Gene Mapping Methods:Linkage Maps & Mapping with Molecular Markers

Prosenjit Bhowmick
M.Sc. 4th Semester(Botany), Roll No. 11
Dept. of Life Science & Bioinformatics, AUS
Linkage Maps & Mapping with Molecular Markers

A ‘gene’ is a sequence of nucleotides in DNA or RNA that encodes the
synthesis of a gene product, either RNA or Protein. The term was coined by Wilhelm
Johannsen(1909).
The charting of the posotions of genes on a DNA molecule or chromosome
and the distance, in linkage units or physical units, between genes is called ‘gene
mapping’.
INTRODUCTION
Gene mapping describes the methods used to identify the locus of a gene and
the distances between genes.
The essence of all genome mapping is to place a collection of molecular
markers onto their respective positions on the genome.
Genetic linkage maps allow to determine the position of a gene even when
nothing about the gene is known except for its phenotypic effect.
 Such maps are often the first step towards isolating the gene responsible for a
disease or for a specific trait.

HISTORY OF GENE MAPPING
Genetics as a branch of biology began in 1866 when Gregor Johann Mendel
(1822-1884) described the inheritance pattern of conceptual hereditary units, now, called
genes.
 In 1902, Boveri-Sutton suggested that chromosomes were the
carriers of genetic material.
Technique of genetic mapping was first described in 1911 by T. H.
Morgan. He noticed that some traits violated Mendel's Law of
Independent Assortment.
Genetic mapping did not start in humans until the 1950s, because it
was hard to know what traits were caused by genetic mutations.
With introduction of RFLP(1980s), effort was undertaken to
generate maps of all the chromosomes & the first maps were made in
1980s but covered only parts of chromosomes and had few markers.
 Maps of whole chromosomes were made by 1980s. By 1990s, a
number of whole-genome (i.e., covering all the chromosomes) genetic
maps were generated. T. H. Morgan( 1866-1945)
Image source: Google images
G. J. Mendel (1822-1884)

Three different strategies have been used to construct genetic maps. All
these strategies offer different levels of resolution at which genomes can be
studied. The strategies are:
TYPES OF GENE MAPS
Gene Mapping
Linkage
Mapping
Cytogenetic
Mapping
Physical Mapping
 Arrangement of gene & DNA
markers on a chromosome.
 Relative distance between position
on a genetic map are calculated using
recombinant frequencies
 Provide physical distance between
landmarks on the chromosome, ideally
measured in nucleotide bases.
 Map is created by fragmenting the
DNA molecule using restriction enzymes
and then looking for overlaps

 The greater the frequency of recombination (segregation) between two genetic
markers, the further apart they are assumed to be.
 Conversely, the lower the frequency of recombination between the markers,
the smaller the physical distance between them.
 Historically, the markers originally used were detectable phenotypes (enzyme
production, eye colour) derived from coding DNA sequences.
 Eventually, confirmed or assumed noncoding DNA sequences such
as microsatellites or those generating RFLPs have been used.
LINKAGE MAPPING
Linkage is the tendency of DNA sequences that are close together on
a chromosome to be inherited together during the meiosis phase of sexual
reproduction.
A linkage map depicts the order of genetic markers and the relative
distances between them as measured in terms of recombination frequencies
between the markers.

 Recombination frequency is a measure of genetic
linkage and it is used in creation of genetic
linkage maps.
 A centimorgan (cM) is a unit that describes a
recombination frequency of 1%.
 Using cM as map units , the genetic distance
between two loci can be measured, based upon
their recombination frequency.
RECOMBINATION FREQUENCY
Recombination frequency is the frequency with which a single
chromosome crossover will take place between two genes during meiosis.
Image source: https://isogg.org/wiki/Recombination

Example:
 Distance between gene b & cn = 9cM (as Recombination frequency is 9%)
 Distance between gene cn & vg = 11cM (as Recombination frequency is 11%)
 Total Distance between gene b & gene vg= 9cM + 11cM = 20cM
MAP UNIT or CENTIMORGAN
A map unit or centimorgan is a unit of measure used to
approximate the distance between genes. The Morgan unit is named in
honour of T. H. Morgan.
1 Map unit or 1 cM = 1% Recombination frequency

