Role of molecular marker play a significant supplementary role in enhancing yield along with conventional plant breeding methods. the result obtain through molecular method are more accurate and at genotypic level. It had wider applications in field of plant breeding, biotechnology, physiology, pathology, entamology, etc. The mapping information obtained from these markers had created a revolution in the sequencing sector and open many pathways for developments, innovations and research.

1. ROLE OF
MOLECULAR MARKER
SHWETA TIWARI
190134008
Ph.D. Research Scholar
DEPARTMENT OF PLANT BREEDING AND GENETICS
JAWAHARLAL NEHRU KRISHI VISHWAVIDYALAYA,
JABALPUR - 482002
CREDIT SEMINAR ONTOPIC

2. An object used to indicate a position, place, or route.
What are Markers?
DNA sequences that
are readily detected
and whose inheritance
can be easily
monitored.
Molecular
Markers
INTRODUCTION
Because it has naturally occurring DNA
polymorphism, which forms the basis for
designing strategies to exploit applied
purpose.
Why DNA used as Molecular
Marker?
Also known as DNA
marker or Genetic
marker

3. If DNA sequences are utilized, then
what is the difference between gene and
genetic markers ?
3
The gene of interest is directly related with production of
protein(s) that produce certain phenotypes
GENE
Markers should not influence the trait of interest but are
genetically linked and therefore remain together during
segregation of gametes due to the concomitant reduction in
homologous recombination between the marker and gene of
interest.
GENETIC MARKER

4. 4
High DNA level
differences among
individuals.
01. POLYMORPHISM
Higher availability and
suitability to be
multiplexed the data
accumulated and
shared between
laboratories
(Un-restricted use)
02. REPRODUCIBLE Heterozygote could
be distinguished from
homozygote
03. CO-
DOMINANT
Not clustered in a
certain region
04. EVEN &
FREQUENT
DISTRIBUTION
Automated and Cost
effective marker
genotyping.
05. EASY, FAST &
CHEAP TO
DETECT
NO SINGLE MARKER MEET ALL THESE REQUIREMENTS SO WE
NEED TO DEVELOP WIDE RANGE OF MOLECULAR MARKERS
Single marker
shouldn’t affect
multiple traits.
06. SINGLE COPY
& NO
PLEIOTROPIC
EFFECT
Especially with
polyploids
07. GENOME-
SPECIFIC
So, different allele
could be easily
identified.
08. DISTINCT
ALLELIC
FEATURE

5. 5
CLASSIFICATION OF MOLECULAR
MARKER
•SSR
•AFLP
•SSLP
•VNTRs
•RAMPO
•CAPs
•ISSR
•ASAP
SECOND GENERATION
MARKER
•EST
•SNP
•MIT
E
THIRD GENERATION
MARKER
•RFLP
• RAPD
•STS
•SCAR
•AP-PCR
•DAF
FIRST GENERATION
MARKER
3.
SNPs : Due to sequence in variation (RFLP)
Non-SNPs : Due to sequence in length (SSR)
Casual similarity of SNPs with some of these marker system and
fundamental difference with other marker system, classify them
into two :-

6. 6
PCR based
RAPD(random amplified
polymorphic DNA)
ISSR(inter simple sequence
repeats)
RGA(resistance gene
analogs
PCR based
AFLP(amplified fragment length
polymorphism)
SSR(simple sequence repeats)
SCAR(sequence characterized
amplified regions)
Non PCR based
RFLP(restriction fragment length
polymorphism- Dominant
DOMINANT MARKER
CO-DOMINANT MARKER

