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
Dr. Trilok Mandal (BVSc & AH)
Department of Animal Breeding and
Genetics (MSc)
Agriculture and Forestry University
(AFU) Rampur, Chitwan, Nepal
Email: trilokmandal97@gmail.com

2. Introduction:
• Molecular marker is identified as genetic marker.
• Molecular marker is a DNA or gene sequence within a recognized location on a chromosome
which is used as identification tool.
• In the pool of unknown DNA or in a whole chromosome, these molecular markers helps in
identification of particular sequence of DNA at particular location.
Quality for a good genetic marker:
• Genetic markers should be largely polymorphic in nature
• They should be selectively neutral
• Assay for detecting markers should be simple and rapid
• Genetic markers should occur frequently within genome
• The genetic marker (gene) should show codominant inheritance pattern
• They should be highly reproducible
• They should not interact with other markers while using multiple markers at a same time

3. Types of molecular markers
Hybridization-based, 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.
A) Hybridization based markers
RFLP (Restriction Fragment Length Polymorphism)
• Botstein and his team created the RFLP technology for the first time in 1980, to observe
variations at the level of DNA structure.
• 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 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.

4. 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

5. B) PCR-based markers
RAPD (Random Amplified Polymorphic DNA)
• The polymerase chain reaction (PCR)-based RAPD approach has been one of the most
widely utilized molecular techniques for developing DNA markers.
• RAPDs are seen as having various advantages over RFLP because of their technological
simplicity and independence from any preexisting DNA sequence information.
• 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.

6. 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

7. AFLP (Amplified Fragment Length Polymorphism)
• AFLP was originally developed by the KeyGene in 1990.
• It is a PCR based technique for fingerprinting. It includes both PCR and RFLP.
• The basis of AFLP is the amplification of selected fragments followed by restriction
digestion of whole genomic DNA of specific organism.
The method requires three steps: (1) DNA restriction and oligonucleotide adapter ligation, (2) selective amplification of
sets of restriction fragments, and (3) gel analysis of the amplified fragments

8. 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.
• Short tandem repeats (STRs) or simple sequence repeats (SSR) are other names for
microsatellites and variable tandem repeats (VNTRs) are other names for minisatellites.

9. Single nucleotide polymorphism (SNP)
•SNP was invented by Lander in 1996.
•SNP is formed when any alteration/mutation occurs in single nucleotide (A, T, C, or G).
•The point mutation as such substitutions, insertions or deletions in single nucleotide it represents
SNP.
•SNPs are based on hybridization of detected DNA fragments with SNP chips (DNA probe arrays)
and the SNP allele is named with respect to the hybridization results.
Applications:
• SNPs are widely used in biomedical research for comparing the case and control groups of
disease.
• It is also used in studying phylogenetics, genetic variation etc.
Demerits:
• The information obtained is low as compared to microsatellites and therefore large
numbers of markers and complete genome sequencing is needed

10. 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.
• 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, and SSRs by sequencing specific genomic areas.
• 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.

11. Marker-assisted selection (MAS)
• Marker-assisted selection (MAS) is a technique used in animal breeding to select individuals
with desirable genetic traits.
• It involves the use of molecular markers, which are specific regions of DNA that are
associated with particular traits or genes of interest.
• By analyzing the presence or absence of these markers, breeders can make more informed
decisions about selecting animals for breeding programs.
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 greatly between individuals.
• They act as genetic markers, allowing animals to be distinguished and familial links to be
established.

12. Parentage analysis is classified into three types:
Identifying the father in the absence of the mother.
When the mother is known, identify the father.
Identifying both the father and mother at the same time.
• 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.

13. Application of Molecular Markers in Breed Improvement
• Enhancing genetic gain
• Improving Disease Resistance
• Enhancing Reproductive Performance
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.
• The use of molecular markers in breed conservation refers to assessing and
managing genetic variability within and between populations of a certain breed.
• RFLP, RAPD, AFLP, microsatellites, and minisatellites are the most often used
molecular tools for studying genetic changes at the DNA level.

14. Advantages of Molecular Markers
• High Genetic Resolution
• Cost-Effectiveness
• Rapid and High-Throughput Analysis
• Preservation of Genetic Diversity
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.
• It is challenging to develop marker-trait relationships, which necessitate extensive data
collecting, statistical analysis, and validation across many populations.
• 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.

15. Emerging Trends and Future Directions
• NGS technologies have transformed DNA sequencing and made it possible to
analyze animal genomes more quickly, accurately, and affordably.
• High-density genotyping arrays are now able to genotype thousands to millions
of markers throughout the genome because of advancements in genotyping
technology.
• Epigenetic markers offer hope for bettering breeding techniques and
understanding the underlying biological mechanisms of complex characteristics.
• To improve breeding tactics, functional genomics methods including
transcriptomics, proteomics, and metabolomics will continue to be used in
addition to molecular marker studies.
• Gene editing techniques, such as CRISPR-Cas9, can be used to alter an animal's
genome and test the functionality of potential genes.

16. 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.
• 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, CRISPR/Cas9, gene editing
technologies, and sequencing technologies, resulting in faster genetic gains.
• 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.

17. THANK YOU