LINKAGE AND
GENETIC MAPPING
Lecture 7
Molecular Genetics
INTRODUCTION


Eukaryotic genomes contain hundreds to thousands
of genes
 Yet most species have fewer than 50
chromosomes in a haploid set
 Humans have only 23 chromosomes per
haploid set but have ~25,000 genes
Therefore, each chromosome carries hundreds or
even thousands of different genes
 The transmission of such genes may go against
Mendel’s law of independent assortment
LINKAGE & CROSSING OVER
Genetic linkage is the tendency of genes that are
located close to each other on a chromosome to be
inherited together during meiosis.
Genes whose loci are nearer to each other are less
likely to be separated onto different chromatids
during chromosomal crossover, and are therefore
said to be genetically linked.
Chromosomes are called linkage groups
 They contain a group of genes that are linked together
on the same DNA molecule
The number of linkage groups is equal to the number of
types of chromosomes of the species
 For example, in humans

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22 autosomal linkage groups
An X chromosome linkage group
A Y chromosome linkage group
If no crossing over, the alleles of
all genes located on the same
chromosome would be inherited
together
Crossing Over May Produce
Recombinant Phenotypes


In diploid eukaryotic species,
linkage can be “undone”
during meiosis as a result of
crossing over
Crossing over
 Occurs during prophase I
of meiosis
 Non-sister
chromatids of
homologous chromosomes
exchange DNA segments
IF NO CROSSING
OVER IN REGION
BETWEEN THE TWO
GENES
= 100% NonRecombinants
The arrangement of linked
alleles has not been altered
IF CROSSING OVER IN
REGION BETWEEN THE
TWO GENES
= 50% Non-Recombinants
and 50% Recombinants
Non-recombinant
gametes
Recombinant gametes
Recombination Frequency

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Recombination fraction or frequency 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 carry out independent assortment
have recombination frequency of 50 percent and are
located on nonhomologous chromosomes or far apart
on the same chromosome = un-linked

Genes with recombination frequencies less than 50
percent are on the same chromosome = linked
Calculation of Recombination Frequency

The percentage of recombinant progeny (off-springs)
produced in a cross is called the recombination
frequency, which is calculated as follows:
Recombinant frequency =
Number of recombinant offspring
Total number of offspring
X 100
Recombination Frequency
Genetic map
Genetic maps allow us to estimate the
relative distances between linked
genes, based on the probability that a
crossover will occur between them

Experimentally, the percentage of recombinant offspring is
correlated with the distance between the two genes


If the genes are far apart  many recombinant offspring
If the genes are close  very few recombinant offspring
Recombinant frequency =

Number of recombinant offspring
X 100
Total number of offspring
The units of distance are called map units (mu)
 They are also referred to as centiMorgans (cM)
One map unit is equivalent to 1% recombination frequency

Genetic mapping experiments are typically accomplished
by carrying out a testcross

A mating between an individual that is heterozygous for two or more
genes and one that is homozygous recessive for the same genes

This cross concerns two linked genes affecting bristle length and
body color in fruit flies
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s = short bristles
s+ = normal bristles

e = ebony body color
e+ = gray body color
One parent displays both recessive traits
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It is homozygous recessive for the two genes (ss ee)
The other parent is heterozygous for the two genes
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The s and e alleles are linked on one chromosome
The s+ and e+ alleles are linked on the homologous chromosome
Chromosomes are
the product of a
crossover during
meiosis in the
heterozygous parent
Recombinant
offspring are fewer
in number than
nonrecombinant
offspring

Estimate the distance (recombinant frequency) between the
two genes
Recombinant frequency =
Number of recombinant offspring
X 100
Total number of offspring
=
76 + 75
542 + 537 + 76 + 75
X 100
= 12.3 map units

Therefore, the s and e genes are 12.3 map units apart
from each other along the same chromosome
Trihybrid Crosses
Data from trihybrid crosses can also yield information about map
distance and gene order. The following experiment outlines a
common strategy for using trihybrid crosses to map genes

In this example, we will consider fruit flies that differ in
body color, eye color and wing shape

b+ = gray body color > b = black body color

pr+ = red eye color > pr = purple eye color

vg+ = normal wings > vg = vestigial wings

Step 1: Cross two true-breeding strains that differ
with regard to three alleles.
Female is mutant
for all three traits

Male is homozygous
wildtype for all three
traits
The goal in this step is to obtain a F1 individuals that
are heterozygous for all three genes

Step 2: Perform a testcross by mating F1 female
heterozygotes to male flies that are homozygous
recessive for all three alleles
During gametogenesis in the heterozygous female F1 flies,
crossovers may produce new combinations of the 3 alleles

Analysis of the F2 generation flies will allow us to map
the three genes
The three genes exist as two alleles each
3
 Therefore, there are 2 = 8 possible combinations of offspring
 If the genes assorted independently, all eight combinations
would occur in equal proportions
 It is obvious that they are far from equal

In the offspring of crosses involving linked genes,
I.
II.
III.
Non-recombinant (phenotypes of grandparents) occur most
frequently
Double crossover phenotypes occur least frequently
Single crossover phenotypes occur with “intermediate”
frequency

