Molecular markers have revolutionized the field of genetics and genomics by providing valuable tools for studying genetic diversity, identifying individuals, and characterizing traits of interest. This review paper aims to explore the applications of molecular markers in breed improvement and conservation. We discuss the various types of molecular markers commonly used, such as microsatellites, single nucleotide polymorphisms (SNPs), amplified fragment length polymorphisms (AFLPs), and many more. Additionally, we examine their applications in genetic diversity assessment, parentage analysis, marker-assisted selection (MAS), and conservation efforts. The paper highlights the importance of molecular markers in accelerating breed improvement programs and enhancing conservation strategies for maintaining genetic diversity within a population.Molecular markers have had a significant impact on breed development and conservation efforts, transforming genetics and offering vital insights into genetic diversity, lineage tracing, and genotype characterization. The importance of molecular markers in improving genetic gains, facilitating breeding programs, and preserving genetic diversity for the long-term sustainability of the animal population has been underlined in this review paper. Emerging advancements in molecular marker technology show enormous potential for improving and conserving breeds. Deeper insights into the genetic basis of complex traits will be provided through GWAS, CRISPR/Cas9, gene editing technologies, and sequencing technologies, resulting in faster genetic gains. Breeders and conservationists will be able to make more informed judgments thanks to these technologies. In conclusion, molecular markers have had a significant impact on breed conservation and enhancement. Their innovations have changed the industry and given both conservationists and breeders vital knowledge. We can pave the road for more effective and sustainable genetic improvement and the preservation of biodiversity for future generations by combining the power of molecular markers with conventional breeding and conservation techniques.

1. Molecular Markers and Their Application in Animal Breed
Improvement and Conservation: A Review
Dr. Trilok Mandala
a
Agriculture and Forestry University (AFU) Rampur, Chitwan, Nepal
c
Faculty of Animal Science, Veterinary Science, and Fisheries (FAVF)
b
Department of Animal Breeding and Biotechnology
E-mail address:trilokmandal97@gmail.com Phone No: 9819979467
Target Audience: Breeders, Government policymakers, academics
Abstract
Molecular markers have revolutionized the field of genetics and genomics by providing valuable tools
for studying genetic diversity, identifying individuals, and characterizing traits of interest. This review
paper aims to explore the applications of molecular markers in breed improvement and conservation.
We discuss the various types of molecular markers commonly used, such as microsatellites, single
nucleotide polymorphisms (SNPs), amplified fragment length polymorphisms (AFLPs), and many more.
Additionally, we examine their applications in genetic diversity assessment, parentage analysis,
marker-assisted selection (MAS), and conservation efforts. The paper highlights the importance of
molecular markers in accelerating breed improvement programs and enhancing conservation
strategies for maintaining genetic diversity within a population.
Keywords: Molecular Markers, Microsatellite, Molecular Markers, Genome, Polymorphism
Introduction:
Nepal has 17 domestic livestock species, including diverse bovids, birds, equines, pigs, rabbits, and
elephants. The most frequent quadrupeds are cattle, goats, buffaloes, and sheep, while the most common
poultry species are domestic fowl, pigeons, and ducks(Wilson, 1997). However, very less effort is seen
here in Nepal regarding genetic diversity assessment, breed identification and characterization, and
conservation of endangered species. In the 1990s, the primary focus of animal breeding activities shifted
from quantitative to molecular genetics. Historically, the primary focus of the conventional breeding
system, which includes progeny testing and numerous selection procedures, has been selective
breeding. To maximize the animal breeding program, molecular genetic approaches must be combined
with traditional animal breeding procedures. Over the last three decades, advances in DNA technologies
have enabled the discovery of a large number of genetic polymorphisms at the DNA sequence level and
their use as markers for evaluating the genetic basis for observed phenotypic variability, which plays
an important role in animal breeding(Athe et al., 2018). A molecular marker is a unique and detectable
DNA sequence or genetic variation used to track or identify a specific feature or gene of interest inside
an organism's genome. It acts as a genetic landmark, guiding researchers and scientists to certain genes
or sections of the genome linked to specific features, diseases, or situations. A genetic marker could be
a long DNA sequence like mini and microsatellites, or it could be a short DNA sequence like the
sequence surrounding a single base-pair change (single nucleotide polymorphism, or SNP)(Al-samarai
& Al-kazaz, 2015). Marker-Assisted Selection (MAS) is the most often employed marker technique in
breeding. The use of marker-based information for animal genetic improvement depends on the

2. selection of the right marker. In the future, molecular markers may be used as a potential tool for the
assessment and modification of current germplasm to choose and develop desired traits in animals,
resulting in the genetic improvement of those species(Reshma & Das, 2021).
