This document provides an introduction and overview of molecular characterization of livestock breeds in Ethiopia. It discusses the importance of characterizing livestock breeds to document genetic diversity, establish conservation priorities, and inform breeding programs. The document then reviews different types of characterization, including phenotypic, morphological, biochemical, cytogenetic, and molecular characterization. It describes the principles, methodologies, advantages, and limitations of each approach. The overall purpose is to discuss methods and criteria for molecular characterization of livestock breeds in Ethiopia based on past research experiences.

1. Molecular Characterization of Livestock
Breeds of Ethiopia: A review
Mohammed Endris Seid
ID. No: PhD/ 059/2012
Department of Animal science, Haramaya University
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2. 1.INTRODUCTION
Ethiopia has served as a gateway to domestic animals from Asia to
Africa and its diverse ecology favored diversification of these resources
There are many indigenous breeds of livestock in Africa adapted to a
wide range of ecological conditions
Ethiopia stands first in Africa and 10th in the world in terms livestock
population
Maintaining such resource requires the conservation of diversity among
indigenous livestock,
In order to cope with unpredictable future genetic reserves that are
capable of readily responding to directional forces imposed by a broad
spectrum of environments
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3. Introduction…
According to FAO (2013), maintaining genetic diversity is an insurance
against future adverse conditions and thus, can serve as a genetic pool for
selection of suitable strains and lines
There is a need to characterize the livestock species and breeds, their
Population sizes
geographic distribution and
importantly their genetic diversity
which is generally undertaken as a preliminary step in any national
programme for the management of Livestock genetic resources for food
and agriculture
The primary purpose of characterization:- is
- to document the current state of knowledge in terms of a population’s
ability to survive, reproduce, produce and provide services to farmers
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4. Introduction...
- provides the baseline information as well as the criteria that will be
used to establish and update the inventory or records
- provides data on present and potential future uses of the Livestock
genetic resources under consideration, and
- establishes their current state as distinct breed populations and
their risk status
 management of livestock genetic resources are dynamic processes,
monitoring the status of a population has to be done on a regular
basis
 Thus, risk status indicators for use during the monitoring process
need to be defined following the characterization steps
- Phenotypic
- Morphological
- Cytogenetic and
- Molecular or genetic characterization
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5. Introduction…
Genetic improvement of livestock depends on:-
- access to genetic variation and effective methods of exploiting
this variation
Genetic diversity፡-
- constitutes a buffer against changes in the environment
- is a key in selection and breeding for adaptation and
production on a range of environments
Principally in the development of polymerase chain reaction (PCR) :-
- Amplifying (DNA), DNA sequencing and data analysis, have
resulted in powerful techniques which are used for the screening,
characterization and evaluation of genetic diversity
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6. Introduction…
The extensive number of research information-: describing the use
of these techniques on wide range of animal species and diversity
problems, is a testimony to their increasing impact in this field (Karp
and Edwards, 1996)
Thus, this seminar paper is intended to review and discuss the
methods and criteria that currently available, from research and past
experiences concerning molecular characterization of livestock
breeds in Ethiopia
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7. 2. Characterizations of Livestock and Its
Importance
 The distillation of all the information/knowledge on & about a livestock
breed/population
 Characterization is the primary assessment or baseline survey, which
should include collection of data on population size and structure,
geographical distribution, production systems in which the breed is
found,
 phenotypic attributes (physical features, performance levels and any
unique features), historical development of the breed through exchange,
upgrading and selection, and the genetic connectedness of populations
when these are found in more than one country
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Definition of characterization

8.  The within-population genetic diversity is measured both at the phenotypic
level (phenotypic breed diversity) and at molecular level; the two are
complementary
 All these data are needed to inform decisions on the utilization,
improvement and conservation of the population (Tixier-Boichard et al.,
2011).
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9. Why characterize?
In order to better understand & comprehensively describe the
animal and production environment (Dessie, 2012):-
– Provides options for informed utilization & management of AnGR
- develop informed improvement/conservation programs
- determine population trends & levels of threats etc.
- advocate for, make & supportive/relevant policies
- Support/inform negotiations for transfers/exchange etc.
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10. Importance of Characterization in livestock genetic
resources
Based on the guideline report of FAO (2015), the importance of
Characterization in AnGR :-
- When a breed/population/species is found to be at risk, active
conservation strategies have to be implemented or the potential loss of the
breed must be accepted or known
- To allocate the limited resources that are available for conservation
programs, the characterized breeds need to be prioritized
- These decisions may be based on the genetic distinctiveness, adaptive
traits, relative value for food and agriculture, or historical and cultural
values of the breeds in question –
- Characterization- is contributes to the objective and reliable prediction
of animal performance in a defined environment
- It is, therefore, important that relevant information on breed
characteristics is made accessible to decision-makers at all levels
through characterization
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11. What if there is no characterization?
If characterization is not achieved:-
 Information about the particular breed population could
not be available, whether it is self-sustainable or is at risk
 It also causes a missing of strategic and coherent approach
to identification, description and documentation of breed
populations.
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12. 2.1. Classification of Characterization
2.1.1. Phenotypic characterization:-
Description of external characteristics, associated pests/pathogens etc.
