers 13
2.4.3.2.2. Amplified fragment length polymorphism (AFLP) markers 13
2.4.3.2.3. Microsatellites or Simple sequence Repeat (SSR) markers 14
2.4.3.2.4. Inter Simple Sequence Repeat (ISSR) Markers 15

viii
 TABLE OF CONTENTS (continued)

3. MATERIALS AND METHODS 18

3.1. Plant Material 18
3.2. DNA Extraction 20
3.3. ISSR Primers Screening and Optimization 20
3.4. PCR Amplification and Gel Electrophoresis 21
3.5. Data Scoring and Analysis 22
4. RESULTS AND DISCUSSION 25
4.1. ISSR Markers and Banding Patterns 25
4.2. Genetic Diversity of P. edulis Populations 26
4.3. Analysis of Molecular Variance (AMOVA) 29
4.4. Cluster Analysis 30
4.5. Genetic Similarity 36
4.6. Principal Coordinate Analysis (PCO) 37
5. SUMMARY, CONCLUSIONS AND RECOMMENDATIONS 40
5.1. Summary and Conclusions 40
5.2. Recommendations 41
6. REFERENCES 42
7. APPENDICES 50
7.1. Appendix 51
7.2. Tables in the Appendix 53
7.3. Figures in the Appendix 54

ix
 LIST OF TABLES

Table Page

Table 1. Nutritional content of Plectranthus edulis, Solanum tuberosum and Ipomoea

batatas 8
Table 2. Regions and sites of Ethiopia from where the samples were collected 18
Table 3. List of ISSR primers tested for polymorphism and the reproducibility of the
amplified bands. 21
Table 4. Number of scorable bands, number of polymorphic bands, percent of
polymorphic loci, Nei’s genetic diversity (h) and Shannon's information index
(I) for each primer. 26
Table 5. The Number of polymorphic loci (NPL), Percent polymorphic loci (PPL), gene
diversity (h) and Shannon‘s information (I) Index for Ten Populations 28
Table 6. The Number of polymorphic loci (NPL), Percent polymorphic loci (PPL), gene
diversity (h) and Shannon‘s information (I) Index for Groupings 29
Table 7. Analysis Molecular Variance among and within ten populations of P. edulis. 30
Table 8. Similarity matrix for Jaccard’s coefficients based on the 46 bands obtained 36

x
 LIST OF FIGURES

Figure Page

1. ISSR- PCR 16
2. Map of Ethiopia showing where samples of P. edulis (Vatke) Agnew collected 19
3. PCR amplification product of 12 individuals of P. edulis 25
4. Dendrogram generated based on UPGMA analysis demonstrating the genetic similarity
between ten populations of P. edulis 32
5. UPGMA Dendrogram generated based on Jaccard’s coefficients of similarity 33
6. Neighbor-joining analysis of 98 individuals based on Jaccard’s coefficient 36

7. Two dimensional representations of 98 P. edulis individuals 38

8. Three dimensional representation of principal coordinate analysis 39

xi
 LIST OF TABLES IN THE APPENDIX

Appendix Table Page

1. Polymerase Chain Reaction (PCR) mixture (master mix and DNA template) in
this study 53

xii
 LIST OF FIGURES IN THE APPENDIX

Appendix Figure Page

1. Banding profiles of some P. edulis individuals selected using ISSR primer UBC-835
exhibiting fourteen individuals 54
2. Banding profiles of some P. edulis individuals selected using ISSR primer UBC- 841
exhibiting fourteen individuals 54
3. Banding profiles of some P. edulis individuals selected using ISSR (UBC-844) 55

xiii
 Genetic Diversity of Oromo Potato (Plectranthus edulis (Vatke) Agnew) as revealed by
Inter Simple Sequence Repeat Markers (ISSR)

Ijara Shiferaw, Haramaya University, Dire Dawa, Ethiopia

ABSTRACT

Oromo Potato (Plectranthus edulis Vatke Agnew) is an ancient Ethiopian indigenous, under-
utilized and annual tuber crop grown widely in the central, southern, western, northwestern
and southwestern parts of Ethiopia. It is a perennial shrub and one of the economically
important tuber crops. In spite of its importance as food security crop, there is no information
available on molecular genetic diversity of this crop. Therefore, the aim of this study was to
assess genetic diversity within and among 10 populations of Oromo potato using ISSR
markers. Using four ISSR primers, a total of 46 scorable bands were generated of which 42
were polymorphic. The only penta-nucleotide primer used in the study, primer UBC-880
(GGAGA)3, showed a unique band in some individuals and showed 100% PPL. Within
populations diversity based on polymorphic bands ranged from 41.30% to 65.22% with
overall PPL of 91.30%, Nei’s genetic diversity of 0.18 to 0.30 with overall Nei’s of 0.33,
Shannon information index of 0.25 to 0.39 with overall index of 0.48, and analysis of
molecular variation (AMOVA) of 74.23% were detected within populations. AMOVA showed
25.77% among populations variability which significantly lower than that of within
population variation. With all diversity parameters, the highest diversity was obtained from
Sodo Zuria, Gojam-II and Areka populations, whilst the lowest was from Nekemte. From
Jaccard’s pairwise similarity coefficient, Ambo and Sodo Zuria were most related populations
exhibiting 0.648 similarity and Gojam-I and Sodo Zuria were the most distantly related
populations with similarity of 0.489. Wolaita and Wollega populations exhibited highest
genetic diversity so that the populations should be considered as the primary sites in
designing conservation areas for this crop.

Keywords: Indigenous, ISSR, Lamiaceae, Molecular markers, Oromo Potato, Underutilized

Crop

xiv
 1. INTRODUCTION

Oromo Potato (Plectranthus edulis (Vatke) Agnew) (Lamiaceae) is an ancient Ethiopian

indigenous annual tuber crop grown widely in the central, southern, western, northwestern and
southwestern parts of Ethiopia (Edward, 1991; Edossa Etissa, 1996; GRIN, 2005). It is an
ancient Ethiopian tuber crop (Siegenthaler, 1963) which is locally known as Wolaita dono,
Gamo dinich, Dinicha Oromo, Agew dinich, etc., and also as Ethiopian potato (Mulugeta
Taye et al., 2007; Yeshitila Mekbib, 2007).

Plectranthus edulis (Vatke) Agnew is a perennial shrub and one of the economically
important tuber crops of the genus Plectranthus. The genus Plectranthus consists of mainly
tropical old world herbs and shrubs. A recent molecular study of the genus revealed the
presence of two major clades, the ‘‘Coleus’’ clade, which comprises most of the species
which are used to be called Coleus, and a second clade contains the remaining species of
Plectranthus (Paton et al., 2004).

This plant is one of the four economically important tuber crops of the genus Plectranthus,
together with Plectranthus esculentus (Livingstone potato) (Tindall, 1983; Allemann et al.,
2003; Allemann and Hammes, 2006), Plectranthus parviflorus (Sudan potato) (Tindall, 1983)
and Plectranthus rotundifolius (Madagascar potato) (Jansen, 1996). It is also primarily
cultivated for its tubers. These tubers are consumed after cooking. It is believed that P. edulis
sometimes serves as medicinal plant, eating the boiled root can avoid loss of appetite. To
some extent, also, the leaves are used as a cooked vegetable in some western Ethiopian
regions (Zemede Asfaw and Zerihun Woldu, 1997; Mulugeta Taye et al., 2007).

The genus Plectranthus has been distributed all over the tropical and subtropical regions of
India, Pakistan, Sri Lanka, Tropical East Africa, Brazil and Egypt. It occurs both as wild and
cultivated species in Ethiopia. However, P. edulis is one of the native tuber crops in Ethiopia.

1
Additionally, sixty-two species of Plectranthus are reported to be of economic and medicinal
interest and some are grown as ornamental plants (Lukhoba et al., 2006). They are used in
folk medicine for a variety of diseases including infectious diseases (Rivera Nun˜ez and
Obo´n de Castro, 1992).

Analysis of genetic structure at intra-specific level of an endangered species is important to

development of conservation strategies, exploration of plant genetic resources, and future
breeding programs of wild plants. Knowing the patterns of genetic variability among and
within populations is a valuable tool that may help to develop more efficient practices related
to conservation, serve as a basis for suitable management techniques of fragments, and
support in situ conservation measures (Frankel et al., 1995).

Genetic diversity assessment has potential uses in evolution, breeding and conservation of
genetic resources (Wu et al., 1999). The extent of diversity in the accessions is paramount for
improvement and utilization of genetic resources. Genetic diversity is therefore the backbone
of conservation of plant genetic resources for both present and future use (Quedraogo, 2001).
Genetic diversity of species also helps formulate appropriate sampling strategies for both in
situ and ex situ conservations, with the aim to protect the variability of taxa so as to preserve
ecological processes, rate of establishment, survival and fecundity (Millar et al., 2000).

Phenotypic characterizations of genotypes of this plant species were made (Yeshitila Mekbib,
2007 and Weyessa Gamtessa et al., 2009), but the molecular genetic diversity of this plant
have not been studied yet although its genetic diversity has long been based mainly on
morphological traits. However, morphological variability is often restricted; characters may
not be obvious to study its genetic diversity since morphological characters may be affected
by environment. Nowadays, a variety of different genetic markers has been proposed to assess
genetic variability as a complementary strategy to more traditional approaches in genetic
resources management (Somasundaram and Kalaiselvam, 2004).

Molecular markers currently available for the study of genetic diversity have their own
advantages and disadvantages. The recent DNA marker systems are based on PCR technology,

2
and for this reason are more suitable for routine cultivar identification, due to the small
amount of DNA required, and generally fast and simple tests. Polymerase chain reaction
(PCR) amplification of Inter simple sequence repeat (ISSRs) markers has been widely used
for genetic analyses of plants (Gupta & Varshney, 2000).

The objective of this study is therefore to analyze the genetic diversity of Oromo Potato
(Plectranthus edulis) populations grown in different regions of Ethiopia using ISSR markers
in an effort to provide some information for future research to improve and conserve this
species.

General Objective
The general objective of this research was to analyze genetic diversity of Oromo Potato
(Plectranthus edulis (Vatke) Agnew) populations in Ethiopia using ISSR Markers.

Specific objectives
• To evaluate the level of genetic diversity among and within P. edulis populations
growing in Ethiopia.
• To reveal the genetic structure within and among populations of P. edulis.
• To identify populations of P. edulis with the highest genetic diversity for sustainable
use, conservation and improvement programs.

3
 2. LITERATURE REVIEW
2.1. Origin, Taxonomy and Botanical Description of Plectranthus edulis

Plectranthus edulis is said to be originated in East Tropical Africa, Ethiopia. It has been
grown in different mid and high altitude areas (Siegenthaler, 1963) and its cultivation dates
back to about 3000 BC. Westphal (1975) reported it to be a major highland tuber and
indigenous crop in the southwestern parts of Ethiopia.

Plectranthus edulis grouped under a large family called Lamiaceae that occurs worldwide and
has species that are adapted to almost all habitats and altitudes. It is a diploid, dicotyledonous
plant and belongs to the family Lamiaceae; subfamily Nepetoideae and tribe Ocimeae (GRIN,
2005). In Plectranthus, the upper lip of the flower is unusually four-lobed and the large shoe-
shaped lower lip is formed from a single lobe, while in Labiatae the upper lip often consists of
two lobes and the lower consists of three lobes (Mulugeta Taye et al., 2012).

P. edulis, therefore, consists of similar structural components as other crops producing stem
tubers on stolons, like the Irish potato (Solanum tuberosum) (Struik and Wiersema, 1999). P.
esculentus differs from P. edulis because P. esculentus produces stem tubers that are sessile
and clustered around the stem (Allemann et al., 2003). The stolons of P. edulis are much
longer than those of Irish potato and particularly the aerial stolons behave differently from
those of Irish potato. They are very vigorous, growing fast over the soil surface, and could
achieve a length of more than two meters. The aerial stolons produce several branches and
bear tubers after the branches entered into the ground.