7. 7
Genetic and
physical mapping
are two distinctive
types of genome
mapping use a
collection of
molecular markers
with respective
positions on the
genome.
GENOME MAPPING
Identification of
existing DNA or
the introduction
of new DNA that
can function as a
tag or label for
the gene of
interest.
GENE TAGGING
Marker assisted
selection or marker
aided selection (MAS)
is an indirect
selection process
where a trait of
interest is selected
based on a marker
linked to a trait of
interest rather than
on the trait itself.
MAS
A.FOR CROP
GERMPLASM Establishes the
syntenic
relationships
between
genomes of
different
species.
COMPARATIVE
MAPPING
Used to clone a
gene (e.g.,
disease gene)
from its known
closest markers
CHROMOSO
ME WALKING
ROLE OF MOLECULAR
MARKERS
Unique
approach that
identifies the
underlying
genetic cause of
a variation..
MAP BASED
CLONNING OF
GENE
PREDICTIN
G
HETROSIS
BASED ON
MOLECULA
R
DIVERSITY
B. FOR PATHOGEN
POPULATION
Used to identify the
actual pathogen
causing disease.
Cultivar Identification and
Analysis of Seed Purity.
DNA FINGERPRINTING

8. GENOME
MAPPING
• The identification and recording of location of genes and their
distances on a chromosomes of an individual is termed as genome
mapping.
• The two methods of genome mapping includes molecular markers
are physical mapping and genetic mapping.
• Molecular markers have three-fold applications in gene mapping:
(1) A marker allows the direct identification of the gene of
interest instead of the gene product, and consequently, it
serves as a useful tool for screening somatic cell hybrids;
(2) Use in several DNA probe and easy-to-screen
techniques, a marker also helps in the physical mapping
of the genes using in situ hybridization.
(3) Molecular markers provide sufficient markers for
construction of genetic maps using linkage analysis

9. PHYSICAL MAPPING
9
GENETIC MAPPING
Genetic maps depend on the genetic linkage information, but physical
maps are based on the actual physical distances as measured by the
number of base pairs.
Genes (genetic markers) are the markers used in genetic mapping, but
restriction recognition sites (DNA markers) are the markers used in
physical mapping

10. AFFILIATED RESEARCH FINDINGS
OF PHYSICAL MAPPING
• Using phenotypic data of four biparental spring wheat populations
evaluated at multiple environments under two management systems,
we discovered 152 QTL and 22 QTL hotspots, of which two QTL
accounted for up to 37% and 58% of the phenotypic variance,
consistently detected in all environments, and fell within genomic
regions harboring known genes
• QSv.dms-1A and QPht.dms-4B.1 individually explained up to 37% and
58% of the variation in sedimentation volume and plant height,
respectively, and had very large LOD scores that varied from 19.0 to
35.7 and from 16.7 to 55.9, respectively. (Semagn et al., 2021)

11. AFFILIATED RESEARCH
FINDINGS OF GENTIC MAPPING
• GBS-SNP and SSR based genetic mapping and QTL analysis for
drought tolerance in upland cotton
• Chromosomes 3 and 8 harbored important drought tolerant QTLs for
chlorophyll stability index trait while for relative water content trait,
three QTLs on chromosome 8 and one QTL each on chromosome 4,
12 were identified. One QTL on each chromosome 8, 5, and 7, and two
QTLs on chromosome 15 linking to proline content were identified. For
the nitrate reductase activity trait, two QTLs were identified on
chromosome 3 and one on each chromosome 8, 13, and 26
• chromosome 8 harbored a drought tolerance QTL hotspot with two in-
house QTLs for chlorophyll stability index (qCSI01, qCSI02) and three
public domain QTLs (qLP.FDT_1, qLP.FDT_2, qCC.ST_3). (Shukle et
al., 2021)

12. CHROMOSOME
WALKING
Chromosome walking is a tool which explores the unknown sequence
regions of chromosomes by using overlapping restriction fragments.
In chromosome walking, a part of a known gene is used as a probe and
continued with characterizing the full length of the chromosome to be mapped
or sequenced. This goes from the marker to the target length.
(Ends of each overlapping fragments are used for hybridization to identify the next
sequence)
The probes are
prepared from the end
pieces of cloned DNA
and they are sub
cloned.
Then they are used to find the next
overlapping fragment.
All these overlapping sequences are used to construct
the genetic map of the chromosome and locate the target
genes

