What is a marker???

What is a marker???
Marker in life sciences is
defined as a trait or an
allele or a DNA
sequence or a
cytogenetic segment or
a chromosome
fragment or a protein or
an enzyme used as an
experimental probe to
keep track of an
individual, a tissue, a
cell, a nucleus, a
chromosome or a gene.
Ideal genetic marker
1.Polymorphic & multi allelic
2.Codominant
3.Not epistatic
4.Not pleotropic
5.Not influenced by environment
6.Marker locus should be neutral- should not
affect the fitness of the individual
7.Abundant & distributed throughout the
genome
 Morphological Markers
Morphological descriptors of
plant/animal/
microorganism/ human,
correspond to qualitative
traits that can be scored
visually, with discrete
phenotypes-direct
measurement of phenotype
(purple leaf sheath
associated with BPH
resistance in rice, blue eye
color, bacterial cell shape
etc).
 Advantages
Simple inheritance and inexpensive to score
Found in nature or the result of mutational
experiments (used to develop maps), maps
and markers to speed up process of plant
breeding
Identification & selection of minor genes of
interest by linkage with major genes
Leaf-tip burning is associated Black chaff is associated with stem
with leaf rust resistance gene rust resistance gene Sr2.
Lr34 in wheat

Brown glumes in the case of wheat variety HD-2329

Crooked neck (peduncle) in the case of Kalyan Sona wheat

 Demerits of morphological markers
The delay of marker expression until late into the
development of the organism- Time taking and
developmental stage dependent
Dominance
Deleterious effects- dominant lethals or recessive lethals,
mutants may not scorable
Pleotropy - single gene governing more than one
genotype – complex genetic inheritance
Confounding effects of genes unrelated to the gene or
trait of interest but which also affect the morphological
marker (epistasis)- complex genetic inheritance
Visible polymorphisms relatively rare- Limited in number
Dominant lethals
 Demerits of morphological markers
Most genetic polymorphism/variation not so easily observable
Frequent confounding effects of environmental factors cause
large effects on phenotype that they are undesirable in breeding
programme
 Mask the effects of linked minor genes
 Impossible to identify desirable linkages for selection - many have
undesirable linkage
Few of these markers can be analyzed in a single cross/mapping
population
Can be scored only on whole plants and that to during specific
developmental stages
Many traits require threshold limit for their expression Eg: Dis.
Res.
Maintenance of suitable genetic stocks - necessary
 Biochemical markers
 Isozymes, allozymes, proteins and secondary
metabolites are successful biochemical markers.
 First true molecular markers – derive their name
from allelic variation of enzymes- Pyruvate
dehydrogenase, Esterase, Peroxidase etc.
Biochemical markers are proteins produced by
gene expression. -Protein and isozyme markers
were developed by Markert and Moller, 1959
Biochemical markers are the products of various
alleles of one or several genes
 Variants in proteins, enzymes can be
resolved electrophoretically- migrational
properties based on their size, shape and
charge difference caused by amino acid
differences, Easily assayed for many
individuals- SDS PAGE (denaturing gels)
 Protein/Enzyme polymorphism can be
revealed on electrophoregrams through a
colored reaction associated with enzymatic
activity
Resolved through starch gel/SDS PAGE (denaturing or non-
denaturing gels)/Chromatogram techniques

Histochemical
staining

 Crude protein extract is made from some tissue sources-usually leaves.

 The extracts are separated by an electrophoresis system/ chromatogram. The
gel is then placed in a solution that contains reagents required for the
enzymatic activity of the enzyme, for which it is monitoring -Isozyme variation
revealed in characteristic banding patterns is called zymogram.
 The solution contains a dye that the enzyme can catalyze into a color reagent
that stains the protein/enzyme or gel may be assayed by western blot
technique.
 Most Common protein markers are Isozymes
 Isozymes are the different molecular forms of the same enzyme
that catalyze the same reaction, the enzyme is coded by alleles at
more than one gene locus.
Allozyme: the enzyme that is coded by different alleles at one
gene locus.
Isozymes were first described by R. L. Hunter and Clement
Markert (1957) who defined them as different variants of the same
enzyme having identical functions and present in the same
individual.
Isozymes are usually the result of gene duplication, but can also
arise from polyploidisation or nucleic acid hybridization.
Tanksley and Rick (1980) used isozyme markers for the first time
to speed up introgression of a monogenic trait into adapted
tomato cultivars.
Isozyme variation revealed in characteristic banding patterns
is called zymogram.
A schematic representation of the isozyme banding patterns seen in
F1 generation.