P. edulis differs also in other aspects from P. esculentus, like the flower colour which was
violet - blue tubers in P. edulis (Yeshitila Mekbib, 2007) while P. esculentus has yellow
flowers (Tindall, 1983), but also by the fact that tubers of P. esculentus show a clear positively
gravitropic growth (Allemann et al., 2003). There is no record that the exact growth direction
of P. edulis tubers and stolons, but the wide spread of the stolons suggests it was mainly
plagiotropic for the stolons (with the exception of the branches from aerial stolons entering the
soil).

4
P. edulis is small hairy, succulent herb about 50-60 cm high, with ovate and shallowly serrate
leaves. The inflorescence is a raceme of small flowers which are usually purplish-blue. The
leaves vary from dark-green to purplish-green. There are also different local cultivars that
have been identified by farmers (Mulugeta Taye et al., 2006).

Plants produce main stems and primary, secondary and tertiary branches, with primary and
secondary branches and their leaves constituting the main part of the canopy. Plant
components were the seed tuber pieces, sprouts, main stems, branches, leaves, flowers, fruits,
seeds, roots, stolons and tubers. Five, partly overlapping, ontogenetic vegetative phases were
observed during one crop cycle: emergence, canopy development, stolon initiation and
development, tuber initiation and growth, and a phase of canopy senescence (Mulugeta Taye
et al., 2012).

Stolons are formed on main stems and primary branches and originated below ground or
above ground (aerial stolons). Aerial stolons are initiated later than below-ground stolons and
are much longer (up to 2.5 m). Tubers usually are produced as a swelling on the tip of the
stolon and sometimes as a swelling of the middle part of stolons. Tubers are stem tubers with
pairs of ''eyes'' (compound buds) being arranged in the same decussate pattern as the auxillary
buds on stolons and stems (Mulugeta Taye et al., 2012).The leaves are oval, elliptical in shape,
dentate, sessile, pubescent, slightly bent outward at the tip and on the margin, and with
conspicuous veins. The phyllotaxis is opposite, with leaf pairs decussate. Leaves started to
appear from the first node of the branches. The matured leaf length is about 10 cm long and
the width of the widest middle part was 5 cm. Seeds are brown/black, ovoid-shaped and
smaller than 1 mm. The fruit consists of four seeds included in the persistent calyx (Mulugeta
Taye et al., 2012).

Tubers are formed at the tip of a stolon - as in Irish potato - or by swelling of a middle part of
a stolon. In Irish potato, tubers in the middle of the stolon are only observed when secondary
growth occurs, that is, the growth of the apex changes from tuber-like to stolon-like due to a
switch in external conditions to those that are not conducive to tuberization and tuber bulking,

5
for example, warm temperatures or rainfall after a dry period. For P. edulis, the exact nature
of the tuber formation in the middle of the stolon still has to be established, but most likely it
results from thickening of an existing stolon part.

P. edulis is propagated vegetatively using the edible parts, i.e. the tubers and grown as clones.
The tuber pieces, which can be planted as sprouted or unsprouted are principally obtained
from the previous crop or market (Yeshitila Mekbib, 2007).

2.2. Importance of P. edulis

Root and tuber crops are main stays for millions of people and occupy an important position
in world agriculture. Like the major crop species, potato, cassava, taro and yams, there are
about 100 root and tuber species of significance for agricultural or medicinal purposes. Most
of these may be important only locally, but play a significant role in the subsistence
economies and crop diversification (MAFF Research Council, 1994). For instance, the yield
of P. edulis is comparable to that of other root and tuber crops.

P. edulis, one of the crops that originated in Ethiopia, is inexpensive and accessible source of
essential nutrients to a country like Ethiopia where the people experience malnutrition
(Dandena Gelmesa, 2010). At present, there is a huge interest from the farmers, government
and nongovernmental organizations to maintain the crop and increase its production, firstly
because it contributes significantly to household food security (Yeshitila Mekbib, 2007),
secondly because it is also seen as a traditional food (Mulugeta Taye, 2008).

P. edulis is cultivated primarily for its tubers and is consumed after cooking (Westphal, 1975;
Zemede Asfaw and Zerihun Woldu, 1997). To some extent, also, the leaves are used as a
cooked vegetable in some western Ethiopian regions (Zemede Asfaw and Zerihun Woldu,
1997; Mulugeta Taye et al., 2007). The raw tuber is rich in carbohydrate energy (6 per
calories). The boiled tuber has a slightly higher carbohydrate concentration than Irish potato,
but both have comparable low protein and fat concentrations. The leaves of P. edulis are
frequently used as medicines to treat a range of ailments, particularly skin, infectious and
respiratory problems and digestive problems.

6
The P. edulis culture matches the definition of traditional vegetables as defined by FAO
(1999), which refers to the categories of plants whose leaves, fruits or roots are acceptable and
used as vegetables by rural and urban communities through custom habit or tradition.
Moreover, farmers highly appreciate P. edulis and indicate it satisfies one’s hunger better than
other tuber crops.

Weyessa Garedew et al. (2009) reported that P. edulis has comparable yield to potato with
yield range of 536.9 to 1008.9 gram per plant. Indigenous vegetables and tubers have rescued
thousands of hungry Ethiopians during famine period and it is a bridge during periods of grain
shortage, crucial to food security (Dandena Gelmesa, 2010). For instance, P. edulis has been
grown and used as a major source of food in many parts of Ethiopia and is liked as a tasty
source of carbohydrates (Mulugeta Taye, 2008). They are species that have not been fully
exploited or whose potential has not been fully taken advantage of possibly because their
cultivation is restricted and their use is localized.

The nutritional content of one hundred gram edible portion of both raw and cooked tubers of
P. edulis showed that it has high amounts of micro and macro-nutrients. While it has relatively
higher food energy when cooked than Solanum tuberosum, the fat and calcium contents are
almost twice as high as that of S. tuberosum. The protein content is similar to that of S.
tuberosum and is almost twice as high as that of Ipomoea batatas when cooked. The cooked
tubers have more amounts of energy, fiber and carbohydrate compared to the raw tuber (Table
1). However, the latter is richer in nitrogen, protein, calcium, phosphorous, iron and niacin
than cooked ones (EHNRI, 1997).

7
 Table 1. Nutritional content of Plectranthus edulis, Solanum tuberosum and Ipomoea batatas
(All values are per 100 gm edible portion).

Composition P. edulis S. tuberosum I. batatas

Raw Cooked Raw Cooked Raw Cooked
Food energy 69.00 100.60 103.7 89.7 136.00 134.20
(Calories)
Moisture (%) 81.90 73.80 73.10 76.80 67.40 65.60
Nitrogen (gm) 0.30 0.24 0.30 0.26 0.30 0.13
Protein (gm) 1.50 1.00 1.30 1.10 1.30 0.50
Fat (gm) 0.20 0.20 0.10 0.10 2.00 0.20
Carbohydrate 15.30 23.70 24.4 21.10 28.20 32.60
(gm)
Fiber (gm) 0.70 1.00 1.40 0.90 1.10 1.50
Ash (gm) 1.10 1.30 1.10 0.90 1.10 1.10
Calcium (mg) 29.00 19.00 14.00 9.00 52.00 53.00
Phosphorous 90.00 62.00 57.00 49.00 34.00 54.00
(mg)
Iron (mg) 9.30 1.10 2.30 1.50 3.40 0.90
Thiamin (mg) - 0.11 0.08 0.05 0.08 0.06
Riboflavin - 0.32 0.08 0.09 0.05 0.01
(mg)
Niacin (mg) 0.70 0.30 1.00 0.80 0.90 0.40

Source: Ethiopian Health and Nutrition Research Institute (EHNRI, 1997)

2.3. Genetic Diversity

Genetic diversity is referred to as the sum total of genetic variations found in a species or
population and it is source for ecological biodiversity. Measuring genetic diversity is
significant to examine variations present among the organisms on the basis of genetic markers
at phenotypic, biochemical and genotypic level. It is a prerequisite for efficient breeding plans,
collection expeditions or germplasm exchange in order to acquire specific characteristic
(Abebe Demissie and Bjornstrand, 1996).

More generally, individuals with different genetically determined trait values can interact in
unpredictable ways. Thus, despite the obvious presence of genetic variation for ecologically
important traits, we know relatively little about the range of potential ecological effects of

8
genetic diversity for population dynamics, species interactions and ecosystem processes. Early
interest in the ecological effects of genetic diversity occurred in several fields in addition to
evolutionary biology. For instance, in agronomy, there have long been efforts to increase crop
yield by planting genetically diverse varieties within a single field (Smithson & Lenne, 1996).

Although not universal, there is evidence that increasing varietal diversity can lead to greater
yield, as well as decreased damage by herbivores and pathogens (Smithson & Lenne, 1996).
The significance of these effects is apparent in current agricultural practices: the planting of
genetically diverse crops is now being applied on a large scale in some areas as an effective
means to maximize the yield by minimizing damage by pathogens (Zhu et al., 2000). There is
no work done on the genetic diversity based on molecular markers, even it’s based on the
morphological mainly.

2.4. Genetic Markers and Their Application in Genetic Diversity Analysis

Genetic markers fall into one of the three broad classes: those based on visually assessable
traits (morphological and agronomic traits), those based on gene product (biochemical
markers), and those relying on a DNA assay (molecular markers). The idea of using genetic
markers appeared very early in literatures but the development of electrophoretic assays of
isozymes, and molecular markers (Zietkiewicz et al., 1994) have greatly improved our
understanding in biological sciences.

2.4.1. Morphological markers

Traditionally, diversity within and among populations was determined by assessing

differences in morphology. Gomez et al., (2004) reported the complementarities of molecular
and phenotypic markers and recommended the use of both markers for a complete description
of the level and pattern of genetic diversity. Therefore, diversity analysis using morphological
characters is still relevant for evaluation of genetic diversity.

9
The limitation of morphological markers is often restricted, characters may not be obvious at
all stages of the plant development and appearance may be affected by environment. De
Vicente and Fulton (2003) also stated that they are subject to changes due to environmental
factors and may vary at different developmental stages and their number is limited. However,
morphological determinations need to be taken by an expert in the species. For example using
morphological characteristics, Weyessa Garedew (2013) showed the accessions of P. edulis
collected from geographically similar areas were grouped in different clusters. Similarly, a
variety of different genetic markers has been proposed to assess genetic variability as a
complementary strategy to more traditional approaches in genetic resources management.

2.4.2. Biochemical markers

Monoterpenes are a subgroup of the terpenoid substances found in resins and essential oils of
plants. Although the metabolic functions of monoterpenes are not fully understood, they
probably play an important role in resistance to attack by diseases and insects (Hanover, 1992).
The concentrations of different monoterpenes, such as alpha-pinene, beta-pinene, myrcene, 3-
carene and limonene are determined by gas chromatography and are useful as genetic markers.
Monoterpene genetic markers have been applied primarily to taxonomic and evolutionary
studies. However, they have also been used to a limited extent to estimate genetic patterns of
geographic variation within populations.

Allozymes have been the most important type of genetic markers in forestry and are used in
many species for many different applications. Allozymes are allelic forms of enzymes that can
be distinguished by a procedure called electrophoresis. Most allozyme genetic markers have
been derived from enzymes of intermediary metabolism, such as enzymes in the glycolytic
pathway; however, conceivably an allozyme genetic marker could be developed from any
enzyme. Allozymes are particularly useful at the level of conspecific populations and closely
related species, and are therefore useful to study diversity in crops and their relatives
(Hamrick & Godt, 1997).