Step 3: Collect data for the F2 generation
Phenotype
Gray body, red eyes, normal wings
Gray body, red eyes, vestigial wings
Gray body, purple eyes, normal wings
Number of Observed
Offspring (males and
females)
411
61
2
Gray body, purple eyes, vestigial wings
30
Black body, red eyes, normal wings
28
Black body, red eyes, vestigial wings
Black body, purple eyes, normal wings
Black body, purple eyes, vestigial wings
1
60
412
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Step 4: Calculate the map distance between adjacent pairs
of genes
The combination of traits in the double crossover tells us which
gene is in the middle. A double crossover separates the gene in
the middle from the other two genes at either end

Thus, the gene for eye color lies between the genes for body
color and wing shape
We know that pr is in the middle, so map b-pr and then pr-vg
b- -----pr ------vg
Body color / eye color
Non-recombinant
offspring
Gray body, red eyes
(411 + 61)
Black body, purple eyes
(412 + 60)
Total
recombinant Offspring
Total
472
Gray body, purple eyes
(30 + 2)
32
472
Black body, red eyes
(28 + 1)
29
944

61
The map distance between body color and eye color is
Recombinant frequency =
61
X 100 = 6.1 map units
944 + 61
Eye color / wing shape
Parental offspring
Red eyes, normal wings
(411 + 28)
Purple eyes, vestigial wings
(412 + 30)
Total
Nonparental Offspring
Total
439
Red eyes, vestigial wings
(61 + 1)
62
442
Purple eyes, normal wings
(60 + 2)
62
881

124
The map distance between eye color and wing shape is
Recombinant frequency =
124
881 + 124
X 100 = 12.3 map units

Step 5: Construct the map

Based on the map unit calculation the body color and
wing shape genes are farthest apart
 The eye color gene is in the middle

The data is also consistent with the map being drawn
as vg – pr – b (from left to right)
Total distance b – vg = 18.4 mu
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Mapping Function
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When mapping two genes that are relatively far map
distance underestimated because most doublecrossovers cannot be detected
Four types of double crossovers possible
 Two strand
 Three strand
 Four strand
Two strand counted as no crossovers
Three strand counted as single crossover
Four strand is the only one counted as a double crossover
Overall, double crossovers result in 50% recombinants
and 50% non-recombinants, as if they had not occurred at
all!
Types of Crossing over according to the
number of strands involved in crossing
over
1. Two Stranded crossing over

Result in new
recombination
2. Four stranded crossing over
Result in 50% new
recombination and 50%
parental
or
non
recombinant
A chiasma
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A chiasma (plural: chiasmata), in genetics, is thought to be the
point where two homologous non-sister chromatids exchange
genetic material during chromosomal crossover during meiosis
(sister chromatids also form chiasmata between each other, but
because their genetic material is identical, it does not cause any
change in the resulting daughter cells)
. The chiasmata become visible during the diplotene stage of
prophase I of meiosis, but the actual "crossing-over" of genetic
material is thought to occur during the previous pachytene
stage.
When each tetrad, which is composed of two pairs of sister
chromatids, begins to split, the only points of contact are at the
chiasmata.
chiasma frequency
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chiasma frequency = 2 x recombination frequency where
recombination frequency is:
recombination frequency = (no. of recombinants x 100) /
(total no. of progeny)
The phenomenon of genetic chiasmata (chiasmatypie) was
discovered and described in 1909 by Frans Alfons Janssens,
a Jesuit professor at the University of Leuven in Belgium.
[1][2] A bivalent refers to the two homologous chromosomes
(4 chromatids).
The chiasmata refers to the actual break of the
phosphodiester bond during crossing over. The larger the
number of map units between the genes, the more crossing
over occurs.
Kinds of Crossing-over:
Depending upon the number of chiasmata appeared, kinds of
crossing-over of can be
(i) Single cross-over:
 In this case, only one chiasma is formed which leads to formation
of single cross-over gametes. It is most common type of cross-over.
(ii) Double cross-over:
 In double cross-over, two chiasmata develop. These chiasmata may
appear between the same chromatids or between different
chromatids. This type of crossing over forms double crossing-over
gametes.
(iii) Multiple cross-over:
 Here, ‘more than two chiasmata are constituted. It may be further
classified into triple (3 chiasmata), quadruple (4 chiasmata) and so
on. Multiple crossing-over is of rare occurrence.

Factors influencing crossing-over:
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Distance between the genes. More the distance between two
genes on same chromosome, higher the frequency of crossing
over.
Significance of crossing-over:
(i) This process provides a vast store of genetic variability in
sexually reproducing organisms.
(ii) Useful recombinations are used by plant and animal
breeders. Breeders try to break up the linkages by crossing-over
to get combinations of useful traits in the progeny.
(iii) This process produces new combination of genes
(recombination). Green revolution is mainly due to selective
picking up of useful genetic recombination developed by the
process of crossing-over.
Asignment
The following experiment outlines a data from trihybrid crosses
of fruit flies that differ in body color, eye color and wing shape
define
1- single crossing over
2- double crossing over
3-calculate a map distance and draw gene order

S+ = normal bristles
> s = short bristles

W + = red eye color > w = white eye color

Cu + = normal wings > cu = curly wings
Back cross result
Phenotypes
normal wings normal bristles red eyes
normal wings normal bristles white eyes
normal wings short bristles red eyes
normal wings short bristles white eyes
curly wings normal bristles red eyes
curly wings normal bristles white eyes
curly wings
short bristles red eyes
curly wings
short bristles white eyes
Genotypes
Cu +
S+
Cu +
Cu +
Cu
Cu
Cu
W+
S+
S
Cu +
Cu
Frequency
S
S+
S+
S
S
W
435
1
W+
16
W
45
W+
38
W
17
W+
1
W
425