Molecular markers are DNA sequences that can be used to identify specific locations in the genome. They allow detection of differences between individuals. Common types of molecular markers include RFLP, RAPD, AFLP, SSR, and SNP. RFLP uses restriction enzymes and probes but requires a large amount of high quality DNA. RAPD uses PCR with random primers and needs little DNA but has low reproducibility. AFLP combines restriction enzymes and PCR for higher reproducibility. SSR and SNP detect differences in repetitive DNA sequences and single nucleotides, respectively. Molecular markers have various applications including measuring genetic diversity, fingerprinting, marker-assisted selection, and identifying genotypes.

1. WELCOME
ON
PRESENTATION

2. AGRICULTURE
AND FORESTRY
UNIVERSITY
FACULTY OF AGRICULTURE
DEPARTMENT OF GENETICS AND PLANT
BREEDIG

3. PRESENT TO
ASSISTANT PROFFESSAR
Dr. MADHAV PANDEY
DEPARTMENT OF BIOTESHNOLOGY
AFU
PRESENTED BY
SHIV SHANKAR LONIYA
MSC.AG 2nd SEM
ROLL NO:PLB-13M-2022
AN ASSIGNMENT ON MARKER SYSTEM
COURSE TITLE:MOLECULAR MARKER AND
RESISTANCE BREEDING
COURSE CODE :

4. Definition
A molecular marker is a DNA sequence that is readily detected and
whose inheritance can easily be monitored .
What are Molecular Markers?
Specific fragments of DNA that can be identified within the whole
genome.
Molecular markers are the general assays that allow detection of the
sequence differences between two or more individual.
Molecular markers are found at specific locations of the genome.
 They are used to 'flag' the position of a particular gene or the
inheritance of a particular characteristic or desired characteristics

5. Markersystemsaretoolswhich is used to marka trait in living organism
MORPHOLOGICAL MARKER:
Classical markers
MOLECULAR MARKERS:
Variation in macro-molecules
BIOCHEMICAL MARKERS
ISOENZYME
PROTEIN
GENETIC MARKERS
RFLP
AFLP
RAPD
They are protein produced by expression of gene
Depend upon sequence of DNA
•Low polymorphism
•Requires expression of trait / gene
•Dominance effect
•Expression sex limited
•Expressed late in life

6. DNA markers
Non-PCR Based,
 RFLP- Restriction fragment length polymorphism.
PCR Based
RAPD- Random amplification of polymorphic DNA.
AFLP-Amplified fragment length polymorphism.
 SCAR-Sequence characterize amplified region.
STS- Sequence tagged sites.
EST-Express sequence tags.
SNP-Single nucleotide polymorphism.
SSR-Simple sequence repeats
CAPS-Cleaved amplified polymorphic sequences.

7. ⚫ it must be polymorphic.
⚫ Co-dominant inheritance.
⚫ A marker should be evenly and frequently distributed throughout the
genome.
⚫ It should be easy, fast and cheap to detect.
⚫ It should be reproducible.
⚫ High exchange of data between laboratories.
Properties of Ideal Genetic Marker

8. Applications of molecular markers
►MEASURE OF GENETIC DIVERSITY
►FINGER PRINTING
►GENOTYPIC SELECTION
►GENOTYPIC PYRAMIDYING AND
INTROGRESSION
►INDIRECT SELECTION USING QUANTITATIVE
TRAITS LOCI (QTLS)
►MARKER-ASSISTED SELECTION
►IDENTIFICATION OF GENOTYPE

9. Feature RFLP RAPD AFLP SSR or
Microsatellite
DNArequired (µg) 10 0.02 0.5-1.0 0.05
DNAquality High High Moderate Moderate
PCR-based No Yes Yes Yes
No. of polymorphic loci
analysed
1.0-3.0 1.5-50 20-100 1.0-3.0
Ease of use Not easy Easy Easy Easy
Reproductibily High Unreliable High High
Development cost Low Low Moderate High
Cost per analysis High Low Moderate Low
Table 1 : Comparision of the most broadly used techniques
of molecular markers
Cont…..

10. Definition
The variation(s) in the length of DNA fragments produced by a specific
restriction endonuclease from genomic DNA s of two or more
individuals of a species
RFLP

11. Principle
Restriction fragment length polymorphism (RFLP) technology was first developed in
the 1980s for use in human genetic applications and was later applied to plants.
By digesting total DNA with specific restriction enzymes, an unlimited number of
RFLPs can be generated.
RFLPs are relatively small in size and are co-dominant in nature.
If two individuals differ by as little as a single nucleotide in the restriction site, the
restriction enzyme will cut the DNA of one but not the other. Restriction fragments of
different lengths are thus generated.
All RFLP markers are analyzed using a common technique. However, the analysis
requires a relatively complex technique that is time consuming and expensive.