10
The main advantages of protein markers are their co-dominant inheritance and the technical
simplicity and low cost of the assay. Disadvantages include the restricted number of suitable
allozyme loci in the genome, the requirement of fresh tissue, and the sometimes limited
variation (Weising et al., 2005).

2.4.3. Molecular markers

Molecular markers are based on naturally occurring polymorphisms in DNA sequences (i.e.:
base pair deletions, substitutions, additions or patterns) (Gupta, 1999). Molecular markers are
superior to both morphological and biochemical markers because they are relatively simple to
detect, abundant throughout the genome even in a highly inbred cultivars, completely
independent of environmental conditions and can be detected at virtually any stage of plant
development.

According to Vithanage et al. (1995) molecular markers are "land marks" which can be
identified on the genome and, therefore, offer the best possible means of identifying
individuals from biological samples. Molecular markers can be applied in the identification of
cultivars and clones, genetic mapping, marker assisted selection (MAS), population genetics,
molecular systematics, etc. (Weising et al., 2005).

Different marker systems such as Restriction Fragment Length Polymorphisms (RFLPs),

Random Amplified Polymorphic DNAs (RAPDs), Sequence Tagged Sites (STS), Amplified
Fragment Length Polymorphisms (AFLPs), Simple Sequence Repeats (SSRs) or micro-
satellites, Single Nucleotide Polymorphisms (SNPs) and others have been developed and
applied to a range of crops. The DNA based marker systems are generally classified as
hybridization-based (non-PCR) markers and (PCR)-based markers (Weising et al., 2005).

11
2.4.3.1. Non-PCR based molecular markers

Molecular markers based on restriction- hybridization techniques were employed relatively

early in the field of plant studies and combined the use of restriction endonucleases and the
hybridization method.

2.4.3.1.1. Restriction fragment length polymorphism (RFLP) markers

Restriction Fragment Length Polymorphism (RFLP) is the most widely used hybridization-
based molecular marker in 1980s. It was used for human genome mapping (Botstein et al.,
1980), and later adopted for plant genomes (Helentjaris et al., 1986; Weber and Helentjaris,
1989). The technique is based on restriction enzymes that reveal a pattern difference between
DNA fragment sizes in individual organisms. RFLP is a co-dominant marker and are more
informative than dominant markers. However, this technique requires relatively large amounts
of purified DNA, time consuming, laborious and uses probe that is difficult to handle.

2.4.3.2. PCR based molecular markers

The Polymerase chain reaction (PCR) is a molecular biology technique for enzymatically
replicating (amplifying) small quantities of DNA without using a living organism. It is used to
amplify a short (usually up to 10 kb), well-defined part of a DNA strand from a single gene or
just a part of a gene. The invention of this technique enabled the development of various types
of PCR-based techniques.

The commonly used PCR-based DNA marker systems are random amplified polymorphic
DNA (RAPD), amplified fragment length polymorphism (AFLP) and simple sequence repeats
(SSRs) or microsatellites (Staub et al., 1996; Gupta & Varshney, 2000). The major limitations
of these methods are low reproducibility of RAPD, high cost of AFLP and the need to know
the flanking sequences to develop species specific primers for SSR polymorphism.

12
2.4.3.2.1. Random amplified polymorphic DNA (RAPD) markers

Random Amplified Polymorphic DNA (RAPD) is a PCR-based technology. The method is

based on enzymatic amplification of target or random DNA segments with arbitrary primers.
This procedure detects nucleotide sequence polymorphisms in DNA by using a single primer
of arbitrary nucleotide sequence. In this reaction, a single species of primer anneals to the
genomic DNA at two different sites on complementary strands of DNA template. If these
priming sites are within an amplifiable range of each other, a discrete DNA product is formed
through thermo cyclic amplification. On an average, each primer directs amplification of
several discrete loci in the genome, making the assay useful for efficient screening of
nucleotide sequence polymorphism between individuals (William et al., 1993). However, due
to the stochastic nature of DNA amplification with random sequence primers, it is important
to optimize and maintain consistent reaction conditions for reproducible DNA amplification.

RAPDs are DNA fragments amplified by the PCR using short synthetic primers (generally 10
bp) of random sequence. These oligonucleotides serve as both forward and reverse primer,
and are usually able to amplify fragments from 1–10 genomic sites simultaneously. Amplified
products (usually within the 0.5–5 kb size range) are separated on agarose gels in the presence
of ethidium bromide and view under ultraviolet light (Jones et al., 1997) and presence and
absence of band was observed.

2.4.3.2.2. Amplified fragment length polymorphism (AFLP) markers

AFLP analysis is able to detect high levels of polymorphism and has high repeatability and
speed of analysis. These markers have a very high diversity index, resulting in a limited
number of primer combinations required to screen a whole genome and has been applied to
develop a system for the fingerprinting of an organism and for map expansion (Castiglioni et
al., 1998).

Among the molecular marker techniques, AFLP is robust and provides a powerful tool in
studies of genetic variation, genotype identification and phylogeny. This method is based on

13
the detection of genomic restriction fragments by PCR amplification, and it can be used for
DNAs of any origin or complexity (Vos et al., 1995). Large number of loci, high levels of
polymorphism, high reproducibility without prior sequence knowledge and genome-wide
marker distribution are the most important advantages of this technique (Powell et al., 1996).

AFLP is essentially intermediate between RFLPs and PCR. The use of AFLP in genetic
marker technologies has become the main tool due to its capability to disclose a high number
of polymorphic markers by single reaction (Vos et al., 1995). The need for purified, high
molecular weight DNA, the dominance of alleles, and the possible non-homology of co-
migrating fragments belonging to different loci are its demerit. In addition, due to the high
number and different intensity of bands per primer combination, there is the need to adopt
certain strict but subjectively determined criteria for acceptance of bands in the analysis.

AFLPs can be applied in studies involving genetic identity, parentage and identification of
clones and cultivars, and phylogenetic studies of closely related species because of the highly
informative fingerprinting profiles generally obtained. Their high genomic abundance and
generally random distribution throughout the genome make AFLPs a widely valued
technology for gene mapping studies (Vos et al., 1995).

2.4.3.2.3. Microsatellites or Simple sequence Repeat (SSR) markers

Microsatellites or Simple sequence Repeat (SSR) are sections of DNA, consisting of tandemly
repeating mono-, di-, tri-, tetra- or penta-nucleotide units that are arranged throughout the
genomes of most eukaryotic species (Powell et al., 1996). Microsatellite sequences are
especially suited to distinguish closely related genotypes; because of their high degree of
variability, they are, therefore, favoured in population studies and for the identification of
closely related cultivars (Vosman et al., 1992). Microsatellite polymorphism can be detected
by Southern hybridisation or PCR.

Microsatellites, like minisatellites, represent tandem repeats, but their repeat motifs are shorter
(1–6 base pairs). If nucleotide sequences in the flanking regions of the microsatellite are

14
known, specific primers (generally 20–25 bp) can be designed to amplify the microsatellite by
PCR. Polymerase slippage during DNA replication, or slipped strand mispairing, is considered
to be the main cause of variation in the number of repeat units of a microsatellite, resulting in
length polymorphisms that can be detected by gel electrophoresis. Other causes have also
been reported (Matsuoka et al. 2002).

In general, microsatellites show a high level of polymorphism. As a consequence, they are

very informative markers that can be used for many population genetics studies, ranging from
the individual level (e.g. clone and strain identification) to that of closely related species.
Conversely, their high mutation rate makes them unsuitable for studies involving higher
taxonomic levels.

2.4.3.2.4. Inter Simple Sequence Repeat (ISSR) Markers

Inter Simple Sequence Repeat (ISSR) are DNA fragments of about 100–3000 bp located
between adjacent, oppositely oriented microsatellite regions. This technique was reported by
Zietkiewicz et al., 1994). Primers based on microsatellites are utilized to amplify inter-SSR
DNA sequences. ISSR technique involves amplification of DNA segment present at an
amplifiable distance in between two identical microsatellite repeat regions oriented in opposite
direction. The technique uses microsatellites, usually 16–25 bp long, as primers in a single
primer PCR reaction targeting multiple genomic loci to amplify mainly the inter- SSR
sequences of different sizes. The microsatellite repeats used as primers can be di-nucleotide,
tri-nucleotide, tetra-nucleotide or penta-nucleotide. The method uses a single oligonucleotide
primer composed of 4 to 10 tri or di-nucleotide repeats and ending with 3’- or 5’- anchor
sequence. ISSR targets the highly variable microsatellite regions of the nuclear genome
providing a large number of polymorphic fragments (Gupta et al., 1994). It provides more
reproducibility than similar methods viz., RAPD, AFLP and SSRs (Awasthi et al., 2004).

The primers used can be either unanchored (Gupta et al., 1994; Meyer et al., 1993; Wu et al.,
1994) or more usually be anchored at 3’ or 5’ end with 1 to 4 degenerate bases extended into
the flanking sequences (Zietkiewicz et al., 1994). About 10–60 fragments from multiple loci

15
are generated simultaneously, separated by gel electrophoresis and scored as the presence or
absence of fragments of particular size.

Figure 1. ISSR PCR: A single primer targeting a (CA)n repeat, anchored either at 3’ (light arrows) of
the repeat, is used to amplify geneomic sequence flanked by 2 inversely oriented (CA)n
elements(Zietkiewicz et al., 1994).

ISSR is a technique that overcomes most of the limitations of other molecular markers such as
RAPD, AFLP and SSR (Zietkiewicz et al., 1994; Gupta et al., 1994; Wu et al., 1994; Meyer
et al., 1993). It is rapidly being used by the research community in various fields of plant
improvement (Godwin et al., 1997). The technique is useful in areas of genetic diversity,
phylogenetic studies, gene tagging, genome mapping and evolutionary biology in a wide
range of crop species. ISSR analysis can be also applied in studies involving genetic identity,
parentage, clone and strain identification, and taxonomic studies of closely related species.

The technique combines most of the benefits of AFLP and microsatellite analysis with the
universality of RAPD. ISSRs have high reproducibility possibly due to the use of longer
primers (16–25 mers) as compared to RAPD primers (10- mers) which permits the subsequent
use of high annealing temperature (45– 60 ◦C) leading to higher stringency. The studies on
reproducibility show that it is only the faintest bands that are not reproducible. About 92–95%

16
of the scored fragments could be repeated across DNA samples of the same cultivar and
across separate PCR runs when detected using polyacrylamide (Fang & Roose, 1997; Moreno
et al., 1998).

ISSRs segregate mostly as dominant markers following simple Mendelian inheritance (Gupta
et al., 1994; Tsumura et al., 1996; Wang et al., 1998). However, they have also been shown to
segregate as co-dominant markers in some cases thus enabling distinction between
homozygotes and heterozygotes (Wu et al., 1994; Akagi et al., 1996; Wang et al., 1998;
Sankar& Moore, 2001).

The annealing temperature of ISSR depends on the GC content of the primer used and ranges
from 45 to 65 C. The primers are not proprietary and can be synthesized by anyone. The
technique is simple, quick, and the use of radioactivity is not essential. ISSR markers usually
show high polymorphism (Kojima et al., 1998) although the level of polymorphism has been
shown to vary with the detection method used.

17
 3. MATERIALS AND METHODS

3.1. Plant Material

The plant material used in the study includes 10 populations each consisting of 10 individuals
of Plectranthus edulis (Oromo Potato) (Vatke) Agnew except Areka and Sodo Zuria
consisting nine individuals each. Each of the 10 populations was collected from four Oromo
Potato growing Zones in Ethiopia and each geographical location was positioned by GPS
(Table 2).