Alfred Henry Sturtevant, a Columbia University undergraduate who was
working with Morgan constructed the first Linkage map in 1913.
The First Linkage Map
Image source: https://www.nature.com/scitable/topicpage/thomas-hunt-morgan-genetic-recombination-and-gene-496/
He has taken four genes & these were present on the
X chromosome of the Drosophila melanogaster.
By making experimental crosses, he had observed the
number of parental and recombinant genotypes among
the progenies.
The calculated recombination frequencies between
these four genes were used to depict the distance
between the investigated genes.
Recombination frequencies
Between m & v = 3.0% Between m & y = 33.7%
Between v & w = 29.4% Between w & y = 1.3%

As linkage map or genetic map is the outcome of crossing over
studies, it is also called as cross over map.
The linkage mapping includes the following processes:
1. Determination of Linkage Groups (Mapping Populations).
2. Determination of Map Distance.
(a) Two point test cross
(b) Three point test cross
3. Determination of Gene Order.
4. Combining Map Segments.
5. Interference & Coincidence
CONSTRUCTION OF A LINKAGE MAP

Mapping population is a group of individuals used for gene
mapping. Selection of appropriate mapping population is fundamental
to the success of a gene mapping project.
1. DETERMINATION OF LINKAGE GROUPS
Before starting a gene mapping of the chromosome of a species, the
exact number of chromosomes must be known.
The total number of genes of that species by undergoing hybridization
experiments in between wild & mutant strains have to be determined.
By the same hybridization technique it can also be determined that how
many phenotypic traits remain always linked and consequently their
genes during the course of inheritance.
And thus the different linkage groups of a species can be determined.

 The intergene distance on the chromosome cannot be measured in the
customary units employed in light microscopy.
 Geneticists use an arbitrary unit to measure the map units, to describe distance
between linked genes.
2. DETERMINATION OF MAP DISTANCE
A map unit is equal to 1% crossover, i.e.
It represents the linear distance along the
chromosome for which a recombinant
frequency of 1% is observed.
Example:
In this cross the crossover of gamates are
Ab(2%) and ab(2%) .
Total crossover = 4% (aB%+Ab%)
So, distance between A & B will be 4 cM
or map unit.

2(a) TWO POINT TEST CROSS
The % of crossing over between two linked genes is calculated by a
two point test cross.
Example:
In this dihybrid test cross the
Ac/ac(13%) & aC/ac(13%) were
produced from the crossover gametes
from the dihybrid parents.
Thus 26 % (13% + 13%) of the
gamates are cross over types.
So distance between the loci A & C is
estimated to be 26 cM
As double crossover do not occur
between genes less then 5cM apart, so
for genes farther apart 3 point test
cross is used.

2(a) THREE POINT TEST CROSS
A three point test cross gives us the linear order in which three genes
should be present on chromosome.
Example:
In addition to the previous A & C genes, let us assume gene B, located in close
proximity in the same linkage group.
A trihybrid cross of ABC/ABC & abc/abc produced F1 hybrid ABC/abc and its
test cross with recessive parent abc/abc produced following offsprings:
Three Point test cross results (Btw. F1 hybrid ABC/abc & Recessive parent abc/abc)
36% ABC/abc
36% abc/abc
9% Abc/abc
9% aBC/abc
4% ABc/abc
4% abC/abc
1% AbC/abc
1% aBc/abc
72% Parental
Type
18% Single crossovers
(Btw. A & B)-Region1
8% Single crossovers
(Btw. B & C)-Region 2
2% Double
crossover
To measure distance between two genes we add all double and single crossovers(CO)
Distance between A-B: 18% single C.O.+ 2% double C.O.= 20% = 20 cM
Distance between B-C: 8% single C.O.+2% double C.O.= 10% = 10 cM
Distance between A-C: A-B(20cM) + B-C(10cM) = 30 cM apart A-C