Various types of molecular markers used in animal breed improvement and conservation
Hybridization-based and PCR-based markers, DNA chips, and sequencing-based DNA markers have
been classified into three categories based on the methods used to identify molecular markers.
Hybridization based markers
RFLP (Restriction Fragment Length Polymorphism)
In hybridization-based markers, DNA digested by restriction enzymes is hybridized to a labeled probe,
which can be a DNA fragment with a known origin or sequence, to visualize DNA profiles. The first
hybridization-based marker technique was restriction fragment length polymorphism(R. R. Dhutmal
2018). Later, numerous marker probes were created as hybridization-based markers, including
microsatellites, minisatellites, and STS. With the development of easier cloning and sequencing
techniques and the availability of sequence databases, these markers have been converted into PCR,
which is easy to assay. Botstein and his team created the RFLP technology for the first time in 1980, to
observe the variations at the level of DNA structure (Mburu & Hanotte, 2005). Procedure for RFLP (1)
Individuals A and B's DNA should be extracted. (2) To cut DNA, use restriction enzymes. (3) DNA
fragments are separated by size using an agarose gel electrophoresis. (4) By using a Southern blot,
transfer the DNA from the gel to a nylon membrane. (5) To hybridize the DNA, use radioactively
labeled DNA fragments as probes. (6) Wash the nylon membrane to remove non-specifically bound or
unbound probes. (7) Place the cleansed membrane on an X-ray film. (8) X-ray film should be developed
to detect DNA polymorphism(Mishra et al., 2019). The advantages of RFLPs are that they show
codominant alleles and have good repeatability. However, they have some disadvantages time-
consuming, labor-intensive, and inconvenient for high throughput screening(Mburu & Hanotte, 2005).
PCR-based markers
RAPD (Random Amplified Polymorphic DNA)
The discovery of the polymerase chain reaction (PCR) technique resulted in the development of a new
generation of PCR-based DNA markers(B. D. Singh & Singh, 2015). The polymerase chain reaction
(PCR)-based RAPD approach has been one of the most widely utilized molecular techniques for
developing DNA markers. The basic RAPD technique requires (i) the extraction of highly pure DNA,
(ii) the addition of a single arbitrary primer, (iii) polymerase chain reaction (PCR), (iv) fragment
separation by gel electrophoresis, (v) visualization of RAPD-PCR fragments after ethidium bromide
staining under UV light, and (vi) fragment size determination using gel analysis software(Arif et al.,
2010). RAPDs are seen as having various advantages over RFLP because of their technological
simplicity and independence from any preexisting DNA sequence information(Al-samarai & Al-kazaz,
2015). The fact that RAPD markers only identify polymorphisms as the presence or absence of a band
with a specific molecular weight and without providing information on heterozygosity is a drawback of
these markers(Brumlop & Finckh, 2011).
AFLP (Amplified Fragment Length Polymorphism)
AFLP is a PCR-based method for creating and comparing unique fingerprints for genomes of interest
by selective amplification of digested DNA segments(Mir et al., 2023). The method requires three steps:

3. (1) DNA restriction and oligonucleotide adapter ligation, (2) selective amplification of sets of restriction
fragments, and (3) gel analysis of the amplified fragments(Vos et al., 1995). The strength of this
approach originates primarily from the fact that no prior information about the targeted genome is
required, as well as its high repeatability and sensitivity for detecting variation at the DNA sequence
level(Mueller & Wolfenbarger, 1999).