Table 2. Regions and sites of Ethiopia from where the samples were collected

No Population Pop. Region Zone

Code Geographical Locations
Latitude(N) Longitude Altitude
1. Sodo Zuria SZ SNNPR Wolaita 6 ⁰55' 05.33"N 37⁰48'49.33"E 2217m
2. Damot Gale DG SNNPR Wolaita 6 ⁰54 '33.42"N 37⁰45'27.64"E 2459m
3. Areka AR SNNPR Wolaita 7 ⁰03 46.38"N 37⁰40'19.38"E 1816m
4. Ambo AM Oromia W. Shewa 8 ⁰54 '59.51"N 37⁰50'01.39"E 2327m
5. Wadessa WD Oromia W. Shewa 9⁰ 01' 29.35"N 37⁰49'00.42"E 2115m
6. Gute GT Oromia E. Wollega 9 ⁰01 16.76"N 36⁰40'10.99"E 1892m
7. Nekemte NK Oromia E. Wollega 9⁰05' 03.62"N 36⁰31'47.79"E 2059m
8. Digga DA Oromia E. Wollega 9⁰ 01' 57.77N 36⁰2654.63"E 2210m
9. Gojam-I Go-I Amhara E. Gojam 10⁰25'46.18"N 37⁰32'55.67"E 2289m
10. Gojam-II Go-II Amhara E. Gojam 10⁰24'52.63"N 37⁰35'59.41"E 2300m

Key: SNNPR=Southern Nations, Nationalities and Peoples Region; W. Shewa= West Shewa;
E. Wollega=East Wollega; E. Gojam=East Gojam; SZ= Sodo Zuria; DG= Damot Gale; AR
=Areka; AM=Ambo; WD=Wadessa; GT= Gute; N= Nekemte; DA=Digga; GO-I= Gojam -I;
Go-II=Gojam-II.

18
As P. edulis is grown only in localized areas and in much smaller scales in some of the
regions such as West Shewa and East Gojam, only two populations were collected from each
of them. Three populations were collected from each of Wolaita and Wollega. The tuber of P.
edulis populations were harvested and collected from the farms without mixing them together
and planted at College of Natural Science, Addis Ababa University glasshouse. The sample
collection was done randomly from the farms which are atleast 15 km far apart from each
other to get the representative sample for each population.

Figure 2. Map of Ethiopia showing where samples of P. edulis (Vatke) Agnew were collected.

Key: DG- Damot Gale; SZ- Sodo Zuria; AR- Areka; AM-Ambo; W-Wadessa; G-Gute; NK;
Nekemte; DA-Digga; GJI-Gojam I ; GJII-Gojam II

19
3.2. DNA Extraction

Genomic DNA was extracted following the CTAB (cetyltrimethyl ammonium bromide)
method of Borsch et al. (2003) from young leaves employing triple extractions to yield
optimal amount of DNA. Young leaves of 98 individuals of total populations were cleaned
with tap water and kept in the dark overnight. About 300 mg fresh leaves were used to extract
DNA with minor modifications (Appendix 7.1).

After the extraction of genomic DNA, it was run on 1% agarose gel to check for the presence
of genomic DNA. The concentration and quality of isolated DNA was measured by nanodrop
spectrophotometer.

Genomic DNA (2μl) and 2μl of 6x loading dye were mixed and loaded on agarose gel in 1x
TBE running buffer and electrophoresed at 80 volt for 45 min. The gels were photographed
with digital camera mounted on BioDoc. Analyze apparatus and connected to desk top
computer. The gel picture was documented.

3.3. ISSR Primers Screening and Optimization

Fourteen ISSR primers (from University of British Colombia, Primer kit UBC 900) were
obtained from Institute of Biotechnology, Addis Ababa University were screened for
polymorphism and reproducibility of PCR by using a total of twenty individuals by random
selection of two individuals from each population trying for reproducibility at least three times
for each primer. Four primers that produced clear, reproducible and polymorphic band
patterns were selected for the present study (Table 3).

20
Table 3. List of ISSR primers tested for polymorphism and the reproducibility of the amplified
bands.

Primers Annealing Sequence Amplification pattern Repeat motif

temperature (C)

UBC-810 45 (GA)8T Not Reproducible Di-nucleotide

UBC-812 45 (GA)8A Not Reproducible Di-nucleotide

UBC-818 48 (CA)8G Not reproducible Di-nucleotide

UBC-824 48 (TC)8G Not amplified Di-nucleotide

UBC-825 48 (AC)8T Not amplified Di-nucleotide

UBC-827 48 (AC)8G Not amplified Di-nucleotide

UBC-834 45 (AG)8YT Not amplified Di-nucleotide

UBC-835 45 (AG)8YC Polymorphic, Reproducible Di-nucleotide

UBC-841 48 (GA)8YC Polymorphic, Reproducible Di-nucleotide

UBC-844 48 (CT)8RC Polymorphic, Reproducible Di-nucleotide

UBC-860 45 (TG)8RA Not amplified Di-nucleotide

UBC-866 48 (CTC)6 Not amplified Di-nucleotide

UBC-873 48 (GACA)4 Not amplified Tetra-nucleotide

UBC-880 45 (GGAGA)3 Polymorphic, Reproducible Penta-nucleotide

R = (A, G); Y = (C, T).

3.4. PCR Amplification and Gel Electrophoresis

PCR was performed by using four ISSR primers that were selected out of fourteen screened
primers (Table 3) based on the degree of polymorphism and the distinctness of the bands they
produced. Each primer was screened for reproducibility for different PCR products of DNA
samples of the same population and subjected to separate runs on agarose gels and the ones
producing consistent DNA fragments across the different samples and PCR runs were selected.
The polymerase chain reaction was conducted in GENE AMP PCR thermocycler.

21
PCR reactions were carried out in the mixture of buffer(10 mM Tris-HCl, pH 8.0, 50 mM
KCl), 25 mM MgCl2, 100 mM dNTP, 0.2 M of primer, 10 ng of genomic DNA per 25 μl
reaction volume with 0.3μL of Taq DNA polymerase and deionized water to make up the
reaction volume constant(Appendix 7.2.). PCR amplifications were performed in a GENE
AMP PCR thermocycler, with initial denaturation at 94°C for 4 min, then 94°C for 15 sec,
annealing at 45-50 for 25 sec depending on the annealing temperature of the primers, and
extension at 72°C for 1 min and 30 sec for 40 cycles, with a 7 min final extension at 72°C.

PCR products were run on 1.67% agarose gel. Agarose gel was prepared by mixing
appropriate amounts of agarose gel powder with 1X TBE running buffer. The PCR products
of each genomic DNA sample was mixed with 2 µl of 6x loading buffer and mixed well. The
genomic samples of 8-12 µl were loaded into the wells and electrophoresed at constant
voltage of 80 V for 1min and 30 seconds. The gel was stained for 30 min with 50µl ethidium
bromide in 450 ml distilled water and de-stained with distilled water for 30 minutes. Gel
picture was taken under UV transilluminator by Biodoc. analyse 2.00 with digital canon
camera. DNA band sizes were estimated by comparing the DNA bands with a 100 base pair
ladder marker loaded in the peripheral wells of the gel.

3.5. Data Scoring and Analysis

ISSR bands were considered as a unit character and scored as present (1), absent (0) and
missing or unclear (‘?’). The bands that were not clearly shown were scored as a missing data
(‘?’). Intensity variations among bands having approximately the same molecular size were
not considered although in some cases intensity differences of the bands were observed. A
binary matrix was constructed with the individuals in the row and the ISSR markers (loci) in
the column. Each amplified fragments was named by the code of the primers across the
column followed by the Arabic numbers starting from the fragment having high molecular
weight to the fragments with low molecular weight. Since ISSR markers are multi-locus
markers, a locus was considered to be polymorphic if the presence and absence of bands were
observed in various individuals for a given locus, and monomorphic if the bands were present

22
in all individuals. On the basis of the recorded band profiles, different software were
employed for the analysis of the data.

The resulting presence/absence data matrix of the ISSR markers was analyzed using
POPGENE version1.32 software (Yeh et al., 1999) to calculate the following genetic diversity
parameters: percentage of polymorphic loci (PPL), gene diversity (h), and Shannon’s
information index (I).

The mean Shannon weaver diversity indices will be calculated as H= -∑pi logpi where pi is
the frequency of a given band for each population (Lewontin, 1972). The proportion of
diversity between populations will then be calculated as (Hsp-Hpop/Hsp) where Hsp is total
diversity and Hpop is the mean intra-population.

The genetic structure was estimated using Analysis of Molecular Variance (AMOVA). The
AMOVA analysis was done using the software ARLEQUIN version 3.01 (Excoffier et al.,
2006) to estimate genetic variability within and among P. edulis populations without grouping.

Dendrograms were constructed on the basis of Jaccard’s similarity coefficient using

unweighted pair group method with arithmetic average (UPGMA) (Sneath and Sokal, 1973)
and neighbor joining (NJ) method (Saitou and Nei, 1987; Studier and Keppler, 1988) to
illustrate the genetic relationship of individual samples of P. edulis. NTSYS- pc version 2.02
(Rohlf, 2000) and Free Tree 0.9.1.50 (Pavlicek et al., 1999) software’s were used for these
cluster analysis respectively. Jaccard's similarity coefficient was calculated with the formula
a
S ij 
a  b  c

Where,

‘a’ is the total number of bands shared between individuals i and j,

‘b’ is the total number of bands present in individual i but not in individual j and

‘c’ is the total number of bands present in individual j but not in individual i.

23
To further examine the patterns of variation among individual samples, a principal coordinate
analysis (PCO) was performed based on Jaccard’s coefficient. The calculation of Jaccard’s
similarity coefficient was made with PAST software version 1.18 (Hammer et al., 2001). The
first three axes were later used to plot with STATISTICA version 6.0 software (Hammer et al.,
2001; Statistica soft, Inc.2001).

24
 4. RESULTS AND DISCUSSION

4.1. ISSR Markers and Banding Patterns

Out of 14 screened ISSR primers, only four primers in which three of them were di-nucleotide
(UBC-835, UBC-841, and UBC-844) and only one was penta-nucleotide (UBC-880) that
produced clear, reproducible and polymorphic bands were selected as informative primers
(Table 4). The other ten ISSR primers that resulted in either smeared, failed to give
amplification products, poor or non-reproducible bands were excluded from the analysis. The
amplicon (band) size of the four primers used in this study varied from 200 bp to 1400 bp.

1400bp

800bp

200bp

M 1 2 3 4 5 6 7 8 9 10 11 C 12

Figure 3. PCR amplification product of 12 individuals of P. edulis produced with ISSR primer
UBC-835. Lane M is 100bp DNA ladder and lanes 1–12 represent different P. edulis
individuals. Lane C is control.

25
A total of 42 polymorphic bands were scored using four primers with an average band number
of 10.5 per primer across 98 individuals assayed. Primers UBC-835 and UBC-880 produced
the highest number of polymorphic bands of 12 and 11, respectively, while primers UBC-841
and 844 produced 10 and 9 polymorphic bands. Among the 46 total number of scorable bands
were 42 polymorphic bands, even though monomorphic loci were observed using primers
UBC-835, UBC-841 and UBC-844 (Table 4).

4.2. Genetic Diversity of P. edulis Populations

From all populations, 46 discernible DNA fragments were generated among which 42
(91.30%) were polymorphic in ten populations (Table 4). The only penta-nucleotide primer
880 (GGAGA)3 generated highest percent of polymorphic loci and unique bands. The
percentage of polymorphism ranged from 84.62% for UBC-841 primer to 100% for UBC-880
primer with an average of 91.30% polymorphism.

Table 4. Number of scorable bands, number of polymorphic bands, percent of polymorphic

loci, Nei’s genetic diversity (h) and Shannon's information index (I) for each primer.