3. DETERMINATION OF GENE ORDER
After determining the relative distance between genes of a linkage
group, it becomes easy to place genes in their proper linear order.
Example:
If we suppose the distance between genes A-B=12cM, B-C=7cM, A-C= 5cM, we
can determine the order of gene in the following manner:
B 12 cM A A 5 cM C
B 7 cM C
A 12 cM B
A 5 cM C
B 7 cM C
A 12 cM B
A 5 cM C C 7 cM B
Case 1: Gene order = B-A-C
Incorrect: Distance between
B-C are not equitable
Case 2: Gene order = A-B-C
Incorrect: Distance between
A-C are not equitable
Case 3: Gene order =B-C-A
Correct: Distance between
A-B are equitable & gene C
must be in centre.

4. COMBINING MAP SEGMENTS
Finally, the different segments of maps of a complete
chromosome are combined to form a complete genetic map.
Example:
We can combine the different segments of the previous example
into a single map by using simple calculation.
A 12 cM B
A 5 cM C C 7 cM B
Case 3: Gene order =B-C-A
Correct: Distance between
A-B are equitable & gene C
must be in centre.
As A-C distance = 5 cM & B-C distance = 7 cM; and by ordering
the genes we know gene C is in between A & B, so we can combine the
map putting C in betwwen(5cM from A & 7cM from B)
A 5 cM C 7cM b

5. INTERFERENCE & COINCIDENCE
In higher organisms , one chisma formation reduces the probability of
anthother chisma formation in an immediately adjacent region.
The tendency of one crossover to interfere with the other is called
interference. Centromere shows similar interference effect.
The net result of this interference is, fewer double crossover then
expected according to map distances.
Interference is expressed in terms of coefficient of coincidence.
When interference is complete(1.0), no double crossover will be
observed and coincidence becomes 0.
Coincidence + Interference = 1.0

A molecular marker is a DNA sequence used for chromosome
mapping as it can be located at a specific site in a chromosome.
MOLECULAR MARKERS
Molecular Marker
Biochemical
Isozymes
DNA Based
Markars
Hybridization
Based
1. RFLP
2. Minisatellite
3. Microsatellite
PCR
Based
1. RAPD
2. AFLP
3. STS
First Generation: 1980s
Based on DNA-DNA
hybridizations, such as RFLP.
Second Generation: 1990s
Based on PCR:
•Using random primers: RAPD
•Using specific primers: STS
Based on PCR and
restriction cutting: AFLP
Third Generation: recently
Based on DNA point
mutations (SNP)

 This has been particularly important with two types of organisms
- microbes and humans.
 Microbes, such as bacteria and yeast, have very few visual
characteristics so gene mapping with these organisms has to rely
on biochemical phenotypes.
 A big advantage is that relevant genes have multiple alleles.
 For example- Gene called HLA-DRB1 has at least 290 alleles
and HLA-B has over 400 alleles.
BIOCHEMICAL MARKERS IN LINKAGE
MAPPING

 Genes are very useful markers but they are by no means ideal.
 Mapped features that are not genes are called DNA markers.
 A DNA marker must have at least two alleles to be useful.
 DNA sequence features that satisfy this requirement are-
DNA MARKERS FOR LINKAGE
MAPPING
Restriction Fragment Length Polymorphism (RFLPs)
Simple Sequence Length Polymorphism (SSLPs)
Single Nucleotide Polymorphism (SNPs)

RFLP TECHNIQUE IN GENE MAPPING
RFLPs(Restriction fragment length polymorphisms) were the first type of
DNA marker to be studied for linkage mapping..
Restriction enzymes cut DNA molecules at specific recognition
sequences.
But restriction sites in genomic DNA are polymorphic and exists as two
alleles.
 One allele displaying the correct sequence for the restriction site and
therefore being cut when the DNA is treated with the enzyme.
 The second allele having a sequence alteration so the restriction site is
no longer recognized.
The RFLP and its position in the genome map can be worked out
following the inheritance of its alleles.