13. 1. Isolation of a DNA fragment which contains the known gene or
marker near target gene
2. Preparation of the restriction map of the selected fragment and
subcloning the end region of the fragment to use as a probe
3. Hybridization of the probe with the next overlapping fragment
4. Preparation of the restriction map of the fragment 1 and subcloning
of the end region of the fragment 1 to use as a probe for the
identification of the next overlapping fragment.
5. Hybridization of the probe with the next overlapping fragment 2
6. Preparation of the restriction map of fragment 2 and subcloning of
the end region of the fragment 2 to serve as a probe for the
identification of the next overlapping fragment Above steps should
be continued till the target gene or up to 3’ end of the total length of
the sequence.
AFFILIATED RESEARCH FINDINGS
Identification of the Cystic Fibrosis Gene: Chromosome Walking and
Jumping
Chromosome walking and jumping and complementary DNA
hybridization were used to isolate DNA sequences, encompassing more
than 500,000 base pairs, from the cystic fibrosis region on the long arm
of human chromosome 7 (Rommens, et al., 1989).
STEPS:

14. GENE TAGGING
1. Gene tagging refers to the identification of existing DNA or
the introduction of new DNA that can function as a tag or
label for the gene of interest
2. Marker based gene tagging readily detected and whose
inheritance can be easily monitored.
3. The genome size of most plant species ranges between
108 to 1010 base pairs, so even a small proportion of
variation in DNA can yield a large number of potential
markers
AFFILIATED RESEARCH FINDINGS
Assessment of ISSR Markers for Tagging Genetic Variability for Yield
Components in Small Cardamom (Elettaria cardamomum Maton)
(Prasannan etal., 2021)

15. COMPARATIVE
GENOMICS
It is the comparison of one genome to
another.
What comparative genomics can address?
1.
2.
3.
4.
Organism
evolved
Differentiates
species
Important non-
coding regions
genes required for
organisms to survive
in a certain
environment

16. Example of a Comparative Genomic Study:
Comparative study of three
genome of Sacchromyces
strains
Purpose of study
1
Numerous new regulatory
motifs were found.
Results
2
Similarity in genome would
reveals the uniqueness of
strain
Future
4
1. 85% of the orhologous pairs
have identical number of exons
2. 91% of the orthologous exons
have identical length
3. 99.5% of the orthologous exons
have identical phase
Orthologous human mouse
genes have conserved exonic
structure.

17. Comparative genome sizes of humans and
other organisms
estimated chromosome
gene number number
Homo sapiens
(human)
Rattus norvegicus
(rat)
Mus musculus
(mouse)
Drosophila melanogaster
(fruit fly)
Arabidopsis thaliana
(plant)
Caenorhabditis elegans
(roundworm)
Saccharomyces
cerevisiae
(yeast)
Escherichia coli
(bacteria)
H. influenzae
(bacteria)
organism estimated size
average gene
density
2900 million bases ~30,000
1 gene per 100,000
bases
2500 million bases ~30,000
1 gene per 100,000
bases 40
46
2,750 million bases ~30,000
1 gene per 100,000
bases 42
125 million bases 25,500
1 gene per 4000
bases 5
180 million bases 13,600
1 gene per 9,000
bases 8
12 million bases 6300
1 gene per 2000
bases 16
97 million bases 19,100
1 gene per 5000
bases 6
1.8 million bases 1700
1 gene per 1000
bases 1
4.7 million bases 3200
1 gene per 1400
bases 1

18. Genetic Similarities in cereals
It has been shown that all genomes of grass species can
now be described in terms of their relationships to a
single reference, i.e. rice genome. 1
In cereals, a consensus map of 12 grass genomes
including wheat is now available, representing
chromosome segments of each genome relative to those
in rice on the basis of mapping of anchor DNA markers. 2
Among cereals, using molecular markers, co linearity
was first reported among A, B & D sub genomes of
wheat. 3
Conservation of co linearity between homoeologous A
genome of diploid einkorn wheat and hexaploid wheat
was exploited for cloning of candidate gene for leaf
rust resistance locus Lr10 in bread wheat
4