12. The hybridization results can be visualized by
1. Autoradiography (if the probes are radioactively labeled), or
2. Chemiluminesence (if non-radioactive, enzyme-link methods are used for
probe labeling and detection).
Any of the visualization techniques will give the same results. The visualization
techniques used will depend on the laboratory condition
Principle

13. Principle

14. -simple method as no sequence specific information is required
-codomonant markers
-it is not depend on PCR
-it required large amount of highly pure DNA
-it require constant supply of probes
-it is laborious to identify suitable markers
-it is time consuming
-it requires expertise in autoradiography
Advantages
Disadvantages

15. Applications of molecular markers
► MEASURE OFGENETIC DIVERSITY
► FINGER PRINTING
► GENOTYPIC SELECTION
► GENOTYPIC PYRAMIDYING AND INTROGRESSION
► INDIRECT SELECTION USING QUANTITATIVE TRAITS LOCI (QTLS)
► MARKER-ASSISTED SELECTION
► IDENTIFICATION OFGENOTYPE

16. Any DNA segment that is amplified using short oligodeoxynucleotide primer
of arbitrary nucleotide sequence (amplifier) and polymerase chain reaction
procedure (Khal, 2001)
RAPD also known as,
AP-PCR(Arbitrarily primed PCR),
DAF (DNA amplification fingerprinting)&
MAAP(Multiple arbitrary amplicon profiling)
RAPD are a dominant marker system
Definition
RAPD

17. 1. RAPDs are produce by PCR using genomic DNA and arbitrary primers
2. Taq polymerase is used to amplify DNA segment between closely spaced
sequence (< 2kb) and complementary to the short random oligomers (typically
10-mers)
3. RAPD polymorphism result from change in the primer-binding site in the DNA
sequence
Principle

18. In variety A there are 4 primer binding sites resulting in two RAPD products, variety
B lacks one of the binding sites resulting in only one RAPD marker being produced

19. Template
DNA
Primers
direction,
point in the same
so amplification
won’t happen

20. Template
DNA
Primers too far apart, so
amplification won’t happen
> 2,000 bases

21. Template
DNA
Primers are just the
right distance
so fragment
apart,
is
amplified
100 - 1,500 bases

22. -need small amount of DNA
-it involves non-radioactive assay
-it does not required specific probe libraries
-it provide quick and efficient screening for DNA
sequence based on polymorphism at many loci
-it is inherited as dominant traits
-there is a bands due to relatively short primer
-the production of non-parental bands in the offspring of
known pedigree warrants its use with extreme care
-it is sensitive to change in PCR conditions
Advantages
Disadvantages

23. APPLICATION of RAPD
Application of RAPD

24. AFLP
Definition
Any difference between corresponding DNA fragment from two
organisms A & B that is detected by amplified restriction length
polymorphism technique

25. Principle
1. The amplified fragment length polymorphism technique combines components
of RFLP analysis with PCR technology.
2. Total genomic DNA is digested with a pair of restriction enzymes normally a
frequent and rare cutter.
3. Adaptors of known sequence are then ligation to the DNA fragments.
4. Primer complementary to the adaptors are used to amplify the restriction
fragments.
5. The PCR amplified fragments can then separated by gel electrophoresis and
banding patterns visualized.
6. A range of enzymes and primer are available to manipulate the complexity of
AFLP fingerprint to suit application

26. Principle
Genomic DNA
For restriction digestion we use two type of cutter i.e
•Rare cutter (6bp) EcoRI
•Frequent cutter (4bp) MseI
Interstitial cohesive ends
Adaptor ligation
PCR amplification using EcoRI/MseI

27. -extremely sensitive
-it has a wide scale applicability
-it discriminates heterozygotes from
homozygotes when a gel scanner is used
-used for mapping
-it is highly expensive
-it required more DNA than RAPD
-it required experience of sequencing gels
Advantages
Disadvantages

28. Application of AFLP

29. (Microsatellite)
SSR
Definition
A microsatellite is a tract of repetitive DNA in which certain DNA
motifs are repeated, typically 5–50 times. Microsatellites occur at
thousands of locations within an organism's genome. They have a
higher mutation rate than other areas of DNA leading to high genetic
diversity

30. Microsatellites can be amplified for identification by the polymerase
chain reaction (PCR) process, using the unique sequences of flanking
regions as primers
DNA is repeatedly denatured at a high temperature to separate the
double strand, then cooled to allow annealing of primers and the
extension of nucleotide sequences through the microsatellite.
This process results in production of enough DNA to be visible
on agarose or polyacrylamide gels.
With the abundance of PCR technology, primers that flank
microsatellite loci are simple and quick to use, but the development of
correctly functioning primers is often a tedious and costly process.
Principle

32. -simple and easy to use
-easy to detect via PCR
-co-dominant marker
-perfectly suited for used in map-based cloning
-cost is higher for establishing polymorphic primer sites
and investment in the synthesizing the oligonucleotides
-initial identification, DNA sequence information
necessary
Advantages
Disadvantages

33. Application of SSR
 Assessment of genetic variability and characterization of germplasm.
 Identification and fingerprinting of genotypes.
 Estimation of genetic distances between population, inbreeds and
breeding material.
 Marker assisted selection.
 Identification of sequence of useful candidate genes

34. SNP
(Single Nucleotide Polymorphisms)
• 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.

36. • 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.
• Disadvantages:
 Most of the SNPs are bi allelic and less informative than SSRs.
 Multiplexing is not possible for all loci.
 Some SNP assay techniques are costly.
 Development of SNP markers is labor oriented.
 More (three times) SNPs are required in preparing genetic maps
than SSR markers.