Primer code NSB NPL PPL h±SD I±SD

UBC-835 13 12 92.31 0.36±0.17 0.53±0.22
UBC-841 13 11 84.62 0.33±0.17 0.45±0.26
UBC-844 10 9 90 0.33±0.17 0.48±0.23
UBC-880 10 10 100 0.35±0.16 0.52±0.10
Over all 46 42 91.30 0.33±0.11 0.48±0.22

Key: NPB=Number of Scorable Bands NPL = Number of polymorphic Loci; PPL = Percent
of polymorphic Loci; h = Nei's gene diversity; I = Shannon’s information index; SD=Standard
Deviation

Nei’s gene diversity (H) and the Shannon Information Index (I) calculated for each of the
ISSR Primers showed that Primer UBC-841 and UBC-844 had the lowest gene diversity
(0.33) for each primers and primers UBC-835 had the highest gene diversity (0.36). And

26
UBC-880 had the Nei’s gene diversity of 0.35. The Shannon’s Information index also ranged
from 0.45 to 0.53 for primers UBC-841 and UBC-835, respectively. In the case of genetic
diversity estimation due to gene diversity (h) and Shannon’s information index (I), the highest
values were achieved from ISSR-835 primer and the least from ISSR-841 primer (Table 5).

The P. edulis population with the highest values of Nei’s gene diversity was Areka
population with 0.30 and the Shannon index was Gojam-II population with 0.39, and Nekemte
population being the lowest (0.18 and 0.25, respectively) for gene diversity and Shannon’s
Information index (Table 5). Based on aggregate data from all the four ISSR Primers, the
highest level of percent polymorphism was obtained from samples of Wolaita grouping, Sodo
Zuria population exhibiting 65.22%, followed by Gojam-II showing 65%, Areka and Damot
Gale exhibited 60.87% polymorphism, and similarly Gute and Digga both have shown equal
percentage of polymorphism of 54.35%. Wadessa, Gojam-I, and Ambo populations exhibited
50%, 47.83%, and 45.65% respectively. The least percent polymorphism was obtained from
accessions in Nekemte that had only 19 polymorphic loci which accounted for 41.30%
polymorphism. Beside this, high level of polymorphism was detected when populations of P.
edulis were analyzed the grouping based on geographical origins. Accordingly, Wolaita
grouping showed highest percent polymorphism (84.28%), while the least level of
polymorphism was shown by Shewa Grouping with only 67.39%. The Wollega and Gojam
populations showed 76.09 % and 73.91 % percent polymorphism, respectively (Table 6).
Similarly, polymorphism of 90.62, 83, 85.1, 85.06 and 92.4 % were found in other tubers
(Zhang et al., 2006; Zhou et al., 2008; Lu et al., 2011; Abreham et al., 2014; and Moulin et al.,
2012).

In case of geographical regions based analysis, Wolaita populations showed the highest
variability (0.32) while the least was obtained from Shewa populations (0.27). Similarly, the
highest Shannon diversity index was obtained from Wolaita (0.48) and the least Shannon
diversity index showed by Shewa populations (0.39).

Genetic diversity estimators, PPB, gene diversity (h), and Shannon’s diversity (I) were highest
for Sodo Zuria (65.22%, 0.24, 0.35), Gojam-II (65 %, 0.28, 0.0.39), and Areka (60.87 %, 0.30,

27
and 0.37), respectively, indicating that probably these places are centers of genetic diversity
for P. edulis. The lowest percentages of polymorphic bands, gene diversity and diversity index
were shown by Nekemte (41.30%, 0.18 and 0.25, respectively) indicating that individuals of
Nekemte are uniform. This low genetic diversity might be explained by the loss of wild
relatives (National Research Council,1991) mainly due to anthropogenic destruction of natural
habitats, existence of uniformity or monoculture possibly due to vegetative propagation
prevailed in the area, homogeneity due to single or few seed sources during its introduction to
these area. Various studies showed that endangered and endemic species tend to have low
level of genetic diversity (Li and Xia 2005; Xiao et al. 2006). Contrastingly, others found high
level of genetic diversity (Da-Wei et al., 2004; Ni et al., 2006; Li and Jin, 2007; and Wang et
al., 2012) in endangered and endemic species.

Table 5. The Number of polymorphic loci (NPL), Percent polymorphic loci (PPL), gene
diversity (h) and Shannon‘s information (I) Index for Ten Populations

Populations/groups Using all primers

No. (Code) NPL PPL (%) h±SD I±SD

1 Damot Gale 28 60.87 0.25±0.21 0.36±0.30

2 Sodo Zuria 30 65.22 0.24±0.20 0.35±0.29
3 Areka 28 60.87 0.30±0.21 0.37±0.29
4 Ambo 21 45.65 0.19±0.22 0.28±0.32
5 Wadessa 23 50 0.21±0.22 0.29±0.31
6 Gute 25 54.35 0.20±0.19 0.28±0.27
7 Nekemte 19 41.30 0.18±0.22 0.25±0.31
8 Digga 25 54.35 0.24±0.23 0.34±0.32
9 Gojam-I 22 47.83 0.19±0.21 0.28±0.38
10 Gojam-II 30 65 0.28±0.21 0.39±0.30

Over all 42 91.30 0.33±0.17 0.48±0.22

NPL = Number of polymorphic Loci; PPL = Percent of polymorphic Loci; h = Nei's gene
diversity; I = Shannon’s information index ; SD= Standard Deviation

28
Table 6. The Number of polymorphic loci (NPL), Percent polymorphic loci (PPL), gene
diversity (h) and Shannon‘s information (I) Index for Groupings

Groups NPL PPL H I

1 Wolaita 39 84.28 0.32± 0.18 0.48±0.24
2 Shewa 31 67.39 0.27±0.21 0.39±0.29
3 Wollega 35 76.09 0.29±0.19 0.42±0.27
4 Gojam 34 73.91 0.28±0.19 0.42±0.27
Over all 42 91.30 0.33±0.17 0.48±0.22

4.3. Analysis of Molecular Variance (AMOVA)

AMOVA was carried out using the 46- ISSR bands generated to assess the overall distribution
of genetic diversity within and among populations. The AMOVA results (Table 7) revealed
highly significant genetic differences (P<0.001) among the four groups as well as among the
ten populations of P. edulis samples collected from Ethiopia. The total genetic variation
among P. edulis populations (Gst) was 0.258, which is relatively higher than the mean Gst
value (0.129) reported in the 220 genus, 662 other species. Also, According to Nybom (2004),
the value of 0.258 is most similar with short-lived perennial (0.41), regional geographical
range (0.42), mixed breeding system (0.40), and mid successional taxa (0.39). Therefore, P.
edulis is typically of shortlived perennial, follows a mixed breeding system and is a mid-
successional taxa species.

The within populations component accounted for 74.23% without creating grouping. The
present study agrees with Abreham et al. (2014) who studied the genetic diversity of anchote
(Coccinia abyssinica) populations by using ISSR marker which showed that the within
population was 73.6% within population variation whereas the rest 26.4% was due to among
population variation. As compared to the present study, there was the same gene flow.
According to compiled data of Nybom (2004), genetic diversity is strongly associated with
life form, geographic range, breeding system, seed dispersal mechanism, and successional
status. In both cases, the results of analysis of molecular variance revealed the same patterns

29
of genetic diversity and supports the larger genetic diversity found within the populations
rather than among populations which could be explained by higher level of gene flow.

Table 7. Analysis Molecular Variance among and within ten populations of P. edulis.

Source of variation d.f. Sum of Variance Percentage p-value

squares components of variation
Among populations 9 113.94 0.99161Va 25.77 p<0.001
Within populations 88 253.08 2.88Vb 74.23 p<0.001
Total 97 367.1 3.87

4.4. Cluster Analysis

The UPGMA-based dendrogram (Fig. 4) of the ten populations of P. edulis generated two
major group and two outliers, namely major clusters I, II, III and IV using Jaccard's similarity
coefficient of 0.65. Hollingsworth and Ennos (2004) stated that grouping together of
individuals from different populations in the same cluster of a tree has been interpreted as
evidence for gene flow between populations. UPGMA and NJ showed comparable results
with an indication of the clustering of individuals of the study populations. The analysis also
revealed intermixing of individuals possibly due to admixture that may be resulted from gene
flow migration of people from place to place, short and long distance marketing of tuber of P.
edulis.

The UPGMA-based dendrogram of the ten populations of P. edulis generated three distinct
major clusters. The first bottom cluster contains population of Areka. Gojam-I, Gojam-II,
Nekemte and Gute populations are clustered together on the second group from the bottom.
Digga, Wadessa, Ambo and Sodo Zuria populations are also clustered together while Damot
Gale clustered separately. Digga, Gute and Nekemte are East Wollega populations and also
Gojam-I and Gojam-II are geographically found in close proximity and in one district. The
highest within-cluster similarity was, however, observed between Gute and Nekemte followed
by Gojam and Gojam-II which are also geographically found in close proximity. Even though
Areka population is geographically distant from Gojam-I and Gojam-II but three of them

30
clustered together and resulted from the migration of Amhara people through settlements in
which local farmers Amhara region might took seeds and tubers to South parts of the country
starting in the Meneliks’ ruling period (Ege, S. et al., 2009) . The migration is often because
of multitude of reasons including environmental degradation, vulnerability, food insecurity,
natural disaster, land scarcity, ethnic conflict and other factors they faced (Tesfaye Tafesse,
2009).

The top cluster contained populations of Damot Gale and Sodo Zuria indicating that P. edulis
seed exchange during planting could be attributed to the observed similarity among these
closely located populations since they are closely related in geographical distance. In the
middle of dendrogram, populations of Ambo and Wadessa also found in close proximity were
clustered together. Seed exchange during planting could be attributed to the observed
similarity among these closely located populations.

31
Figure 4. Dendrogram generated based on UPGMA analysis demonstrating the genetic
similarity between ten populations of P. edulis.

32
 DG1
DG2
DG3
DG4
GJ-I4
Sub-I
DG9
DG10
NK2
NK3
NK4
NK5
NK6
NK7
NK8
NK10
AR4
SZ6
GJ-II5 Major-I
GJ-II6
GJ-II7
GJ-II9
GJ-II10
GJ-II8
SZ1
DA2
DA3
DA5
AM3
DA4
AM5
AM6
AM9
AM10
AM8
SZ8 Sub -II
AR2
AR7
DA10
WD4
WD5
GT8
SZ3
AR5
DA6
DA8
WD1
WD2
WD8
WD9
WD6
SZ7
AR1
SZ9
Sub-III
AR6
GT4
GT7
DA1
GJ-I5
WD10
GT6
GT2
GT1
GT5
GT3
NK9
GJ-I10
GJ-I1
GJ-I3 Major-II
GJ-I2
GT9
NK1
GJ-I6
GJ-I7
GJ-I8
GJ-II1
GJ-II2
GJ-II3
GJ-I9
DG5
DA7
DA9
AR9
AM2
AM1
AM4
AM7
GJ-II4
WD3
GT10
SZ2
SZ4 Major -III
SZ5
WD7
DG6
DG8
DG7
AR3
AR8
0.46 0.59 0.73 0.86 1.00
Coefficient

Figure 5. UPGMA Dendrogram generated based on Jaccard’s coefficients of similarity ninety

eight individuals of P. edulis using the four ISSR primers.

Key: DG- Damot Gale ; SZ- Sodo Zuria; AR- Areka; AM-Ambo; WD-Wedessa; GT-Gute;
NK; Nekemte; DA-Digga; GJI-Gojam I ; GJII-Gojam II

33
On the basis of UPGMA (Fig.5), most individuals of the respective populations were observed
to form moderate grouping with few intermixing with other populations. The intermixing goes
with geographic proximity which could facilitate seed flow and population migration. For
instance, the intermixing of Digga, Nekemte, and both Gojam populations at the top of
dendrogram could be because of their geographic location in first two populations and
interaction among peoples i.e. the movement of peoples from Gojam to Nekemte by crossing
Abbay River could be the reasons for intermixing of Gojam with Wollegas’ populations.