Production characteristics within a defined/described production environment
(management practices)
used to identify and document diversity within and between distinct breeds
based on their observable attributes
The process of identifying distinct breed populations and describing
their external and production characteristics in a given environment and
under given management, taking into account the social and economic
factors that affect them (FAO, 2012)
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13. Basic principles of pheynotypic charcterization
1st distinct breed populations and production characteristics within a
production environment should be identified
2nd before, starting population have homogenous features and seems to
be distinct and found in sufficiently larger geographical area
3rd Through baseline survey, a breed/population should be primarily
assessed:-
- include collection of data on population size and structure,
- geographical distribution, production systems in which the
breed/population is found,
- phenotypic attributes (physical features, performance levels
and any unique and historical development of the
breed/population through exchange, upgrading and selection
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14. 4th a breed/population should cover all the activities associated with
- generating information on their identification,
- qualitative and quantitative description,
- geographic and demographic distribution of populations,
- native habitats and production systems including management
practices and socioeconomic profile,
- indigenous traditional knowledge
5th as a part of livestock characterization, assessment of the
population characteristics of identified breed is also an important
component, which includes
- Estimates of population sizes,
- Flock/herd structure, and
- assessment of the level of crossbreeding which are indicators of
threat to the survival of the adapted indigenous AnGR
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15. Advantages:
 It is a prerequisite for effective assessment of animal genetic resources
diversity and determining whether or not it is being erode
 Important to the establishment of national inventories of Animal genetic
resources (FAO, 2012)
 It is Important to the effective monitoring of Animal genetic resources
populations and to the establishment of early-warning and response systems
for animal genetic resource.
Limitations or challenges:
 Phenotypic characterization activities are technically and logistically
challenging ensuring that they are well targeted (collect data that are important
to the country’s priority AnGR- and livestock-development activities)
 Carried out in an efficient and cost effective manner requires thorough planning
and careful implementation
 It requires the development and use of standard practices and formats for
describing their characteristics. Such standards and protocols are also needed
for assessing requests for the recognition of new breeds (FAO, 2012
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16. 2.1.2. Morphological Characterization
Definition:
 refers to external animal characteristics which can be obtained by direct
visual observation and measurement
 used in the identification, classification and characterization of genetic
evolution of different species or populations (Hailu and Getu, 2015)
Principles:
 Morphological description is an essential component of breed
characterization that can be used to physically identify, describe, and
recognize a breed, and also to classify livestock breeds into broad
categories
 Dossa et al. (2007) reported that morphological measurements such as
heart girth, height at withers and body length can be used for rapid
selection of large size individuals in the field to enable the establishment
of elite flocks or herds.
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17. Methodology:
Discriminate analysis
- is a set of methods and tools used to distinguish between groups of
populations and
- to determine how to allocate new observations into groups
Morphometric characters or measurements are continuous characters
describing aspects of body shape (Riva et al., 2004; Cervantes et al.,
2009; Cited in Agaviezor et al., 2012)
Advantages:
- It is readily available; usually require only simple equipment
- Form of the most direct measure of phenotypic characterization
Limitation: - require expertise
- it is subject to environmental influences or affected by
environment
- limited in number and less polymorphisms in when animals
selected based on appearance.
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18. 2.1.3. Biochemical Characterization
Biochemical characterization is the characterization of the biochemical state of
the organism, which is in fact affected by environment, development as well as
physiological state.
Related to variation in protien and amino acids banding pattern are known as
biochemical markers
Biochemical characterization is giving us a picture of the interaction of
molecular state with the other states (environmental, developmental,
physiological) while molecular characterization is solely giving us the sight of
molecular state (Priyanka Siwach, 2014)
Principles:
- are related to the various in protein and amino acid banding pattern
- Gel electrophoretic studies used for identification of biochemical markers
(example, - peroxidase, acid phosphate, esterase etc) (Liang Jiang, 2012)
- Animals in biochemical markers are selected based on biochemical
properties (example, Hb, Amylase, and Blood Groups) (Paraset al.,
2015).
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19. Advantage:
• Biochemical characterization requires simple equipment and a
vigorous complement to the morphological assessment of variation in
plant and animal species.
Limitation:
- It is subjected to environmental influences and
- limited in number (Liang Jiang, 2012).
- In addition, it is sex limited,
- Age dependent and it covers less than 10% of genome (Para et
al., 2015).
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20. 2.1.4. Cytogenetic characterization
 Is the study of chromosomes and related disease states caused by
abnormal chromosome number and/or structure
 Cytogenetic characterization studies are highly useful in genetic
characterization and for effective conservation of the specie seriously at
risk of extinction
 It is the study of chromosomes and its abnormalities; alterations and
structures
 Research strategies involving cytogenetics hold the promise of yielding
insight into the mechanisms underlying chromosome instability in embryos
and the impact of the in vitro environment on the chromosome make-up of
embryos (King, 2008; Cited in Alok et al. 2017)
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21.  cytogenetic characterization of animals will help to identify the
animals with congenital and acquired chromosome abnormalities
whose elimination will help to maintain the herds cytogenetically clean
(Alok et al. 2017).
 As a tool, cytogenetics has an equally important contribution to make
to the various disciplines involved in the study of ways to enhance more
economical production of animal products for human consumption
 The studies conducted by Sara et al. (2019) on cytogenetic analyses in Ewes
with Congenital Abnormalities of the Genital Apparatus reported that the
clinical and cytogenetic findings of four Disorders of Sex Development (DSD)
cases and 13 couples of heterosexual twins in sheep
Principles :
- First , genomic DNA from different individuals is digested into DNA
of varying size, using known restriction enzymes
- Second, the digested fragments are separated via electrophoretic
analysis
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22. Restriction enzymes
 Molecular scissors that cut double stranded DNA molecules at specific points
 Found naturally in a wide variety of prokaryotes
 An important tool for manipulating DNA
Electrophoresis analysis
 Is a method that is used to separate proteins of differing sizes and/or
charges.
 A sample of the protein, often DNA or RNA, is placed in wells at one end of a
gel
 In addition to analyzing proteins or DNA, electrophoresis is also used to
create purified samples of proteins.