The DNA molecule on the left has a polymorphic restriction site (marked with
the asterisk) that is not present in the molecule on the right.
The RFLP is revealed after treatment with the restriction enzyme because one of
the molecules is cut into four fragments whereas the other is cut into three
fragments.
In order to score an RFLP, it is necessary to determine the size of just one or two
individual restriction fragments against a background of many irrelevant
fragments.
Image source: T. A. Brown (2006), GENOMICS 3 (3rd Edition)

There are two methods for scoring an RFLP:
1. Southern hybridization 2. PCR
1. Southern hybridization:
i. The DNA is digested with the appropriate restriction enzyme and
separated in an agarose gel.
ii. The smear of restriction fragments is transferred to a nylon membrane
and probed with a piece of DNA that spans the polymorphic restriction site.
iii. If the site is absent then a single restriction fragment is detected (lane 2)
iv. if the site is present then two fragments are detected (lane3)

2. RFLP typing by PCR:
i. using primers that anneal either side of the polymorphic
restriction site.
ii. After the PCR, the products are treated with the appropriate
restriction enzyme and then analyzed by agarose gel electrophoresis.
iii. If the site is absent then one band is seen on the agarose gel; if
the site is present then two bands are seen.

SSLPs IN GENE MAPPING
SSLPs(Simple Sequence Length Polymorphism) are arrays of repeat
sequences that display length variation.
Here different alleles contain different number of repeat sequences.
Two types of SSLPs are:
1. Minisatellites (VNTRs)
repeat unit is up to 25 bp
in length
2. Microsatellites (STRs)
Shorter units, dinucleotide or
tetranucleotide units
SSLPs can be multiallelic. In allele 1 the motif 'GA' is repeated three
times, and in allele 2 it is repeated five times.
Microsatellites are more popular than minisatellites as DNA markers.

SSLP typing by PCR:
The region surrounding the SSLP is amplified and the products loaded
into lane A of the agarose gel.
Lane B contains DNA markers that show the sizes of the bands given
after PCR of the two alleles.
The band in lane A is the same size as the larger of the two DNA
markers, showing that the DNA that was tested contained allele 2.

SNPs IN GENE MAPPING
There are some positions in the genome where some individuals have
one nucleotide while others have another.
Some SNPs give rise to RFLPs but many do not.
SNPs originate when a point
mutation occurs in a genome
converting one nucleotide to
another.
There are just two alleles-
the original sequence and the
mutated version.
SNPs enable very detailed genome maps to be constructed.

Detecting an SNP by solution hybridization:
The oligonucleotide probe has two end-labels. One of these is a
fluorescent dye and the other is a quenching compound.
The two ends of the
oligonucleotide basepair to
one another, so the fluorescent
signal is quenched.
When the probe hybridizes
to its target DNA, the ends of
the molecule become
separated, enabling the
fluorescent dye to emit its
signal.
The two labels are called
'molecular beacons'.

Detecting an SNP by Oligonucleotide Ligation Assay:
An oligonucleotide is a short single-stranded DNA molecule, usually
less than 50 nucleotides in length, that is synthesized in the test tube.
If conditions are right, then an
oligonucleotide will hybridize with
another DNA molecule only if the
oligonucleotide forms a completely
base-paired structure with the other.
If there is a single mismatch - a
single position within the
oligonucleotide that does not form a
base pair, then hybridization does not
occur.
Oligonucleotide hybridization can
therefore discriminate between the
two alleles of an SNP.

Process of Linkage Mapping Using
Molecular Markers
We can map RFLPs and other molecular markers by making
crosses between two parents differing for two or more RFLPs and scoring
the progeny for these RFLP markers.
Explanation: Lets us suppose that,
1. One RFLP is detected with probe *1 and gives bands of 5kb and 6kb
indifferent strains.
2. Similarly the second RFLP is detected by probe *2 and yields bands of
2kb and 3kb.
The mapping of these two RFLP markers may be done as follows:
1. Two strains that are homozygous for the two RFLP markers, e.g. One
shows bands of 5kb & 3kb, while the other shows 6kb & 2kb bands
are crossed together.