Nekemte population tends to form separate group but with more intermixing from the other
populations. Yeshitila Mekbib (2007) based on morphological diversity study, revealed that
the accessions of P. edulis collected from geographically similar areas were grouped in
different clusters indicating that factors of the local environment were not related to
phenotypic diversity. The present study, based on molecular genetic diversity of P. edulis,
showed similar results to the previous morphological studies. Additionally, Weyessa Garedew
et. al (2013) clearly showed that the accessions collected from Wolaita (Sodo Zuria) were
clustered with Yem, Jimma, and Gedio accessions and also accessions from Gojam (Dangila)
were clustered with Iluabbabor and Jimma accessions which are geographically far apart and
both of them based on morphological traits. To conclude, the intermixing of individuals of the
study indicated that the individuals from different regions of the country might have a similar
genetic background.

In the case of NJ (Fig.6), each individual of the respective populations was observed to form
small clustered groups with highly intermixing with other groups. This lack of association of
the individual plants into their respective populations in case of NJ may indicate the
vulnerability or exposure of the plants to human intensive management, exchange of seeds via
marketing and limited gene flow due to long distance.

34
 Sub-I
GT2 GT3
WD10 Major -I
GT5
GT6
GT4
GJ-I1
GJ-I2
GJ-I3
GJ-I10
NK9
NK10
NK5
NK8
NK2
NK3
NK4
NK6
NK7
AR3
AR8
AR4
DG6AR6
DG8 Sub-II
DG7
AR1 SZ5
SZ7
GJ-I4
GJ-II4
DG1
DG2
DG3
DG4
DG10
DG9 GJ-II10
GJ-II8
GJ-II7
GJ-II9
GJ-II5
GJ-II6
GT9 Sub-III
NK1
GJ-I6
GJ-I7
GJ-I8
GJ-II3GJ-II2
GJ-II1
SZ2
SZ4
AM7
AM4
AM1
AR9
AM2 DA7
DG5 Major- II
DA9
WD3
WD6 WD7
GT1
GT10
DA1
GJ-I5
GT7
GJ-I9
DA2
DA3
DA5SZ1
WD1
WD2
WD8
WD9
AM3
DA4 Major-III
DA6
DA8
AR5
AR7
DA10 GT8
WD5
WD4
AM5
AR2
SZ8
AM6
SZ9
AM8 SZ3
SZ6
AM10
AM9
0.1

Figure 6. Neighbor-joining analysis of 98 individuals based on Jaccard’s coefficient

Key: DG- Damot Gale; SZ- Sodo Zuria; AR- Areka; AM-Ambo; WD-Wadessa; GT-Gute;
NK; Nekemte; DA-Digga; GJI-Gojam I ; GJII-Gojam II

35
4.5. Genetic Similarity

Based on Jaccard’s coefficient, highest genetic similarity was observed between Ambo and
Sodo Zuria with similarity coefficient of 0.648 and the second highest genetic similarity
observed between Digga and Ambo with similarity coefficient of 0.644 (Table 8). On the
other hand, the least genetic similarity was detected between Gojam-I and Sodo Zuria with
similarity coefficient of 0.489. ISSR marker in this study enabled us to separate between
genetically diverse populations based on their geographic location.

Table 8. Similarity matrix for Jaccard’s coefficients based on the 46 bands obtained with four
ISSR primers.

Populatin Damot Sodo Areka Ambo Wadessa Gute Nekemte Digga Gojam Gojam
Gale Zuria I II

Damot 1.000
Gale

Sodo
Zuria 0.497 1.000

Areka 0.533 0.498 1.00

Ambo 0.551 0.648 0.541 1.000

Wadessa 0.534 0.537 0.518 0.622 1.000

Gute 0.526 0.567 0.573 0.571 0.603 1.00

Nekemte 0.579 0.493 0.529 0.565 0.593 0.626 1.000

Digga 0.522 0.577 0.549 0.644 0.634 0.561 0.566 1.00

Gojam I 0.554 0.489 0.520 0.536 0.557 0.618 0.612 0.555 1.000

Gojam-II 0.533 0.572 0.494 0.594 0.528 0.547 0.597 0.556 0.604 1.000

36
4.6. Principal Coordinate Analysis (PCO)

Principal coordinate analysis was carried out using Past and Statistica software packages by
employing Jaccard’s coefficients of similarity to plot the ISSR products (46 scored bands) of
98 individuals of P. edulis on 2D and 3D coordinate planes. The first three coordinates of the
PCO had Eigen-values of 19.682, 7.007 and 4.4785 with percentage of 37.546%, 13.367%,
and 8.5434%, respectively. The analysis of this study showed that the accessions of the ten
populations examined tend to form clusters based on their geographic origin.

On the first principal coordinate, populations having relatively major variation both within and
among them were grouped together. Most individuals from the Sodo Zuria, Gojam-II and
Areka populations formed a close pattern on the first coordinate towards negative side by
intermixing themselves including Digga population. Populations of Damot Gale, Ambo, and
Gojam-I were dispersly clustered on first coordinate towards positive Eigen values. However,
some individuals of these populations were intermixed with other populations. Nekemte,
Wadessa and Gute populations were separated from other populations towards the positive
Eigen values even though not purely isolated from other populations.

On the second principal coordinate, populations having less variation formed some sort of
clustering. The population of Nekemte that showed the lowest within-population diversity
formed a very close pattern on positive Eigen values of the second coordinate. Additionally,
the Digga and Gojam-II populations (from East Wollega and East Gojam respectively)
separated from other populations on this coordinate (Fig. 7).

The three-dimensional PCO (Fig. 8) also revealed similar result with 2D analysis that
individuals of Nekemte population formed very close and clear grouping. Likewise,
individuals of Digga and Gute populations formed their own grouping relative to others.
Similar to the observation from 2D PCO, some individuals were intermixed.

The present study results agree with Abreham et al., 20214 UPGMA based dendrogram of the
12 populations of C. abyssinica generated three distinct major clusters and there were clear

37
divergence among accession of the ten populations that should be exploited for breeding via
heterosis effect (Patrick et al. 2013).

0.5

0.4 AR8
SZ5GJ-II4 AR3
AR9AM1 DG7
DG8
SZ4 DA7 DG5
0.3 DA9 SZ2 DG6
AM7 AM2 GT3
WD7
GT9
0.2 AM4 AR6 GJ-I3 GJ-I10
SZ7 AR1 NK1
WD3
C oordinate 2

GJ-I1
0.1 GJ-II2
GJ-II3 GT4 GT2
GJ-I9 GT6 GJ-I2
NK9
GJ-I6 GJ-I4
GT1
GT10 DA1
GJ-II6 GT5
SZ6 GJ-I5GJ-I7 GJ-II1 GJ-I8
0 NK10 WD10
GT7 GJ-II8
GJ-II7
WD6
DG4
SZ3 GJ-II5 DG3
-0.1 AR5 AR4
DA6 AM3 SZ9 GJ-II9
AR2 AM6 GJ-II10 DG1
SZ8 DA8AR7 DG9
DG2
-0.2 DG10
GT8
AM5 DA10 NK2
NK3 NK5
NK4
-0.3 DA2
AM10
AM8
AM9
WD4DA4
WD5 WD2 WD9 NK8
DA3
SZ1 WD8 NK7
NK6
WD1
DA5
-0.3 -0.2 -0.1 0 0.1 0.2 0.3 0.4 0.5 0.6
Coordinate 1

Figure 6. Two dimensional representations of 98 P. edulis individuals based on Jaccard’s

similarity coefficients.

Key: DG- Damot Gale; SZ- Sodo Zuria; AR- Areka; AM-Ambo; WD-Wedessa; GT-Gute;
NK; Nekemte; DA-Digga; GJI-Gojam I ; GJII-Gojam II

38
Figure 7. Three dimensional representation of principal coordinate analysis of genetic
relationships among 98 individuals of P. edulis as revealed by ISSR marker generated by four
primers.

Key: DG- Damot Gale ; SZ- Sodo Zuria; AR- Areka; AM-Ambo; WD-Wadessa; GT-Gute;
NK; Nekemte; DA-Digga; GJI-Gojam I ; GJII-Gojam II

39
 5. SUMMARY, CONCLUSIONS AND RECOMMENDATIONS

5.1. Summary and Conclusions

The present study is the first report on genetic diversity of Oromo Potato (P. edulis) using
ISSR marker. The genetic diversity data generated by four ISSR primers revealed that high
genetic diversity exists in P. edulis. The penta-nucleotide primer showed high levels of
genetic diversity. This clearly implied that penta-nucleotide ISSR primer is appropriate for
diversity analysis of the populations.

The Sodo Zuria population showed the highest genetic diversity followed by Gojam-II and
Areka populations while Nekemte population showed the least genetic diversity. These
assessments of the levels and extents of genetic diversity indicated that the Sodo Zuria
population is a valuable site for P. edulis conservation.

Additionally, AMOVA analysis showed the presence of high genetic variation within
populations than among populations which may be attributed to limited geographic distance
and increase gene flow. This shows presence of gene flow that caused by human interference
and adaptation of P. edulis to environmental changes being evolved during long evolutionary
period in the regions.

Based on Jaccard’s coefficient, highest genetic similarity was observed between Ambo and
Sodo Zuria and the second highest genetic similarity was observed between Digga and Ambo.
On the other hand, the least genetic similarity was detected between Gojam-I and Sodo Zuria.
The UPGMA and NJ clustering methods of ninety eight individuals form mixed tree topology
of themselves. In two coordinates almost all individuals of each population were spread all
over the plot. Individuals from Nekemte and Gute populations showed some tendency of
grouping while others spread all over the space. However, the result of this study showed that
there is a gene flow among populations by effectors and man-made activities.

40
5.2. Recommendations

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

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

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

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

41
 6. REFERENCES

Abebe Demissie and Bjonstrand. 1996. Phenotypic diversity of Ethiopian barley in relation to
geographic regions, altitudinal range and agro-ecological zones: as an aid to germplasm
collection and conservation strategy. Hereditas 124:17-29.

Abreham Bekele, Tileye Feyissa & Kassahun Tesfaye .2014. Genetic diversity of anchote
(Coccinia abyssinica (Lam.) Cogn.) from Ethiopia as revealed by ISSR markers. Genetic
Resources and Crop Evolution. An International Journal. ISSN. 0925-9864.Volume 61. No.
3.

Akagi, H., Yokozeki Y., Inagaki A., Nakamura A. & Fujimura T. 1996. A co-dominant DNA
marker closely linked to the rice nuclear restorer gene, Rf-1, identified with inter-SSR
fingerprinting. Genome 39: 1205–1209.

Allemann, J. & Hammes, PS. 2006. Effect of photoperiod on tuberization in the Livingstone
potato (Plectranthus esculentus N.E.Br.Lamiaceae).Field Crop Res. 98:76-81.

Allemann J., Robertse PJ., and Hammes PS. 2003. Organographic and anatomical evidence
that the edible storage organs of Plectranthusesculentus N.E.Br. (Lamiaceae) are stem tubers.
Field Crop Res. 83:35ƒ {39.

Awasthi, AK., Nagaraj GM., Naik GV., Kanginakudru S., Thangavelu K., and Nagaraju J.
2004. Genetic diversity and relationships in Mulberry genus Morus as revealed by RAPD and
ISSR markers assays. BMC Genet. 5:1-9.

Botstein D., White RL., Skolnick M., and Davis RW. 1980. Construction of agenetic linkage
map in man using restriction fragment length polymorphisms. Am. J. Hum. Genet.32: 314 331.

Castiglioni P., Pozzi, C., Heun, M., Terzi, V., Muller, K.J., Rohde, W. and Salamini, F. (1998).
An AFLP‐based procedure for the efficient mapping of mutations and DNA probes in barley.
Genetics, 149: 2039‐2056.