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23. Developments of cytological techniques or methods are enables to accurate and detailed
observations of the chromosomes of mammals and birds
- Various applications for cytogenetic methodology in animal science research are
suggested
- Much of the pregnancy wastage in farm animals may be caused by abnormal chromosome
complements of afflicted zygotes
- To reduce embryonic loss, the causal bases of chromosomal abnormalities should be
sought
 Karyological methods are useful to detect chromosomal damage or irregular mitosis or
meiosis which is indicators of toxicity and carcinogenic activity
 Polymorphisms of "banding patterns" of chromosomes are sufficiently widespread to
enable identification of individual animals and authentication of pedigree
 In animal breeding cytogenetic methodology is applied to test assumptions of theory. New
sources of genetic variation may be chromosome rearrangements, duplications,
aneuploidy, and euploidy
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Methodology:

24. Advantages
 It is theoretical interest of chromosome studies for the understanding of the
chromosomal evolution and its role in animal speciation
 It is an effective tool for monitoring the health status of livestock and improving their
genetic value
 It is detecting carriers of hereditary anomalies
 which often have an apparently normal phenotype but come with negative traits such as
 Reduced fertility and allows detecting in a population
 Those animals that are genetically more predisposed to disorders and excluding them
as breeders (Udroiu and Sgura, 2017).
 It is whole genome scan and relatively low cost.
Limitation:
 It requires actively growing tissues or blood samples and autospectro photographic
microscope
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25. 2.1.5. Molecular Characterization
 Molecular characterization:- can be defined as the complementary procedures used to
unravel the genetic basis of phenotypes, their patterns of inheritance from one generation to
the next, within-breed genetic structure and levels of variability, and relationships between
breeds (FAO, 2015;Cited in Gamaniel and Gwaza, 2017)
 It is characterizing at the molecular level without any effect of environment or
development or physiological state of the organism.
 DNA based markers are called molecular markers and characterization (Priyanka Siwach,
2014)
 Molecular genetic characterization: explores polymorphism in selected protein molecules
and DNA markers in order to measure genetic variation at the population level
 Because of the low level of polymorphism observed in proteins, and hence limited
applicability in diversity studies,
 DNA-level polymorphisms are the markers of choice for molecular genetic characterization
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26.  DNA polymorphisms are the different DNA sequences among individuals
groups, or populations
 DNA polymorphism serves as a genetic marker for its own location in the
chromosome
 Thus, they are convenient for analysis and are often used as
in molecular genetic studies
Principle of Molecular characterization:
 Principally Molecular characterization: should ideally be done along with
phenotypic characterization
 Most molecular work was based on the use of neutral genetic marker data,
which served as a proxy or estimate of the likelihood of important functional
genetic variation within breeds or breed groups
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27.  Ideally, molecular characterization should be undertaken as part of a
comprehensive national programme for management of AnGR
 For maximum efficiency, molecular characterization of AnGR should be
done in concert with phenotypic characterization
 Outcomes of morphological or phenotypic characterization need to be
complemented by genetic characterization (FAO 2007; Cited in Gizaw et
al., 2011)
 Molecular or Genetic characterization involves the description of breeds in
terms of
- The relative allelic frequencies,
- Degree of polymorphism using a set of neutral reference markers and
- Classifying livestock breeds using genetic distances between
populations/breeds (Cited by Gizaw et al,. 2011).
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28. Methodologies:
 Comprises field sampling of biological material (often blood or hair
root samples),
 Laboratory extraction of DNA from the samples, DNA storage,
laboratory assaying (e.g. genotyping or sequencing), data analysis,
report writing, and maintenance of a molecular genetic information
database
 Sampling for molecular analysis may be combined with surveying
and/or monitoring, as molecular information on its own cannot be used
for utilization and conservation decisions (FAO, 2012)
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29. The advantages of molecular genetic technologies are:
• It measures the genetic constitution of a breed/population of a species
at a molecular level
• Assesses the genetic uniformity, admixture or subdivisions,
inbreeding, or introgression in the population
• The use of molecular technologies potentially offers a way to select
breeding animal at an early age (even embryos)
• To select for a wide range of traits and to enhance reliability in predicting
mature phenotype of the individual.
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Advantages:

30. 1) Assuming no genotyping errors, molecular genetic information is
not affected by environmental effects and, therefore, has heritability
equal to 1
2) Molecular genetic information can be available at an early age, in
principle at the embryo stage, thereby allowing early selection and
reduction of generation intervals
3) Molecular genetic information can be obtained on
- All selection candidates, which is especially beneficial for sex-
limited traits,
- Sex limited traits: are traits that are expensive or difficult to record,
or traits that require slaughter of the animal (carcass traits)
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The main reasons why molecular genetic information can result in
greater genetic gain than phenotypic information are:-

31. Limitations:
Require more laboratory facilities such as chemicals, reagents and primers
Time consuming and labor- intensive
In developed countries, breeding programs are based upon performance and
molecular evaluation records and this has led to substantial improvement in
animal production.