Minisatellites and Microsatellites
Mini and microsatellites are some of the most potent genetic markers currently available. They have
served as instruments for numerous tasks, including gene mapping, phylogenetic research, and isolate
typing. However, it might be time-consuming to find micro- and minisatellite markers in huge sequence
data sets(Bally et al., 2010). While the faithful transmission of genetic information necessitates the
fidelity and stability of the DNA involved in protein translation, it has become clear that a considerable
portion of noncoding DNA is arranged in repetitive sequences, which frequently exhibit pronounced
instability and dynamics. This holds true for both minisatellites (between 10-100 base pairs) and
microsatellites (between 2-4 base pairs), which are longer repeated sequences(Ramel, 1997). Short
tandem repeats (STRs) or simple sequence repeats (SSR) are other names for microsatellites and
variable tandem repeats (VNTRs) are other names for microsatellites.
DNA chip and sequencing-based DNA marker
A DNA chip, sometimes referred to as a DNA microarray or gene chip is a highly efficient tool for
high-throughput genotyping, comparative genomic hybridization, and gene expression studies. It is
made up of a solid surface, such as a glass slide or a silicon wafer, on which thousands of DNA
fragments or oligonucleotide probes are fixed in an exact array(Schena et al., 1995). Next-generation
sequencing (NGS) technologies are used to identify and describe genetic variants within DNA samples
in sequencing-based DNA markers. Researchers can gather nucleotide-level information regarding
variants such as SNPs, indels, and SSRs by sequencing specific genomic areas(Deschamps et al., 2012).
Both DNA chips and sequencing-based DNA markers provide useful information on genetic variants,
but their approaches differ. The binding of tagged target DNA to specific probes on a solid surface is
detected and quantified by DNA chips, whereas sequencing-based DNA markers employ the direct
sequencing of genomic areas to discover genetic variants(Wang, 2000).
Marker-assisted selection (MAS)
One of the most important strategies for enhancing an animal's performance is selection. Pedigrees and
phenotypes can be used to estimate the Best Linear Unbiased Prediction (BLUP), which integrates these
two forms of data to produce estimated breeding values (EBVs)(Al-samarai & Al-kazaz, 2015). The
first step in improving traits in a population is trait identification. Genetic indicators linked to the desired
qualities must be found, validated, and used for selection. To forecast genetic potential and make
breeding decisions, marker-assisted selection is used. Breeding programs are used to gradually raise the
frequency of desirable alleles in a population. To refine and update genetic prediction models,
genotyping and data analysis are used. Many qualities are controlled by several genetic loci, each of
which contributes to trait variation and is thus referred to as a quantitative trait locus (QTL). While
genetic markers for QTL linked to the trait gene could be used to choose animals for selective breeding
programs, functional mutations within the trait genes are the most effective indicators. The strategies
for identifying trait markers and their use are presented by reference to instances of loci that control a
range of distinct qualities(Williams, 2005).
Parentage Analysis:
Understanding the structure of DNA and using microsatellite markers to determine parentage are two
aspects of DNA-based parentage analysis. Microsatellites are small, repetitive DNA sequences that vary

4. greatly between individuals. They act as genetic markers, allowing animals to be distinguished and
familial links to be established (Parentage, 2019). Parentage analysis is classified into three types:
1. Identifying the father in the absence of the mother.
2. When the mother is known, identify the father.
3. Identifying both the father and mother at the same time(Huang et al., 2018).
By accurately determining parentage through DNA analysis, animal breeders can make informed
decisions about breeding strategies, maintain pedigree records, and prevent inbreeding. Parentage
analysis helps ensure the genetic diversity and integrity of animal populations, contributing to the
improvement of desirable traits and overall breeding programs.