19. AFFILIATED RESEARCH
FINDINGS
Mining QTL and genes for root traits and biochemical parameters under vegetative
drought in South Indian genotypes of finger millet (Eleusine coracana (L.)
Gaertn) by association mapping and in silico comparative genomics (Siddiqui, et
al., 2021)
• To determine the response of 42 finger millet genotypes grown under vegetative
drought and their responses to root and biochemical traits in greenhouse
analysis.
• Total of 42 Indian genotypes of finger millet were used to identify quantitative trait
loci (QTL) associated with root traits and biochemical parameters.
• Totally, five and two QTL were associated with root length and root weight
respectively.
• In-silico comparative genomic analysis was performed with genomes of various
Poaceae family members to find the QTL associated with any candidate gene.
• This study will help to improve finger millet genotypes under both biotic and
abiotic stress conditions
 The qtlUGEP-07 was associated with five candidate genes in the different
Poaceae family members. Similarly, the qtlUGEP-13 was corresponding to two
candidate genes of Panicum hallii. Furthermore, qtlUGEP-16 and qtlUGEP-
95 were also associated with candidate genes in foxtail millet and maize.

20. SELECTION
Definition: Refer to the use of DNA marker that are tightly linked to target
loci as a substitute to assist phenotypic screening
F2
P2
F1
P1 x
large populations consisting of thousands
of plants
Resistant
Susceptible
MARKER-ASSISTED SELECTION (MAS)
MARKER-ASSISTED BREEDING
Method whereby phenotypic selection is based on DNA markers
SELECTION OF
PARENTS
DEVELOPMENT OF
BREEDING
POPULATION
ISOLATION OF DNA
SCORING OF
MARKERS
CORRELATION WITH
MORPHOLOGICAL
TRAITS

21. 1 Improved Pusa Basmati 1 is first variety developed in India through
MAS for BLB
 FOREGROUND: Selection is based on molecular marker tightly
linked with gene of interest rather than gene itself. Gene Xa 13
(CAPs), Xa21 (STS) & Waxy (STMS)
 2. BACKGROUND: Molecular marker distributed throughout
the genome can be used to monitor or aid the recovery of
whole genome except target regin of one parent. It give Pusa
Basmati contribution.
FOREGROUND AND BACKGROUND
SELECTION
2 GENETIC CONTRIBUTION OF PARENTS
3 A statistical method that link phenotype data and genotype data in
a attempt to uncover the genetic basis of variation in complex
traits
QTL MAPPING & ANALYSIS
4
 Assay for resistance to soybean cyst nematode (SCN) is very
difficult due to problem in inoculation and scoring.
 SCN resistance is due to gene rgh1 the SSR marker sat309 is
located at 2cm from this gene. Selection based on this marker
is 99% accurate for SCN susceptibility.
THE IDENTIFICATION OF PUTATIVE RESISTANT
PLANTS IN ABSENCE OF DISEASE TESTS

22. 5 It is process of combining two or more gene from multiple parents to
develop elite lines and varieties
Examples: In rice BLB Resistance gene pyramided: Xa 4, Xa 5, Xa 13,
Xa 21 and in wheat for leaf rust resistance gene pyramided: Lr 42, Lr
42, Lr 43
GENE PYRAMIDING
6 IN IDENTIFYING HETEROTIC COMBINATIONS FOR
USE IN PRODUCTION OF HYBRIDS.
7 Introgression of specific locus from donor genome through
breeding programe
GENE INTROGRESSION
Maize inbreeds B72 and Mo17 : 2 most important heterotic groups
of maize  molecular mapping showed that QTLs contributing to
heterosis for grain yield were located on 9 of the 10 maize
chromosomes. Also found that inbreds Tx303 and Oh43 could
contribute QTLs to enhance heterosis of the cross B73 X Mo17 by
backcrossing.
When the derived inbred were crossed  he single cross yielded
12-15% more than original cross