Developing countries, have distinct disadvantages for setting up successful
breeding programs
Infrastructure needed for performance testing is normally lacking in developing
countries
The infrastructural facilities required for molecular characterization:-
 Refrigerator/freezer
 Polymerase chain reaction (PCR) machines
 Gel electrophoresis
 Autoclave, Chemical and sample collection tubes, laminar flow hood,
 Centrifuge, thermo cycler, power supply units, hotplate or microwave
 PH meter, standard balance, gel electrophoresis units, UV trans illuminator
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32.  Lack of funds to set up facilities to support molecular research
 The existing research facilities, adequate funding for maintenance is
not there
 Inadequacy of scientific equipments to support research and
technical manpower
 Molecular genetics research is highly sophisticated and also required
skilled manpower as well as technologist
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In developing countries the most important resource limitation is:-

33.  Without an established scientific culture, it is almost impossible to
engage in and keep up developments in molecular biology research
 Poor essential utilities like power and water, no support services such as
gene-bank, in vitro storage facilities, storage facilities,
 Animal holding facilities, radiation huddling, disposal facilities and
computing facilities and ICT services (Rashid, 2010).
 Because the well-equipped and adequately funded laboratories doing
research in molecular biology are found almost exclusively in developed
countries, the gap between the industrialized and developing countries in
technical expertise and relevant research capacity is getting wider
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34. 2.2. Molecular Markers
 A sequence of DNA or protein that can be screened to reveal key
attributes of its state or composition and thus used to reveal genetic
variation
 Also known as “Genetic Marker”
 Genetic markers are the sequences of DNA which have been
traced to specific location on the chromosomes and
associated with particular traits
 Molecular Markers are classified as:
1. Protein Based Markers/ Biochemical Markers
2. DNA Based Markers
12/12/2022 34

35. Molecular marker
 Identifiable physical location on a chromosome whose inheritance can be
monitored
 These are a set of DNA-based genetic markers that can detect DNA
polymorphism both at the level of specific loci and at the whole genome
level
 Genetic markers are the biological features that are determined by allelic
forms of genes or genetic loci
 Can be transmitted from one generation to another, and thus
 They can be used as experimental probes or tags to keep track of an
individual, a tissue, a cell, a nucleus, a chromosome or a gene
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36.  According to the reviewed in Liang Jiang (2012) reported that,
depending on application and species involved, ideal DNA markers for
efficient use in marker-assisted breeding should meet the following
criteria:
- High level of polymorphism
- Even distribution across the whole genome (not clustered in certain
regions)
- Co-dominance in expression (so that heterozygotes can be
distinguished from homozygotes)
- clear distinct allelic features (so that the different alleles can be
easily identified),
- Single copy and no pleiotropic effect.
- Low cost to use (or cost-efficient marker development and genotyping)
12/12/2022 36

37. Types of molecular markers:
Restriction fragment length polymorphisms (RFLPs)
Amplified fragment length polymorphisms (AFLPs)
Random amplification of polymorphic DNAs (RAPDs),
Single-starnd conformation polymorphism (SSCP)
Microsatellites or SSRs
Single Nucleotide Polymorphisms (SNPs)
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38. 2.2.1. Restriction Fragment Length
Polymorphisms (RFLPs)
 A molecular method of genetic analysis that allows individuals to be
identified on the basis of unique patterns of restriction enzymes cutting in
specific regions of DNA.
 It is an application of Southern Hybridization Procedure.
 RFLP are Genomic DNA digested with Restriction Enzymes,
 DNA fragments separated via electrophoresis and transfer to nylon
membrane,
 Membranes exposed to probes labeled with P32 via southern
hybridization, Film exposed to X-Ray (Paras et al., 2015).
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39. Advantages of RFLP-
 They are co- dominant.
 Measure variation at the level of DNA sequence, not protein
 sequence.
 RFLP loci are very large so even very small segments of chromosomes can be
mapped and also study phylogenetic relationship
 Very reliable for linkage analysis and for detecting coupling phase of DNA
molecules
Disadvantages of RFLP
Requires relatively very large amount of DNA
Requirement of radioactive probe makes the analysis expensive and
hazardous.
They are not useful for detecting single base change or point mutations
It is time consuming, laborious, and expensive
The level of polymorphism is low
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40. • RAPD is a PCR based molecular marker developed independently by
two different laboratories (Williams et al., 1990; Welsh and McClelland,
1990; Cited in Salisu et al., 2018)
• RAPD is a type of PCR reaction, but the segments of DNA that are
amplified are random
• It creates several short primers (8–12 nucleotides), and then proceeds
with the PCR using a large template of genomic DNA, the fragments will
amplify
• By resolving the resulting patterns, a semi-unique profile can be assembled
from a RAPD reaction (Amber Hassan, 2017)
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2.2.2. Random Amplified Polymorphic
DNA(RAPD)

41. Basic Principle of RAPD:
• It is a PCR based technique for identifying genetic variation.
• It involves use of single arbitrary primer in a PCR reaction, resulting in
amplification of many discrete DNA
• RAPD technology provides a quick and efficient screen for DNA
sequence based polymorphism at a very large number of loci (Amber
Hassan, 2017)
Isolation:
• Physiological, biochemical reactions and RAPD-PCR molecular
techniques were done on native isolates
• The electrophoretic analysis of RAPD band profiles showed the
presence of polymorphism among the studied isolates (Sabir et al.,
2013)
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42. Advantages:
 They are quick and easy to assay.
 Because PCR is involved, only low quantities of template DNA are required.
 No sequence data for primer construction are needed.
 RAPDs have a very high genomic abundance and are randomly distributed
throughout the genome (Amber Hassan, 2017)
Disadvantages:
 Low reproducibility
 RAPD analyses generally require purified, high molecular weight DNA,
and precautions are needed to avoid contamination of DNA samples because
short random primers are used that are able to amplify DNA fragments in a
variety of organisms.
 The inherent problems of reproducibility make RAPDs unsuitable markers for
transference or comparison of results among research
teams working in a similar species and subject
 RAPD markers are not locus-specific, band profiles cannot be interpreted in
terms of loci and alleles (dominance of markers), and similar sized fragments
may not be homologous ((Paras et al., 2015).