Cho YG, Ishii T, Temnykh S, Chen X, Lipovich L, McCouch SR et al. 2000. Diversity of
microsatellites derived from genomic libraries and Gene bank sequences in rice (Oryza sativa
L.). Theor Appl Genet 100: 713–722

Codd, L.E. 1985. Flora of Southern Africa 28, part 4: Lamiaceae. Botanical Research Institute,
Pretoria.

Dandena Gelmesa.2010. Shifting to alternative food source: potential to overcome Ethiopias‟

malnutrition and poverty problems. Agriculture and Food. Montpellier, France.

42
Da-Wei, X., Xue-Jun, GE., Gang, H., and Chang-Qin Z.2004. High genetic diversity in a rare.
Narrowly Endem Primose Species Primula Interjacens ISSR Anal 46(10):1163–1169

De Vicente, M.C. and Fulton, T. 2003. Using molecular marker technology in studies on plant
genetic diversity. IPGRI , Cornell University.

Edossa Etissa. 1996. Root and Tuber Crops: Potential as food crops in the humid areas of
Ethiopia. In: IAR News letter of Agric. Res. 2 (1). IAR, A.A, Ethiopia

Edward S 1991. Crops with wild relatives found in Ethiopia. In: Engels JMM, Hawkes JG,
Melaku W (ed): Plant genetic resource of Ethiopia. Cambridge. pp. 42-74.

Ege S. 2009. Violence Extended into the Middle Omo Valley: A View of Recent Raiding
Conflicts in Malo, Southwest Ethiopia In: Aspen H, Teferra B and Bekele Shiferaw and
Tafesse Tesfaye (eds). In: Proceedings of the 16 th International Conference of Ethiopian Studi
es.

EHNRI. 1997. Food composition table for use in Ethiopia. Ethiopian Health and Nutrition
Research Institute, Addis Ababa, Ethiopia.

ENBSAP. 2005. Ethiopian National Biodiversity Strategy and Action Plan. Institute of
Biodiversity Conservation, Addis Ababa, Ethiopia.

Excoffier, L., Laval, G. and Shneiider,.S. 2006. Areliquin version 3.001: An integrated
software package for population genetics data analysis. Evolutionary Bioinformatics
onlin.1:47-50.

Fang, D.Q. and Roose, M.L. 1997. Identification of closely related citrus cultivars with inter-
simple sequence repeat markers. Theor Appl Genet 95: 408–417.

FAO 1999. Sustaining agricultural biodiversity and agro-ecological function. Rome: Food and
Agric. Organisation of the United Nation (http::www.fao.org/WAICNET/ FAOINFO/
SUSTDEV/ Epdirect/Epre0063.htm)

Frankel, O.H, Brown, A.H.D, and Burdon, J.J, (1995). The Conservation of Plant Biodiversity.
Cambridge University, Cambridge, 299.

Godwin, I.D., Aitken, E.A.B. and Smith, L.W. 1997. Application of inter-simple sequence
repeat (ISSR) markers to plant genetics. Electrophoresis 18: 1524–1528.

Gomez, O.J., M.W. Blair, B.E. Frankow-Lindberg and Gullberg, U. (2004). Molecular and
phenotypic diversity of common bean landraces from Nicaragua. Crop Science. 44: 1412-
1418.

43
GRIN (Germplasm Resource Information Network) [Online database]. (2005). National
genetic resource program. National germplasm resource laboratory, Beltssoille, Maryland
USDA, ARS.[http://www.ars-grin.gov2/cgi-bin/npgs/html/taxon.]

Gupta, M, Chyi, YS., Romero-Severson, J., and Owen, S. 1994. Amplification of DNA
markers from evolutionarily diverse genomes using single primers of simple sequence repeats.
Theory Appl. Genet., 89: 998-1006.

Gupta, P.K. & Varshney, R.K. 2000. The development and use of microsatellite markers for
genetic analysis and plant breeding with emphasis on bread wheat. Euphytica113: 163–185.

Hammer, O., Harper, D.A.T. and Ryan, P.D. 2001. PAST: Paleontological statistics software
package for education and data analysis. Palaeontologia electronic 4:9,http//palaeo-
electronica.org/2001_1/past/issue1-01.htm.
Hamrick, JL. and Godt, MJW. 1997. Allozyme diversity incultivated crops. Crop Science
37:26–30

Helentjaris, T., Slocum, M., Wright, S., Schaefer, A. and Nienhuis, J. 1986. Construction of
genetic linkage maps in maize and tomato usingrestriction fragment length polymorphisms.
Theor. Appl. Genet. 61:650-658.

Hollingsworth PM, Ennos RA (2004) Neighbour joining trees, dominant markers and
population genetic structure. Heredity 92:490–498

Jansen PCM. 1996. Plectranthus rotundifolius (Poiret) Sprengel. In: Flach M, Rumawas F
(eds) Plant Resources of South-East Asia no 9. Plants Yielding Non-Seed Carbohydrates,
Backhuys Publishers, Leiden, Netherlands pp. 141-143.

Jones, C. J., Edwards, K. J., Castaglione, S., Winfield, M. O., Sala, F., Wiel, C., Van de
Bredemeijer, G., Vosman, B., Matthes, M., Daly, A., Brettschneider, R., Bettini, P., Buiatti,
M., Maestri, E., Malcevschi, A., Marmiroli, N., Aert, R., Volckaert, G., Rudea, J., Linacero,
R., Vazquez, A. and Karp, A. 1997. Reproducibility testing of RAPD, AFLP and SSR markers
in plants by a network of European laboratories. Mol. Breed., 3: 381-390.

Kojima,T., Nagaoka T., Noda, K. & Ogihara Y. 1998. Genetic linkage map of ISSR and
RAPD markers in Einkorn wheat in relation to that of RFLP markers. Theor Appl Genet 96:
37–45.

Li, J. and Jin, Z. 2007. Genetic variation and differentiation in Torreya jackii Chun, an
endangered plant endemic to China. Plant Sci. 172:1048–1053.

Li, FG., and Xia, NH. 2005. Population structure and genetic diversity of an endangered
species, Glyptostrobus pensilis (Cupressaceae). Bot Bull Acad Sci 46:155–162.

44
Lu Y, Zhang X, Pu J, Qi Y, Xie Y 2011. Molecular assessment of genetic identity and genetic
stability in banana cultivars (Musa spp.) from China using ISSR markers. AJCS 5(1):25–31.

Lukhoba, C.W., Simmonds, M.S.J., and Paton, A.J. 2006. Plectranthus: A review of
ethnobotanical uses. J. Ethnopharmacol. 103, 1–24.

MAFF Research Council. 1994. The first ministry of Agriculture, forestry and fisheries, Japan
international workshop on genetic resource: Root and tuber crops. Japan International
Research Center for Agricultural Science, Japan.

Matu, E.N. and van Staden, J., 2003. Antibacterial and anti-inflammatory activities of some
plants used for medicinal purposes in Kenya. J. Ethnopharmacol. 87, 35–4.

Matsuoka Y, Mitchell SE, Kresovich S, Goodman M, J Doebley (2002) Microsatellites in Zea

variability, patterns of mutations, and use for evolutionary studies. Theor Appl Genet
104:436– 450

Meyer, W., T.G. Mitchell, E.Z. Freedman & R. Vilgays. 1993. Hybridization probes for
conventional DNA fingerprinting used as single primers in the polymerase chain reaction to
distinguish strains of Cryptococcus neoformans. J Clin Microbiol 31: 2274–2280.

Millar, M.A, Brine, M, Coates, D.J, Stuckely, M.J.C, McComb, J.A., (2000). Mating system
studies in Jarrah, Eucalyptus marginata (Myrtaceae), Australian J. Botany. 48:475-479.

Moreno, S., Martin, J.P. & Ortiz, J.M. 1998. Inter-simple sequence repeats PCR for
characterization of closely related grapevine germplasm. Euphytica.101: 117–125.

Moulin MM, Rodrigues R, Simo˜es L, Gonc¸alves A, Sudre´ CP, Pereira MG (2012) A

comparison of RAPD and ISSR markers reveals genetic diversity among sweet potato
landraces (Ipomoea batatas (L.) Lam.). Maringa´ 34(2):139–147.

Mulugeta Taye, Lommen, WJM, and Struik, PC. 2007. Indigenous multiplication and
production practices for the tuber crop Plectranthus edulis in Chencha and Wolaita, southern
Ethiopia. Exp. Agric. 43:381-400.

Mulugeta Taye 2008. Studies on agronomy and crop physiology of Plectranthus edulis (Vatke)
Agnew. Ph. D. Thesis. Wageningen University, Wageningen.

Mulugeta Taye, W. J. M. Lommen3* and P. C. Struik3. 2012. Ontogeny of the tuber crop
Plectranthus edulis (Lamiaceae). African Journal of Agricultural Research Vol. 7(30), pp.
4236-4249.

45
Murdock, G.P., 1959. Africa, its peoples and their culture history, New York. In: E. Westphal,
1975. Agricultural systems in Ethiopia. Agricultural Research Reports 826. Centre for
Agricultural Publishing and Documentation, Wageningen.

National Research Council .1991. Managing global genetic resources: the U.S. National Plant
Germplasm System, Committee on Managing Global Genetic Resources: Agricultural
Imperatives, Board on Agriculture, National Research Council. National Academy Press,
Washington, DC.

Ni, X., Huang, Y., Wu, L., Zhou, R., Deng, S., Wu, D., Wang, B., Su, G., Tang, T., and Shi, S.
2006. Genetic diversity of the endangered Chinese endemic herb Primulina tabacum
(Gesneriaceae) revealed by amplified fragment length polymorphism (AFLP). Genetica
127:177–183 718 Genet Resour Crop Evol (2014) 61:707–719

Nybom, H.2004. Comparison of different nuclear DNA markers for estimating intraspecific
genetic diversity in plants. Mol Ecol 13:1143–1155

Paton, A.J., Springate, D., Suddee, S., Otieno, D., Grayer, R.J., Harley, M.M., Willis, F.,
Simmonds, M.S.J., Powell, M.P., and Savolainen, V. 2004. Phylogeny and evolution of basils
and allies (Ocimeae, Labiatae) based on three plastid DNA regions. Mol. Phylogenet. Evol. 31,
277–299.

Patrick, S., Schnable and Nathan M. 2013. Progress toward understanding heterosis in crop
plants. Annu. Rev. Plant Biol. 64:71-88.

Pavlicek,A., Hrda,S. and Flegr J. 1999. Free tree free ware program for construction of
phylogenetic trees on the baese of distance data and bootstrap/Jack Knife analysis of tree
robustness. Application in the RAPD analysis of the genus Frenkelia. Folia Biologiica.45:97-
99.

Powell, W., Machray, G. C. and Provan, J. 1996. Polymorphism revealed by simple sequence
repeats. Tren. Plant Sci., 1: 215-222.

Quedraogo, A.S, (2001). Conservation, management and use of forest genetic resources.
Recent Research and development in Forest genetic Resources. Proceedings of training
workshop on the conservation and sustainable use of forest genetic resources in Eastern and
Southern Africa, December 1999, Nairobi, Kenya. pp.1-14.

Rivera Nun˜ ez, D., Obo´n de Castro, C., 1992. The ethnobotany of Old World Labiatae. In:
Harley, R.M., Reynolds, T. (Eds.), Advances in Labiate Science. Royal Botanic Gardens, Kew,
pp. 455–473.

Rohlf, F.J. 2000. NTSYS- pc. Numerical taxonomy and multivariate analysis system, version
2.02. Exeter software. New York.

46
Saitou, N. and Nei, M. 1987. The neighbor joining Method: A new method for reconstructing
phylogenetic trees. Molecular Biological Evolution.4:406-425.

