Gene Mapping
Prof. M. S. Nirgude
Assistant Professor
Dept. of Plant Biotechnology
CABT, Achloli
Gene Mapping
A genetic map is a schematic representation of
various genetic markers in the specific order, in which
they are located in a chromosome along with the distances
between them.
Genetic maps have been constructed by using three
diverse strategies to generate three different types of
maps, viz
1. Linkage map
2. Cytogenetic or cytological maps
3. Physical maps.
Genetic mapping is based on the use of genetic
techniques to construct maps showing the positions
of genes and other sequence features on a genome.
Genetic techniques include cross-breeding
experiments or,
Case of humans, the examination of family
histories (pedigrees).
Physical mapping uses molecular biology
techniques to examine DNA molecules directly in
order to construct maps showing the positions of
sequence features, including genes.
Linkage Maps
A linkage map is a schematic representation of the relative
locations of various genetic markers present in the chromosomes
of an organism as determined from the frequency of
recombination between pairs of markers.
The recombination frequencies between marker pairs are
estimated from suitable mapping populations and are converted to
map or genetic distances.
Based on the genetic distance, the markers are grouped into
linkage groups, and their order in the linkage group is depicted as
the linkage map.
A special category of linkage maps called functional maps
depicts locations of different genes of the concerned species.
The conventional linkage maps also depict the genes
governing different phenotypic traits, but they are developed by
using the concerned traits as genetic markers.
The functional maps are developed by using molecular
markers located within genes or the gene sequences themselves
are used as markers.
Recombination Frequency
Recombination fraction is a measure of the distance between
two loci.
Two loci that show 1% recombination are defined as being 1
centimorgan (cM) apart on a genetic map.
1 map unit = 1 cM (centimorgan)
Two genes that undergo independent assortment have
recombination frequency of 50 percent and are located on
nonhomologous chromosomes or far apart on the same
chromosome = unlinked
Genes with recombination frequencies less than 50 percent are
on the same chromosome = linked
Recombination and linkage maps - Important facts
Linkage is the exception for law of independent assortment. Since
eukaryotic chromosomes are linear, the nucleotide sequences that are
physically close tend to be inherited together more often – when you make a
dihybrid cross you observe less of recombinants and more of parental types
in such cases. Linkage equilibrium does not exist there. However
individually you would obtain perfect Mendelian segregation.
Linkage is broken by recombination – the tighter the linkage the lower is
the frequency of recombination – corollary is that when genes are
physically too close, use a larger population to identify the rare
recombinants.
Linkage Mapping
Measuring distance between genes
The basis of linkage mapping is that since crossing over occurs at random locations,
the closer two genes are to each other, the less likely it is that a crossover will occur
between them. Thus, the percentage of gametes that had a crossover between
two genes is a measure of how far apart those two genes are. It is essential to
understand that, to estimate distance, we are scoring many meiotic events and
hence the gametes that are produced from the two parents, in linkage mapping.
One single crossing over will yield two parental and two recombinant products.
However there are thousands of meiotic events taking place during reproduction and
you are scoring twice as many events as the number of progeny.
In all these events, crossing over may not take place for the genes under analysis. In
some it would and in others it would not occur. This frequency of ‘some’ and
‘others’ is determined by the distance between the genes.
Hence recombination frequency is considered as the measure of the
distance between the genes.
Since for every chiasma there are two cross over products, and the
other two strands would not have involved in c/over, the fraction of
recombinant gametes does not exceed 50%.
T. H. Morgan and Alfred Sturtevant produced the first Drosophila
gene map in 1913. Morgan was the founder of Drosophila genetics, and
in his honor a recombination map unit is called a centiMorgan (cM).
A map unit, or centiMorgan, is equal to crossing over between 2 genes in
1% of the gametes. A test cross is used for this measurement since
here only one of the parents undergo meaningful meiosis.