23. AFFILIATED RESEARCH FINDINGS
Marker-assisted selection of Ms locus responsible for male fertility
restoration in onion (Allium cepa L.) ()
1. Objective: To identify the male-fertility restoration locus (Ms)
among 72 breeding lines of onion (Allium cepa L.) by genotyping,
which is crucial in the development of F1 hybrid onions using
cytoplasmic-genic male sterility (CGMS) system.
2. Marker Used: Thus, two markers were used to identify
the Ms locus, the simple PCR marker namely jnurf20 is dominant
nature cover across the breeding lines genetic backgrounds,
whereas the PsaO gene-specific marker could not spread all
along the breeding lines evaluated in the study. Since all reported
markers are may or may not have marker genotype association
across different genetic backgrounds of onion breeding lines.
However, these molecular markers are highly useful in the marker-
assisted selection of Ms locus.

24. DNA
FINGERPRINTING
24
1. Extracting the DNA
from cells.
2. Cutting up the DNA
using an enzyme.
3. Separating the
DNA fragments on
a gel.
4. Transferring the
DNA onto paper.
5. Adding the
radioactive probe.
6. Setting up the X-
ray film.

25. AFFILIATED RESEARCH FINDINGS
DNA Fingerprinting and Genetic Diversity Assessment of GM Cotton
Genotypes for Protection of Plant Breeder Rights (Jamil et al., 2021)
OBJECTIVE: Present study was designed for DNA fingerprinting
and genetic diversity assessment of 25 GM cotton genotypes
(possessing Cry1Ac gene) using 297 SSR markers through
conventional PCR and Polyacrylamide gel electrophoresis. Out of
297 SSR markers, 25 markers were not amplified, 28 were
monomorphic and 244 were polymorphic. A total of 1537 alleles
were amplified among which 1294 (84.18%) were polymorphic. PIC
value in our study ranged from 0.08 to 0.93 with an average of 0.73.
Unique allelic pattern was observed for nineteen genotypes
whereas six genotypes were identified using two-step identification
methods.
CONCLUSION: The information generated in this study will be helpful
in variety registration and subsequent protection under PBRs.

26. 1. Rommens, J.M., Iannuzzi, M.C., Kerem, B.S., Drumm, M.L., Melmer, G., Dean, M.,
Rozmahel, R., Cole, J.L., Kennedy, D. and Hidaka, N., 1989. Identification of the cystic
fibrosis gene: chromosome walking and jumping. Science, 245(4922), pp.1059-1065.
2. Semagn, K., Iqbal, M., Chen, H., Perez-Lara, E., Bemister, D.H., Xiang, R., Zou, J., Asif,
M., Kamran, A., N’Diaye, A. and Randhawa, H., 2021. Physical mapping of QTL
associated with agronomic and end-use quality traits in spring wheat under conventional
and organic management systems. Theoretical and Applied Genetics, pp.1-21.
3. Shukla, R.P., Tiwari, G.J., Joshi, B., Song-Beng, K., Tamta, S., Boopathi, N.M. and Jena,
S.N., 2021. GBS-SNP and SSR based genetic mapping and QTL analysis for drought
tolerance in upland cotton. Physiology and Molecular Biology of Plants, 27(8), pp.1731-
1745
4. Prasannan, A. and Jose, S., 2021. Assessment of ISSR Markers for Tagging Genetic
Variability for Yield Components in Small Cardamom (Elettaria cardamomum
Maton). Indian Journal of Agricultural Research, 55(2).
5. Siddiqui, M.N., Léon, J., Naz, A.A. and Ballvora, A., 2021. Genetics and genomics of root
system variation in adaptation to drought stress in cereal crops. Journal of experimental
botany, 72(4), pp.1007-1019.
6. Manjunathagowda, D.C. and Selvakumar, R., 2021. Marker-assisted selection of Ms locus
responsible for male fertility restoration in onion (Allium cepa L.). Genetic Resources and
Crop Evolution, 68(7), pp.2793-2797.
7. Jamil, S., Shahzad, R., Iqbal, M.Z., Yasmeen, E. and Rahman, S.U., 2021. DNA
fingerprinting and genetic diversity assessment of GM cotton genotypes for protection of
plant breeders rights. Int. J. Agric. Biol.
.
REFERENC
ES

27. THANK
YOU