Sankar, A.A. & Moore, G.A. 2001. Evaluation of inter-simple sequence repeat analysis for
mapping in Citrus and extension of genetic linkage map. Theor Appl Genet 102: 206–214.

Siegenthaler IE. 1963. Useful Plants of Ethiopia. Experiment Station Bulletin No. 14. Volume
I. Imperial Ethiopian College of Agricultural and Mechanical Arts, Jima Experiment Station,
Alemaya, Ethiopia.

Smithson, J.B. &Lenne, J.M. 1996. Varietal mixtures: a viable strategy for sustainable
productivity in subsistence agriculture. Ann. Appl. Biol., 128, 127–158.

Sneath, P.H.A. and Sokal, R.R. 1973. Numerical Taxonomy . Freeman. San Francisco. 573Pp.

Somasundaram, S.T. and Kalaiselvam, M. 2004. Molecular Tools for Assessing Genetic
Diversity. Centre of Advanced Study in Marine Biology. Annamalai University

Stat soft.Inc. (2001). Statistica data analysis system, Statistica software.

Staub, J.E., F.C. Serquen & M. Gupta, 1996. Genetic markers, map construction, and their
application in plant breeding. Hort. Science 31(5): 729–739.

Struik, PC. and Wiersema, SG. 1999. Seed Potato Technology, Wageningen Pers,
Wageningen, Netherlands.

Studier, J.A. and keppler, K.J. 1988. A note on the neighbor joining algorithm of Saitou and
Nei. Mol. Biol. Evol. 5:729-731.

Tafesse Tesfaye. 2009. The Predicaments of Amhara Migrant settlers in East Wollega Zone,
Ethiopia. In: Ege S, Aspen H, Teferra B and Bekele S (eds). In: Proceedings of the 16 th
International Conference of Ethiopian Studies.

Tindall, HD. 1983. Vegetables in the Tropics. Macmillan Press, London, UK.

Tsumura, Y., K. Ohba& S.H. Strauss, 1996. Diversity and inheritance of inter-simple
sequence repeat polymorphisms in Douglasfir (Pseudotsugamenziesii) and sugi (Cryptomeria
japonica). Theor Appl Genet 92: 40–45.

Vithanage, V., Anderson, K. A. and Thomas, M. (1995). Use of molecular markers in crop
improvement of Macadamia. In: The Sixth Conference of the Australasian Council on Tree
and Nut Crops, 11-15 September 1995, Lismore, Australia.

47
Vos, P., Hogers, R., Bleeker, M., Reijans, M., Lee, T., Hornes, M., Frijters, A., Pot, J.,
Peleman, J., Kuiper, M. and Zabeau., M. 1995. AFLP: a new technique for DNA
fingerprinting. Nucl. Acids Res., 23: 4407- 4414.

Vosman, B., Arens, P., Rus-Kortekaas, W. and Smulders, M. J. 1992. Identification of highly
polymorphic DNA regions in tomato. Theor Appl. Genet., 85: 239-244.

Wang, X., Hou, X., Zhang, Y., Yang, R., Feng, S., Li, Y. and Ren Y. 2012. Genetic diversity
of the endemic and medicinally important plant Rheum officinale as revealed by inter-simpe
sequence repeat (ISSR) markers. Int J Mol Sci 13:3900–3915

Wang, G., R. Mahalingan& H.T. Knap, 1998. (C-A) and (GA) anchored simple sequence
repeats (ASSRs) generated polymorphism in soybean, Glycine max (L.) Merr. Theor Appl
Genet 96: 1086–1096.

Weber, D. and Helentjaris, T. 1989. Mapping RFLP loci in maize using B-A translocations.
Genetics 121:583-590.

Weising, K., Nybom, H., Wolff, K. and Kahl, G. 2nd ed. (2005). DNA Fingerprinting in
Plants: Principles, Methods and Applications. Taylor and Francis Group, USA. 444Pp.
Westphal, E. 1975. Agricultural systems in Ethiopia. Agricultural Research Reports 826.
Centre for Agricultural Publishing and Documentation, Wageningen.

Weyessa Garedew, Admasu Tsegaye, Bezuayehu Tesfaye, and Hussein Mohammed 2009.
Variability and association of quantitative traits in Plectranthus edulis (Vatke) Agnew. East
Afr. J. Sci. 3:61-69.

Weyessa Garedew, Admasu Tsegaye, Bezuayehu Tesfaye and Hussein Mohammed, 2013.
Diversity analysis in Plectranthus edulis (Vatke) Agnew collection in Ethiopia.International
Journal of Biodiversity and Conservation. Vol. 5(9), pp. 561-566,

Williams, J. G., Hanafey, M. K., Rafalski, J. A., Tingey, S. V. 1993. Genetic analysis using
random amplified polymorphic DNA markers. Meth. Enzymol., 218: 705-740.
Wu, K., R. Jones, L. Dannaeberger& P.A. Scolnik, 1994. Detection of microsatellite
polymorphisms without cloning. Nucleic Acids Res 22: 3257–3258.

Wu, J, Krutovskii, K.V, Strauss, S.H, (1999). Nuclear DNA diversity, population
differentiation, and phylogenetic relationships in the Califonia closed-clone pines based on
RAPD and allozyme markers, Genome 42:893-908.

Xiao M, Li Q, Wang L, Guo L, Li J, Tang L, Chen F. 2006. ISSR analysis of the genetic
diversity of the endangered species Sinopodophyllym hexandrum (Royle) Ying from western

48
Sichuan Province. China. J. Integr Plant Biol 48(10):1140– 1146

Yeh,F.C.,Ynag, R-Cai. and Boyle,T. 1999. Population genetic analysis of codominant markers
and qualitative traits. Belgian Journal of Botany.129:157.

Yeshitila Mekbib (2007). Phenotypic variation and local customary use of Ethiopian potato
(Plectranthus edulis (Vatke) Agnew). CBM Master Theses Series 40, Swedish Biodiversity
Centre,Uppsala.

Zhang, L, Li, QJ., Li, HT., Chen, J. and Li, DZ. 2006. Genetic diversity and geographic
differentiation in Tacca chantrieri (Taccaceae): An autonomous selfing plant with showy
floral display. Ann Bot 98(2):449–457

Zhou, Y., Zhou, C., Yao, H., Liu, Y., and Tu, R. 2008. Application of ISSR markers in
detection of genetic variation among Chinese yam (Dioscorea opposita Thunb.) cultivars. Life
Sci J 5(4):6–12

Zemede Asfaw, and Zerihun Woldu 1997. Crop associations of home-gardens in Welayta and
Gurage in southern Ethiopia.SINET.Ethiopian J. Sci. 20:73-90.

Zhu, Y., Chen, H., Fan, J., Wang, Y., Li, Y., Chen, J. et al. (2000). Genetic diversity and
disease control in rice. Nature, 406, 718–722.

Zietkiewicz, E., Rafalski, A. & Labuda, D. 1994. Genome fingerprinting by simple sequence
repeat (SSR) anchored polymerase chain reaction amplification. Genomics 20: 176–183.

49
7. APPENDICES

50
7.1. Appendix

DNA Extraction Procedure

1. Pour CTAB solution (700 μl per sample) in a 15ml-tube and add 0.2 vol % Mercapto-
ethanol (use fume hood!). Mercapto-ethanol is stored at 4°C. Incubate the CTAB at 65°C.
2. Weigh in 100 mg fresh leave material (50mg dry material) per sample. Pulverise thoroughly
using a clean mortar and pestle. For fresh material add liquid nitrogen or quartz sand for
dry material. First grind down slightly, then more powerful (cells have to be crashed). Use
safety goggles!
3. Transfer the powder into a sterile Eppendorf cap (use a new, sterile spatula for each sample)
4. Add 700 μl of warm (65°C) CTAB solution to the powdered sample (open the caps
carefully), dissolve the powder and incubate the sample for 30 minutes at 65°C.
5. Centrifuge for 5 minutes at 15000 rpm, 20°C.
6. Transfer the supernatant (only clear liquid) in a new Eppendorf-cap. Use blue pipette tips
which are cut.
7. Add new CTAB solution (700 μl) to the tissue pellet and stir slightly with a new 1000 μl
pipette tip, incubate 30 min at 65°C. Step 6 and 7 are repeated. The same is carried out for
a third extraction. Each fraction proceeds with step 9 and is treated separately.
8. Add 600 μl Chloroform to the cap with supernatant. (This chloroform step should be carried
out immediately).
9. Shake the samples thoroughly by inverting the Eppendorf caps for approximately 5 minutes.
10. Centrifuge for 5 min at 15000 rpm.
11. Transfer the supernatant (only clear liquid) in a new Eppendorf-cap. Use blue pipette tips
which are cut. Work carefully; do not transfer suspended matter (normally the Chloroform
is covered by a thin layer of fine sediment material). Chloroform has to be disposed of in a
special waste bottle.
12. Repeat the chloroform extraction (step 8-11) to make sure that all impurities are removed
and then proceed with step 13.

51
13. Add cooled Isopropanol (4°C), approximately 2/3 of the solution volume. Shake carefully
by inversing the Eppendorf cap. In most cases DNA becomes visible as white threads.
Freeze for more than 2 h at -20°C. (BREAK POSSIBLE)
14. Centrifuge 10 min at 15000 rpm in a cooled centrifuge.
15. Aspirate liquid using yellow tips (without touching pellet!). If pellet is solid enough the
larger part of the liquid may be poured out. (Alternatively add TE and proceed with Qiagen
kit)
16. Add 200 μl Ethanol 70 % to the pellet.
17. Centrifuge for 10 min at 15000 rpm in a cooled centrifuge.
18. Aspirate Ethanol using yellow tips. Dry the DNA-pellet at room temperature. (Usually 15
min are sufficient; after drying no liquid drops are to be seen)
19. Dissolve pellet in 100 μl TE (1x, p.a. grade) and store at 4°C. (BREAK POSSIBLE)
20. Add cooled 7.5 M NH4Ac-solution (4°C, half of the solution volume). Mix carefully.
21. Add cool Ethanol 100 % (double of the solution volume). Mix carefully. Freeze for more
then 2 h at -20°C. (BREAK POSSIBLE)
22. Centrifuge 30 min at 15000 rpm in a cooled centrifuge. Aspirate fluid carefully.
23. Add 200 μl Ethanol 70%.
24. Centrifuge 10 min. at 15000 rpm in a cooled centrifuge. Aspirate liquid and dry pellet at
room temperature. Dissolve the pellet in 100 μl TE (1x, p.a. grade)
25. Repeat steps 20 to 23 with 3 M NaAC-solution (instead of NH4Ac), then proceed with
step 26.
26. Centrifuge 10 min. at 15000 rpm in a cooled centrifuge. Aspirate liquid and dry pellet at
room temperature. Dissolve the pellet in 100 μl TE (1x, p.a. grade)

52
7.2. Tables in the Appendix

PCR mix µl/sample

H2o 17

10X Taq buffer 2.5

MgCl2 (25mM) 3
dNTPs (100mM) 0.8
Primer (10µM) 0.4
Taq polymerase (5U/µl) 0.3
Sub total 24
10 ng/µl DNA 1

Total 25

Appendix Table 1. Polymerase Chain Reaction (PCR) mixture (master mix and DNA
template) used during this study.

53
7.3. Figures in the Appendix

Appendix Figure 1. Banding profiles of some P. edulis individuals selected using ISSR
primers UBC-835 exhibiting fourteen individuals.

Appendix Figure 2. Banding profiles of some P. edulis individuals selected using ISSR
primers UBC- 841 exhibiting fourteen individuals.

54
Appendix Figure 3. Banding profiles of some P. edulis individuals selected using ISSR
primers (UBC-844)

55