Map distance (cM) = [no. of recombinants/total no. of progeny] x 100 in a test cross
(AB ab) F1 progeny with (ab ab) tester to look for recombination on these
chromosomes. Suppose you Get……
AB ab 583 <parental
ab ab 597 <parental
Ab ab 134 <recombinant
aB ab 134 <recombinant
Total= 1448
so…. 268 recombinants /1448 progeny
= 0.185 recombinants/progeny
= 18.5% recombinants
= 18.5 cM or mu
Now you can find distance between any two genes – but you
cannot construct a linkage map because you do not know
their order or relative positions for which you need to study a
three point test cross. This will tell you at least which gene is
in the middle.
Three Point Test cross
Start with pure breeding lines, Cross Parent 1(AA BB DD) with Parent 2(aa bb dd)
So you know the Parental chromosomes in the F1 have to be ABD and abc
Cross (ABD abd) F1 progeny with (abd abd) tester
Ab + aB = (45+89)+(94+40) recom
Suppose you Get……
268 recom/1448 total =0.185
ABD abd 580 A-B =18.5 mu
ABd abd 3
abD abd 5
Bd + bD = (3+40)+(5+45)
abd abd 592
AbD abd 45 93 recom/1448 total= 0.064
Abd abd 89
B-D =6.4 mu
aBD abd 94
aBd abd 40
Total= 1448 Ad + aD = (3+89)+(5+94)
191 recom/1448 total= 0.132
A-D =13.2 mu
so the order must be A-----D---B -13.2--6.4-
----18.5---
So how come 13.2 + 6.4 does not equal 18.5?
We missed the double recombinants on the first pass…
(45+89)+(94+40)+2(3+5) recom
284recom/1448 total = 0.196
A-B =19.6mu
ABD abd 580
ABd abd 3
Longer the distance, more abD abd 5
potential to underestimate abd abd 592
recomb freq. So it is advisable
to use a third gene or marker AbD abd 45
between the other two when Abd abd 89
the map distance is > 5 cM.
aBD abd 94
aBd abd 40
total= 1448
Chromosome interference: crossovers in one region decrease the probability of a second
crossover closeby.
Coefficient of coincidence = observed number of double recombinants divided by the
expected number of c/o
\
If the two crossovers were independent,
we would expect that the probability of seeing two recombination events occur
would be (Product rule)
0.132 between A-D AND 0.064 between D-B
0.132 X 0.064 = 0.008
For every 1448 progeny, this would be (1448x0.008)=12.23 double recombinants
We actually observed only (5+3)= 8 double recombinants
So the Coefficient of coincidence = observed double recombinants / expected
double recom = 8/12.23 =0.65
Interference = 1-Coefficient of coincidence = 1-0.65 = 0.35
Factors affecting recombination - Age, sex, radiation, chemicals, nutrition, temperature,
chromosomal aberrations, distance from the centromere and recombination hot spots
Problems For Constructing a genetic map
The relationship between recombination frequency and real distance
between the genes is not linear in any species. Genetic distances are
relative and not absolute.
Minimum double cross over distance also varies among species.
Mapping function is employed to correct for such issues.
How many genes - polymorphic pairs do you know between any
two parents?
How many crosses are you planning to make?
How many you are planning to score for recombination?
Mapping Function
Genetic map distances are additive but
recombination frequencies are not. The
relationship is linear only till 20 cM.
Recombination data refers to only features of
chromosomes at end points of intervals
considered.
Odd number of c/o produce recombinants but
even number of c/o do not. Double co frequency is
not uniform across genome and species.
There is also the phenomenon of interference.
To account for interference and to reliably
convert recombn. Frequency to mapping distance
mapping functions are used.
R = M (d) for longer distances.
Haldane; Kosambi; Fisher etc.
Interference is considered in Kosambi but not in
Haldane.
LOD score
The recombination frequency that yields the highest value of LOD
score is taken to be the most likely value of recombination between the
two genes.
LOD score (z) is the log to the base 10 of the ratio of probability of
obtaining the given data assuming linkage between the two genes with
a specified frequency of recombination to the probability of getting the
same data with independent segregation
Cytogenetic Maps
Cytogenetic map depicts the locations of various genes in the
chromosomes of a species relative to specific microscopically observable
landmarks in the chromosomes.