•
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43. 2.2.3. Amplified fragment length polymorphism
(AFLP) marker:
 AFLP is a PCR-based tool developed by Vos et al. (1995), used in genetics
research and fingerprinting
 It is a multi-locus technique resembling RAPD, and is highly popular.
 The technique is based on PCR amplification of restriction fragments
generated by specific restriction enzymes and oligonucleotide adapters of few
nucleotide bases
 The amplified products are radioactively labeled and separated on
polyacrylamide gel. A subset of the restriction fragments is then selected to
be amplified
 Their main strengths include high specificity and reproducibility owing to the
restriction digestion of DNA, selective neutrality, specific adaptors and high
annealing temperatures for selective amplification and no prior sequence
information or probe (Zenger et al., 2006; reviewed in Sabir et al., 2014)
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44. Basic principles
 AFLP technique is based on the principle of selectively amplifying a
subset of restriction fragments from a complex mixture of DNA fragments
obtained after digestion of genomic DNA with restriction endonucleases
Isolation of AFLP
 Genomic DNA isolation and amplification procedures follow similar
protocol with RFLP
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45. Advantages of AFLPs:
 It is labor-intensive, time consuming limitations of RFLP method and solves
the reliability problem caused by non-specific amplifications in RAPDs
 Like RAPD, AFLP also does not require any prior knowledge of the
sequence and it detects the greater number of loci than RAPD does.
 AFLPs are notable for their genetic stability,
 They provides an effective, rapid, reproducible and economical tool for
detecting a large number of polymorphic genetic markers that can be
genotyped automatically (Vos and Kuiper, 1997)
Disadvantage of AFLPs
 They are dominant bi-allelic markers (Paglia and Morgante, 1998),
 Expensive and technically demanding
 Since a dominant markers Presence of a band could mean the
individual is either homozygous or heterozygous for the sequence, but it
is difficult to distinguish between the two
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46. 2.2.4. Single-strand conformation
polymorphism (SSCP)
 SSCP technique was employed to screen individual PCR products for variation
 Most researchers use SSCP to reduce the amount of sequencing necessary to
detect new alleles at loci of interest or to better estimate allele frequencies of
populations
 SSCP may be used to screen PCR products for genes intended to be sequenced
for phylogenetic analysis
 This SSCP screening allows researchers to determine if the gene in question contains
sufficient polymorphism, which portion of the gene is most polymorphic, what level
of intra-specific variation exists, and if there is a polymorphism among multi copy
genes within individuals.
 SSCP is an efficient technique for obtaining information about levels of
polymorphism within anonymous nuclear loci as compared to the restriction
enzyme protocol originally used (Sabiret al., 2014)
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47. Principles:
 SSCP analysis is based on the fact that single-stranded DNA has
a defined conformation
 Altered conformation due to a single base change in the
sequence can cause single-stranded DNA to migrate differently
under non denaturing electrophoresis conditions
Isolation:
 SSCP analysis of PCR products is a genetic screening technique for
rapid detection of nucleotide substitutions in PCR-amplified genomic
DNA or cDNA
 The method was applied to the analysis of Bovine Viral Diarrhea
Virus (BVDV) isolates
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48. Advantages
 PCR amplifications for SSCP are normally smaller and easy to manipulate,
as the amplifications are only 200 - 300 bp, the entire sequence needed for
PCR primer design can be obtained from a single sequencing reaction
 Simply running the amplifications on 2 SSCP gels (e.g., one with glycerol
and one without glycerol), rather than doing 50 restriction digests and
assuming that the restriction sites are independent (a problematic
assumption when dealing with 1 kb amplifications)
 When polymorphisms detected, the amplifications are of optimal size are
sequenced.Thus, getting sequence level information is easy (Teneva, 2009;
Sabiret al., 2014)
 SSCP is widely applied as a diagnostic tool in molecular biology. It is used
in genotyping to detect homozygous individuals of different allelic states, and
with heterozygous individuals that should each demonstrate distinct patterns
in an electrophoresis experiment
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49. Disadvantages:
 The amounts of mobility differences have little, if any, correlation to the
amount of sequence differences.
 The only information gained from SSCP study was-if PCR amplification
is “identical” or “different”.
 The optimal amplification size for detection of most point
mutations is rather small, i.e., around 200 bp (Sabir et al., 2014)
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50. 2.2.5. Microsatellites (SSR)
 The term microsatellite was invented by (Litt and Luty 1989; Cited in
Yang et al., 2013) and to characterize the simple sequence repeats
 Microsatellites, Short Tandem Repeats (STR) or Simple Sequence
Repeats (SSR)]:- The DNA sequences of 1 to 6 nucleotide length
tandem repeats and widely distributed throughout the genome
 The change in the number of repeats caused by unequal crossing over
between homologous tandem repeats or due to gain or loss of repeat
units at a particular locus represents a variety of alleles with different
number of tandem repeats
 Therefore, in the population these loci have variable number tandem
repeats (VNTR) and hence also called as VNTR loci
 Microsatellite loci are amplified using PCR with specific primers and the
different alleles observed are separated using electrophoretic analysis
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51. Advantage
 SSR markers tend to be highly polymorphic
 The genotyping throughput is high
 This is a simple PCR assay. Many SSR markers are multi-allelic and highly
polymorphic
 Most SSRs are co-dominant and locus specific
 No special equipment is needed for performing SSRs assays; however, special
equipment is needed for some assay methods,
 Start-up costs are low for manual assay methods (once the markers are
developed). SSR assays can be performed using very small DNA samples (~100
ng per individual). SSR markers are easily shared between laboratories (Yang et
al., 2013)
Disadvantages:
 The development of SSRs is labor intensive
 SSR marker development costs are very high
 Start-up costs are high for automated SSR assay methods
 Developing PCR multiplexes is difficult and expensive
 Some markers may not multiplex
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52. 2.2..6. Single-nucleotide polymorphism (SNP):
 SNP is a substitution of a single nucleotide that occurs at a specific
position in the genome, where each variation is present at a level of more
than 1% in the population
 In human beings, 99.9 percent bases are same
 Remaining 0.1 percent makes a person unique
– Different attributes /characteristics / traits
• how a person looks,
 These variations can be:
– Harmless (change in phenotype)
– Harmful (diabetes, cancer, heart disease, Huntington's disease, and
hemophilia )
– Latent (variations found in coding and regulatory regions, are not
harmful on their own, and the change in each gene only becomes
apparent under certain conditions e.g. susceptibility to lung cancer)
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53. SNPs are found in
– coding and (mostly) non coding regions
Occur with a very high frequency
– about 1 in 1000 bases to 1 in 100 to 300 bases
The abundance of SNPs and the ease with which
they can be measured make these genetic variations significant
SNPs close to particular gene acts as a marker for that gene.