Application of Molecular Markers in Breed Improvement
Enhancing genetic gain
Breed improvement methods have been transformed by the use of molecular markers, particularly in
conjunction with contemporary breeding and biotechnological platforms. Genetic gain has been greatly
enhanced by the use of marker-based selection techniques such as Marker-Assisted Recurrent Selection
(MARS), Marker-Assisted Selection (MAS), Marker-Assisted Backcrossing (MABC), and Genomic
Selection(M. Singh et al., 2022). Breeders can discover and choose individuals carrying desired features
early on by utilizing molecular markers, which saves time and resources compared to conventional
phenotypic selection. By quickening the breeding process, improved varieties can be developed and
released more quickly. Molecular markers are used to identify features that may not be obvious or easily
measurable by phenotypic observation alone, which improves the accuracy of selection(Bohar et al.,
2020).
Improving Disease Resistance:
Disease resistance is an important characteristic in breeding programs since it directly influences
livestock health and productivity, as well. Molecular markers can aid in the identification of genes or
genomic areas linked to disease resistance. Breeders can selectively produce animals that are more
resistant to specific diseases by genotyping individuals for these markers. This targeted selection aids
in the development of disease-resistant breeds or variations, lowering illness effects and reducing the
need for costly treatments or interventions(Islam et al., 2020).
Enhancing Reproductive Performance:
Molecular markers are genetic polymorphisms that can be detected and examined to better understand
the genetic makeup of an animal and predict its performance. Breeders can choose which animals to
use as breeders with more knowledge thanks to the identification of certain genetic markers linked to
reproductive features, improving reproductive success in succeeding generations(Martínez et al., 2021).
Application of Molecular Markers in Breed Conservation
There has been an irreparable loss of genetic diversity among our local animal breeds as a result of the
uncontrolled crossbreeding of exotic animals with indigenous breeds to exploit heterosis. The
preservation of genetic diversity is crucial because it promotes a high level of heterozygosity in the
population(Gholizadeh et al., 2008). The use of molecular markers in breed conservation refers to
assessing and managing genetic variability within and between populations of a certain breed.
Molecular markers can be used to identify distinct conservation units to preserve unique genetic
characteristics and prevent the loss of genetic diversity(Li et al., 2020). Molecular markers aid in the
determination of conservation priorities among breeds. Conservationists can discover breeds with lesser
genetic diversity and thus higher conservation priority by comparing genetic diversity among different

5. breeds(Toro et al., 2009). RFLP, RAPD, AFLP, microsatellites, and minisatellites are the most often
used molecular tools for studying genetic changes at the DNA level(Gwakisa, 2002).
Advantages of Molecular Markers
High Genetic Resolution: The detection of genetic variants at the DNA level is made possible by the
high level of genetic resolution offered by molecular markers. This makes it possible for researchers to
precisely estimate genetic diversity and locate variations linked to particular traits or
illnesses(Gupta,2014).
Cost-Effectiveness: Molecular marker-based breeding approaches may be less expensive than
conventional breeding techniques. When markers for specific traits are found, they can be utilized in
breeding programs for marker-assisted selection (MAS), which saves time and money compared to
phenotypic selection(Bernardo, 2016).
Rapid and High-Throughput Analysis: Molecular markers enable rapid and high-throughput genetic
information analysis. Polymerase chain reaction (PCR) and next-generation sequencing (NGS)
techniques allow for the simultaneous investigation of many markers across vast populations, allowing
for the rapid screening and characterization of genetic resources(Mammadov et al., 2012).
Preservation of Genetic Diversity: By spotting uncommon and distinct alleles within populations,
molecular markers help to preserve genetic diversity. This knowledge can direct conservation efforts
and aid in preserving the genetic integrity of populations or species that are threatened with
extinction(Allendorf et al., 2010).
Challenges and Limitations
There are several forms of molecular markers, including hybridization-based markers (RFLP), PCR-
based markers (RAPD, AFLP, Microsatellites), and DNA chip and sequencing-based markers (SNPs).