In most cases each chromosome has a characteristic banding pattern,
which may be either naturally present, e.g., in polytene chromosomes of
Drosophila, or is most commonly generated by specific staining protocols
like Giemsa C.
Approaches:
fluorescence in situ hybridization (FISH), including multicolor FISH (McFISH),
using gene sequences as probes;
human–mouse somatic cell hybridization, followed by genetic and cytogenetic
analyses of the hybrid clones; and
analysis of small changes in polytene chromosomes and the genetic alterations
associated with them.
Limitations of genetic Mapping
The resolution of a genetic map depends on the number of
crossovers that have been scored.
Genetic maps have limited accuracy
Physical Mapping
Physical map, the genes/molecular markers are depicted in the
same order as they occur in the chromosomes, but the distances
between adjacent genes/markers are depicted in terms of base
pairs.
The distance in terms of base pairs is known as physical
distance and is determined by either hybridization of appropriate
probes or sequence alignment to a good quality reference genome.
A plethora of physical mapping techniques has been developed to
address this problems of genetic mapping, the most important
techniques being:
Restriction mapping: which locates the relative positions on a
DNA molecule of the recognition sequences for restriction
endonucleases.
Fluorescent in situ hybridization (FISH): in which marker
locations are mapped by hybridizing a probe containing the
marker to intact chromosomes.
Sequence tagged site (STS) mapping: in which the positions
of short sequences are mapped by examining collections of
genomic DNA fragments by PCR and/or hybridization analysis.
Restriction mapping
First, the DNA molecule is digested with just one of the
enzymes and the sizes of the resulting fragments measured by
agarose gel electrophoresis.
Next, the molecule is digested with the second enzyme and
the resulting fragments again sized in an agarose gel.
Double restriction
Partial restriction
Fluorescent in situ hybridization
FISH enables the position of a marker on a chromosome or extended DNA
molecule to be directly visualized.
In FISH, the marker is a DNA sequence that is visualized by hybridization
with a fluorescent probe.
A sample of dividing cells is dried onto a microscope slide and treated with
formamide so that the chromosomes become denatured but do not lose their
characteristic metaphase morphologies.
The position at which the probe hybridizes to the chromosomal DNA is
visualized by detecting the fluorescent signal emitted by the labeled DNA.
A disadvantage of this method is that the highly condensed nature of
metaphase chromosomes means that only low resolution mapping is possible,
two markers having to be at least 1 Mb apart to be resolved as separate
hybridization signals.
Fluorescent in situ
hybridization.
Sequence tagged site mapping
Sequence tagged site, or STS, is simply a short DNA
sequence, generally between 100 bp and 500 bp in length, that is
easily recognizable and occurs only once in the chromosome or
genome being studied.
DNA fragments from a single chromosome or from the entire
genome is needed.
A fragment collection has been prepared from a single
chromosome, with each point along the chromosome represented,
on average, five times in the collection.
The data from which the map will be derived are obtained by
determining which fragments contain which STSs
This can be done by hybridization analysis but PCR is generally
used because it is quicker and has proven to be more amenable to
automation.
The chances of two STSs being present on the same fragment
will, of course, depend on how close together they are in the
genome.
If they are very close then there is a good chance that they will
always be on the same fragment; if they are further apart then
sometimes they will be on the same fragment and sometimes they
will not
The data can therefore be used to calculate the distance
between two markers, in a manner analogous to the way in
which map distances are determined by linkage analysis.
The map distance is based on the frequency at which breaks
occur between two markers
Flow cytometry
A mixture of fluorescently stained
chromosomes is passed through a small aperture
so that each drop that emerges contains just one
chromosome.
The fluorescence detector identifies the signal
from drops containing the correct chromosome
and applies an electric charge to these drops.
When the drops reach the electric plates, the
charged ones are deflected into a separate
beaker.
All other drops fall straight through the
deflecting plates and are collected in the waste
beaker
Genetic vs. Physical Distance
Map distances based on recombination frequencies
are not a direct measurement of physical distance along
a chromosome
Recombination “hot spots” overestimate physical
length
Low rates in heterochromatin and centromeres
underestimate actual physical length