2. The F1 so obtained shows all
the four bands. The F1 is back
crossed with one of the parent
having 6 & 2kb bands.
3. The resulting back-cross
progeny are scored for these
RFLP bands & each progeny
will yield 1 of the 4 band
patterns.
4. If the two RFLP markers were
not linked, equal proportion of
progeny will be present in these
4 categories.
5. But, incase of linkage the
proportion of parental progeny
will be much higher then the
recombinant progeny.
Image source: P.S. Cerma(2012), Cell biology and Genetics

The LOD Score
In actual analysis of RFLP data, linkage is detected by a statistical test
called the lod [Logarithm(base 10) of odds] score method developed by
Morton in 1955.
The LOD score is calculated as follows:
If the lod score equals +3 or is greater, the two RFLP markers are
considered to be linked.
A LOD score less than -2.0 is considered evidence to exclude linkage.
If the lod score indicates the two RFLP markers to be linked, then the
recombination frequency is calculated to obtain the map distance in terms of
map units or cM.
Using this map distance data the linkage map (RFLP map) is prepared.

PHYSICAL MAPPING
In a physical map, genes are depicted in the same order as they occur
in chromosome.
The distance between genes are shown as number of base pairs
separating genes. It uses techniques such as Restriction mapping, FISH etc.
Example: Genes sc & w are separated by 1.5x106 bp.
Physical mapping usually requires the:
1. Cloning of many pieces of chromosomal DNA.
2. Characterization of these fragments of size, i.e. Length in base pairs &
genes present in them.
3. Determination of their relative location along a chromosome (e.g., by
FcFISH).

CYTOGENETIC MAPPING
A cytogenetic map depicts the location of various genes in a
chromosome relative to specific microscopically visible landmarks.
Each chromosome has a banding pattern, which may be either naturally
present or generated by specific staining protocol.
In most species, cytogenetic mapping is accurate within limits of approx.
5 megabase pairs(Mb=1000000 bp) along a chromosome.

Application/Uses of Linkage Maps
Studying genome structures, organization and evolution.
Estimation of gene effects of important agronomic traits.
Tagging genes of interest to facilitate marker assisted selection (MAS)
programs.
Map based cloning.
Identify genes responsible for traits:
Plants or Animals
Disease resistance.
Meat or milk production, etc.

Limitations of Linkage Mapping
A map generated by linkage technique is rarely sufficient for directing
the sequencing phase of a genome project. This is for two reasons:
1. The resolution of a genetic map depends upon the number of
crossovers that have been scored.
 Genes that are several kb apart may appear at the same
position on the linkage map.
2. Genetic maps have limited accuracy.
Presence of recombination hotspots means that crossover
are more likely to occur at same points rather than at others.
Physical mapping technique have been developed to
address this problem.

CONCLUSION
 Linkage mapping is entirely based upon recombination frequency and
LOD analysis. Analysing a LOD score is a critical problem in case of
large maps.
Although by using linkage maps we can determine the distance between
two genes and by using three point test cross we can determine the relative
locations of genes.
The accuracy of linkage maps is a big question and to overcome this
issue introduction of physical and cytogenetic mappings were introduced.
All molecular markers are not equal and none is ideal. Some are better
for some purposes than others.
However, all are generally preferable over morphological markers for
mapping and marker assisted selection.

REFERENCES
B. D. Singha (2012), Biotechnology: Expanding horizons (4th Revised
edition); Kalyani Publishers, New Delhi; ISBN: 9789327222982; Pages(112-
146).
Boris M. Sharga et. Al (2018), Practical 6. Genetic linkage and mapping.
P. K. Gupta(2015), Cytology & Genetics (7th Edition), Rastogi Publisher;
ISBN: 9788171330473; Pages 72-97).
Paul H Dear(2001), Genome Mapping, ENCYCLOPEDIA OF LIFE
Sciences, Macmillan Publishers Ltd, Nature Publishing Group.
P. S. Verma & V. K. Agarwal (2012), Cell biology, Genetics, Molecular
biology, Evolution & Ecology(14th Edition);S. Chand (P) Ltd.; ISBN:
8121924421; Pages(84-113).
T. A. Brown (2006), GENOMICS 3 (3rd Edition);Garland Science; ISBN-
10:0815341385; Pages(255-286).

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