Each variety has its own set of advantages and disadvantages, necessitating careful selection and
improvement depending on specific aims and species of interest and insufficient marker density can
lead to inaccurate trait prediction and population genetic analyses(Reshma & Das, 2021). It is
challenging to develop marker-trait relationships, which necessitate extensive data collecting, statistical
analysis, and validation across many populations. The generalizability of some marker systems is
constrained by the variability in the transferability of DNA markers across various animal populations
or species(Hasan et al., 2021). For the analysis of molecular markers, it is necessary to use specialized
tools, reagents, and personnel, all of which have substantial setup and running expenses. The gathering
of genetic material for molecular marker research poses ethical concerns in the enhancement and
conservation of animal breeds. To maintain the welfare and conservation of animal populations, proper
ethical norms and regulations must be followed(Li et al., 2020).
Emerging Trends and Future Directions
Molecular markers have revolutionized animal breeding because they offer a strong tool for genetic
study and selection. Genomic selection uses dense marker information to predict the genetic merit of
animals, with the potential for improved accuracy and efficiency(Meuwissen et al., 2001). NGS
technologies have transformed DNA sequencing and made it possible to analyze animal genomes more
quickly, accurately, and affordably. Comprehensive views of genetic variants and functional
components can be obtained using whole-genome sequencing and targeted sequencing techniques. NGS
will keep being a key tool for locating and describing molecular markers linked to complex
characteristics(Shendure & Ji, 2008). High-density genotyping arrays are now able to genotype
thousands to millions of markers throughout the genome because of advancements in genotyping
technology. These arrays make it easier to conduct genome-wide association studies (GWAS) and find

6. new markers that are connected to significant traits. As they develop, high-density genotyping arrays
will give ever more comprehensive marker coverage and resolution(Daetwyler et al., 2008). Gene
expression and phenotypic diversity can be affected by epigenetic changes such as DNA methylation
and histone modifications, or epigenetic markers. Epigenetic markers offer hope for bettering breeding
techniques and understanding the underlying biological mechanisms of complex characteristics.
Epigenetic data can be included in animal breeding systems to increase selection precision and yield
new insights(Skvortsova et al., 2018). Functional Genomics: Combining genomic data with functional
genomics information, such as gene expression levels and protein interactions, can help us better
understand the molecular processes behind complex features. To improve breeding tactics, functional
genomics methods including transcriptomics, proteomics, and metabolomics will continue to be used
in addition to molecular marker studies(Kadarmideen et al., 2006). Gene editing techniques, such as
CRISPR-Cas9, can be used to alter an animal's genome and test the functionality of potential
genes(Pennisi, 2013). Non-coding RNAs are important for gene regulation and genetic manipulation,
providing potential insights for improved breeding programs. These future perspectives and emerging
tools in molecular marker applications demonstrate the ongoing advancements and potential in animal
breeding.
Conclusions:
Molecular markers have had a significant impact on breed development and conservation efforts,
transforming genetics and offering vital insights into genetic diversity, lineage tracing, and genotype
characterization. The importance of molecular markers in improving genetic gains, facilitating breeding
programs, and preserving genetic diversity for the long-term sustainability of the animal population has
been underlined in this review paper. Emerging advancements in molecular marker technology show
enormous potential for improving and conserving breeds. Deeper insights into the genetic basis of
complex traits will be provided through GWAS, CRISPR/Cas9, gene editing technologies, and
sequencing technologies, resulting in faster genetic gains. Breeders and conservationists will be able to
make more informed judgments thanks to these technologies. In conclusion, molecular markers have
had a significant impact on breed conservation and enhancement. Their innovations have changed the
industry and given both conservationists and breeders vital knowledge. We can pave the road for more
effective and sustainable genetic improvement and the preservation of biodiversity for future
generations by combining the power of molecular markers with conventional breeding and conservation
techniques.
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