SNPs in coding regions may alter the protein structure made by that coding
region
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54. Principles:
 The basic principles of SNP array are the same as the DNA microarray.
 These are the convergence of DNA hybridization, fluorescence microscopy and solid
surface DNA capture.
 The three mandatory components of the SNP arrays are:
(1) an array containing immobilized allele-specific oligonucleotide (ASO) probes
(2) Fragmented nucleic acid sequences of target, labelled with fluorescent dyes
(3) A detection system that records and interprets the hybridization signal (Laframboise,
2009)
 The allele-specific oligonucleotide (ASO) probes are often chosen based on sequencing
of a representative panel of individuals:
 Positions found to vary in the panel at a specified frequency are used as the basis for probes
 SNP chips are generally described by the number of SNP positions they assay. Two probes
must be used for each SNP position to detect both alleles; if only one probe is used,
experimental failure would be indistinguishable from homozygosity of the non-probed allele
(Rapley and Harbron, 2004).
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55. Isolation:
 locus-specific amplification (LSA) and comparative sequencing from
multiple individuals,
 expressed sequence tags (ESTs) sequencing,
 whole-genome shotgun sequencing and, more recently, reduced
representation shotgun sequencing (RRS).
 Finally, high-density oligonucleotide arrays have been shown to be useful
for efficient SNP discovery and screening in humans. Unfortunately,
 All these procedures are currently too expensive and too time consuming,
particularly for small- to medium-scale DNA sequencing laboratories (Jean-
Claude and Carlo, 2003)
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56. Advantages-
 SNP markers are useful in gene mapping
 SNPs help in detection of mutations at molecular level.
 SNP markers are useful in positional cloning of a mutant locus.
 SNP markers are useful in detection of disease causing genes (John et al.,
2007)
Disadvantages:
 Most of the SNPs are bialleleic and less informative than SSRs
 Multiplexing is not possible for all loci
 Some SNP assay techniques are costly
 Development of SNP markers is labour oriented
 More (three times) SNPs are required in preparing genetic maps than SSR
markers
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57. 3. Molecular Marker wise characterization in
Livestock Species and its challenges
 Progress in sequencing techniques and the opportunities offered by the
development of high-density marker arrays have considerably improved the
availability of DNA information over the last ten years, both in terms of
number of markers and in cost of genotyping (FAO, 2015)
 Several researches have been conducted concerning genetic
characterization of various livestock populations//breeds using molecular
markers in developed and developing countries
 Molecular characterization of animal genetic resources though efficient in
developed countries
 Its applications in developing countries are hindered due to mainly
shortage of well-trained personnel, inadequate high-throughput capacity,
poor phenotyping infrastructure, lack of information systems or adapted
analysis tools, or simply resource-limited breeding programs
 The magnitude of these challenges is exacerbated where there is an
imperative to breed for biotic (pests and diseases) and a biotic stresses
(drought, heat, cold and salinity), making accurate phenotyping challenging
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58.  FAO estimates that there more than 6,300 breeds of livestock in the world
belonging to 30 domesticated species (FAO,2015)
 These breeds were developed following domestication and natural and human
selection over the past 12,000 years
 Different livestock populations have evolved unique characteristics for
adaptation to their production systems and agro-ecological environments.
 Their genetic diversity has provided the material for the very successful breeding
programmes in the 19 and 20th century
 These livestock breeds represent a unique resource to respond to the present
and future needs of livestock production, both in developed and developing
countries
 However, livestock diversity is shrinking rapidly
 Loss of biological diversity is a serious problem or potential problem in many
places of the world
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59. 3.1.Measurement of genetic
variation
 Genetic variation can be measured at various levels (i.e. morphological,
protein and DNA),
 Molecular genetic characterization explores polymorphism in selected
protein molecules and DNA markers in order to measure genetic
variation at the population level
 Protein polymorphisms were the first molecular markers used in genetic
characterization of livestock breeds (1960s - 1970s)
 This involved the characterization of blood group and allozyme systems
(allelic variants of enzymes encoded by structural genes ) of livestock
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60.  However, the level of polymorphisms observed in proteins is low, this has
reduced the applicability of protein typing in genetic diversity studies
 Furthermore, allozymes are phenotypic markers, hence, they may be
affected by environmental conditions
 DNA-level polymorphisms are the markers of choice for molecular genetic
characterization (FAO, 2015)
 Because they are based on differences in the DNA sequence, DNA markers
are not environmentally influenced, which means that the same banding
profiles can be expected at all times for the same genotype.