ffAAa
f

The enzyme molecule may be (1) monomer, (2) homodimer, (3)

homotrimer, or (4) homotetramer. A and a are the polypeptides
encoded by the alleles A and a
Strengths
 co-dominant
 robust
 reproducible
 most cost-effective tools for data point
generation
 the turnaround time is relatively rapid
 rarely exhibit epistatic interaction so
that a genetic stock containing an infinite
number of markers could be constructed.
Limitations
Polymorphism within a cultivated species is poor- originates from inter-
specific crosses – less coverage of genome
Many DNA variants do not result in changes in amino acid sequence
(e.g., synonymous substitutions-silent mutations).
Some changes in amino acid sequence do not result in changes in
mobility on the gel - they detect mutation which produce functional
enzymes
Dimeric and multimeric enzymes add complexity
Limited enzyme systems available (eg: 15 – 20), limited no. of loci and
systems differed with plant species/crops
The enzymes are not active in all the organs and all the stages- spatial
expression of genes and developmental stage of plant.
Many different biochemical procedures are required
Their expression may usually neutral (may influenced by environment
in terms of amount.)
 Advantages of biochemical Markers
 Germplasm classification
 Gene mapping
 Selection
 Monitoring genetic segregation and recombination in
distance crosses
 Characterization F1 hybrid
 Nature of calli and somatic hybrid plants
 Identification of sexual hybrid
 Identification of plants from pollen or anther wall
 Variety/hybrid purity, and
 Determination of phylogenic relationship in plant
The chromosomal banding in humans
An Ideal DNA Marker
1. It should generate a very large number of single-copy
neutral effect markers that are polymorphic and, preferably,
evenly distributed throughout the genome.
2. It should be codominant and have multiple alleles
3.The detection of marker alleles, i.e., genotyping, should be
simple, easy, quick, inexpensive, reproducible, and
amenable to automation and have high throughput.
4. Only small amount of DNA should be needed for
genotyping, and the error in genotyping should be near zero.
5.Finally, the marker system should not require prior
information about the genome of an organism.
Features/Advantages of DNA Markers
1. They represent polymorphism in the actual base sequence of DNA
distributed over the entire genome.
(2) The number of different marker loci is very large so that all the genomic
regions can be mapped at very high marker densities.
(3) The sequence variation detected by these markers is
generally neutral, except when it is located in coding sequences and affects
the functions of concerned genes.
(4) Scoring for one DNA marker usually has no effect on that of the others,
so that multiple markers can be evaluated simultaneously.
(5) Molecular markers show simple Mendelian inheritance.
(6) Marker genotyping is independent of the prevailing environment
and (7) the developmental stage of the plant,
and (8) the marker assays are nondestructive.
Therefore, MAS can be effectively used under any environment and at any
stage of development
(9) MAS can also be used for an allele that is not expressed in the available
genotypes, e.g., a recessive allele in heterozygotes.
(10) The DNA samples can be stored for future use, and
(11) Specific marker stocks are not required.
Applications of DNA Markers
(1)Fingerprinting of strains/varieties for unequivocal identification
(2) Mapping of genes and QTLs
(3) Efficient MAS for tightly linked QTLs and such oligogenes, direct selection for
which may be costly or problematic
(4) Positional cloning of genes/QTLs
(5) Identification of chromosome segments that would contribute to improvements
in the target traits
(6) Establishing phylogenetic relationships among different strains/species;
(7) Selection of parents for hybridization
(8) Assessing the basis of somaclonal variation
(9) identification of pathogen races and biotypes
(10) Prediction of heterotic cross combinations
(11) Identification of wide hybrids
(12) Gene pyramiding
(13) Management and utilization of genetic resources.
(14) MAS allows the use of off-season nursery and greenhouse facilities to
reduce the time needed for variety development