 A variety of DNA markers are used for the study of genetic variation at the
DNA level. The most widely used are (RFLP, SSRs, RAPD, AFLP, SNPs)
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61.  These markers provide the means to examine directly nucleotide
sequence differences in the DNA and to determine the amount of
genetic variation present in the populations
 Variation at DNA level is identified by gel electrophoresis
 Among the DNA markers, microsatellites are very useful for the study of
genetic variation within and between closely related populations such
as livestock breeds or strains.
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62. 3.2. Measuring genetic diversity within apopulation
 Within population diversity is measured by allelic diversity, which is the
average number of alleles at a locus across all loci analysed
 Another measure of within population genetic diversity is observed
heterozygosity, which is the total number of heterozygotes divided by the
total number of animals that have been analysed
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63. 3.3. Measuring genetic diversity between
populations
 Genetic variation between populations (species, breeds or strains)
is measured by assessing the genetic uniqueness of the breeds.
 To know the uniqueness of a breed or strain, one must study the
genetic variation in a set of breeds
 Genetic uniqueness of breeds or strains is measured by the relative
genetic distances of such breeds or strains from each other
(AFAO,2015)
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64. 4. Polymorphisms
 Genetic polymorphism is the existence of at least two variants with
respect to gene sequences, chromosome structure, or a phenotype (gene
sequences and chromosomal variants are seen at the frequency of 1% or
higher), typical of a polymorphism, rather than the focus being on rare
variants (Daly, 2010).
 DNA polymorphisms are the different DNA sequences among individuals,
groups, or populations
 Polymorphism at the DNA level includes a wide range of variations from
single base pair change to many base pairs, and repeated sequences.
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65.  Genomic variability at DNA level can be present in many forms including:
single nucleotide polymorphisms, variable number of tandem repeats
(e.g., mini- and microsatellites), transposable elements (e.g., Alu repeats),
structural alterations, and copy number variations. It can occur in the
nucleus or mitochondria
 There are two major sources: (1) mutations that may result as chance
processes or have been induced by external agents such as radiation and
(2) recombination during meiosis. Once formed, it can be inherited from
parent to child (Ismail and Essawi, 2012)
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66. 5. Polymorphic Information Content
(PIC)
 For constructing gene map, the polymorphism information content (PIC)
developed by Botstein et al. (1980) is the one most often used for linkage
analysis
 The PIC represents the probability that a given offspring of a random mating
between a carrier of a rare dominant gene and a non carrier is informative for
linkage between the locus of the dominant gene and a codominant marker
 If grandparents are not examined for the first child, the PIC is the probability of
establishing linkage phase. Once phase is established, it is the probability that an
offspring is informative for accepting or rejecting linkage
PIC = 1 - S2- S2
2 + S4. However, this formula has been developed for the co-
dominant system; a new formula may be desirable in order that could assess the
effect of recessiveness on PIC in genetic marker
PIC = (1 - 2r + 2r2+ r3) S1 _ (1 - r) 2 S2' + rS3' - (S2')2 + S4'
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67. 6. Molecular characterization of livestock species in
Ethiopia, their objectives and work done so far
Cattle:
 There have been some attempts to characterize populations at the genome level (Taye
et al., 2018; Edea et al., 2017; Zerabruk et al., 2011)
 African cattle are believed to have developed a wide range of adaptations to tropical
environments
 Hailu et al. (2008), evaluated the genetic diversity, population structure and degree of
admixture of 10 Ethiopian cattle populations using 30 microsatellite markers
 The main target was to find out if the current uncontrolled mating practices resulted a
high risk of becoming genetically homogeneous.
 The study revealed that the various levels of admixture and high genetic diversity make
Ethiopian cattle populations suitable for future genetic improvement, and utilization under
a wide range of agro-ecologies in Ethiopia.
 The genetic variability and extent of population substructures in five indigenous cattle
breeds of North-Western Ethiopia were also studied using 22 microsatellite markers
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68.  Controlling gene flow between breeds by adopting effective breeding and
management practices to maintain variability and overcome within-breed
substructures is suggested to facilitate the conservation and utilization of
each breed (Zewdu et.al., 2010)
 Zerabruk et al. (2011), considered microsatellite variation to determine
genetic diversity, population structure and admixture of seven North Ethiopian
cattle breeds by combining multiple microsatellite data sets from other cattle
populations abroad (Cited in Dessie et al.2019)
 Overall, North Ethiopian cattle showed a high level of within‐population
genetic variation and indicated their potential for future breeding applications
 Taye et al. (2017) identified 583 candidate genes involved in 41 biological
processes and pathways which include heat shock response, feed intake
and metabolism, and reproduction functions in African cattle
populations whole genom scan
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69.  Results of the analysis identified important putative genes and gene
regions that are involved in different biological processes and pathways
associated with different tropical environment adaptation traits including
thermo tolerance, disease and parasite resistance and feed
utilization
 Meseret et al. (2020) conducted genetic diversity and population
structure of six Ethiopian cattle breeds from different geographical
regions using high density single nucleotide polymorphisms
 A genome-wide SS analysis revealed 816 loci (p < 0.01) associated to
candidate genes to adaptation (Edea et al., 2014)
 The candidate genes are involved in metabolism, hypoxia response
and heat stress
 Noyes et al. (2011) detected ARHGAP15 gene for trypanotolerance in
Sheko cattle.
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70. Camel
 Camel is an important animal in arid areas of the country
 The pressure due to shortages of feed causing movement
 DNA sequences from mitochondrial cytochrome-b gene and
genotyping of 6 nuclear microsatellite loci were examined
to assess genetic diversity and phylogenetic relationship of
Ethiopian camels (Yoseph et al., 2018)
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71. Donkeys
 Genetic diversity and matrilineal genetic signature of native Ethiopian
donkeys (Equus asinus) inferred from mitochondrial DNA (mtDNA) sequence
polymorphism was conducted (Kefena et al., 2014).
 In the study, mtDNA sequence polymorphisms of six morphologically
diverse domestic donkeys (Equus asinus) populations in Ethiopia was
investigated
 The result suggested that Ethiopia could be one of the centers of diversities
for domestic donkeys in the Horn of Africa.
 The present study also overrides some previous reports that claimed
donkeys were solely an Egyptian domesticate
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72. Goats
 The genetic diversity within and among 11 indigenous Ethiopian goat
populations/types was investigated using microsatellite markers
(Tesfaye, 2004)
 Solomon (2014) studied the molecular genetic diversity and homozygous
segments of two goat breeds of Ethiopia using 47K genome-wide SNPs
markers to understand the within and between breed diversity for future
breed improvement and conservation planning
 Getnet et al. (2017) also used mtDNA markers to characterize the genetic
diversity and population structure of Ethiopian goats
 Another study by Getnet et al. (2017), identified genetic variants associated
with fecundity traits in some Ethiopian goat populations, and this could be
used in Marker Assisted Selection
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73. Sheep
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 The genetic and morphological diversity and population structure of 14
traditional sheep populations originating from four ecological zones in
Ethiopia (sub‐alpine, wet highland, sub‐humid lowland, and arid lowland)
were studied by Gizaw et al., (2007)
 The study showed a strong indication of adaptive divergence in
morphological characters, patterns of morphological variation being highly
associated with ecology
 The genetic diversity and population structure of Ethiopian sheep
populations were characterized using high-density SNP markers to reveal
their genetic diversity for improving breeding strategies and mapping
quantitative trait loci associated with productivity (Zewdu et al. 2017).
 The high-density SNP data generated in the study can be used to identify
genes and pathways relevant for physiological adaptation to extreme
environments and variation in phenotypic traits (Zewdu et al. 2017)

74. Chickens
 Genetic improvement of indigenous chicken exercises in Ethiopia, as in
other developing countries, have been towards the use of exotic chicken
strains to improve the local chicken.
 Extensive crossbreeding has been common over the 5 decades of poultry
research in Ethiopia. An exception is a single selective breeding program in
indigenous chicken in Ethiopia with the application of quantitative genetics
approaches
 The earlier research on the genetic characterization was by Tadelle (2003)
who have characterized five chicken ecotypes of Ethiopia using
microsatellite markers. The result has led to the discovery of some unique
alleles that are believed to be involved in production traits.
 Later, Halima (2007) has characterized some indigenous ecotypes from
Northwestern parts of Ethiopia using microsatellite markers to reveal the
between- and within-population genetic variations
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75.  A genome-wide association study (GWAS) conducted by Psifidi et al
(2016) using single nucleotide polymorphism (SNP) markers to reveal
the association of markers with phenotypic traits.
 SNPs significantly associated with immune system, disease resistance, and
production traits in indigenous village chickens were identified.
 Adebabay (2019), studied the genetic diversity and population
structure of Ethiopian chicken strains.
 The study further investigated the signature of artificial selection with a
whole-genome analysis that showed positive genetic selection
through a short-term selective breeding program (Cited by Dessie et
al. 2019)
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76. 7. Suggested Solutions and Recommendations to
Overcome the Problems
• The tools of molecular genetics are likely to have considerable impact in the
future.
• For example, DNA-based tests for genes or markers affecting traits that are
difficult to measure currently, such as meat quality and disease resistance, will
be particularly useful (Leakey 2009).
• Genomic selection should be able to at least double the rate of genetic gain in
the dairy industry (Heins et al, 2009), as it enables selection decisions to be
based on genomic breeding values, which can ultimately be calculated from
genetic marker information alone, rather than from pedigree and phenotypic
information
• Key factors hampering the use of molecular technologies in most developing
countries include- poor infrastructure; inadequate capacity and operational
support; and lack of an enabling policy, statutory and regulatory framework at
country level, which in turn affects research institution
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77.  Advanced molecular techniques being used to some extent in various species,
mainly focusing on the characterization of genetic diversity and population
structure (Dessie et al. 2019)
 They all are academic efforts and not supported by phenotypic information
 Conventional breeding approaches which would have provided an opportunity
to make use of the pieces of knowledge and information generated from the
molecular technique exercises seemed to be given less attention.
 Very poor commitment from researchers is also hampering the application of
the knowledge in the design of the breeding programs. Researchers were not
committed enough to further peruse the avenue of breeding programs that
are to be set up in villages
 Among the molecular technique studies, some of the efforts help in better
understanding of the domestication but practically less important
 Breeding programs to improve their traction ability might be more relevant but
needs to be based on farmers interests.
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78.  Ethiopia has faced with the challenge to rapidly increase agricultural
productivity to help feed its growing population without depleting the natural
resource base.
 Marker identification will help to enhance selection of superior genotypes for
breeding to improve important traits as tolerance to diseases and resistance
to environmental stresses.
 Thus, developing nations, like Ethiopia, must take the advantage of utilizing
molecular biology for better advancement of livestock productivity.
 To this end, the following recommendations are suggested to overcome the
challenges on effective utilization of molecular technology:
- Capacity-building
- Communication and public awareness
- The Role of the Public and Private Sector
- Livestock Related Policy and Regulations
- Regulatory Systems and Safety
- Intellectual Property Management
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79. Than you!
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