Includes information regarding Linkage and crossing over

1. 1
June 27, 2020
Linkage and Crossing Over
Dr. A. S. Wabale
Assistant Professor and Research Guide
Post Graduate Department of Botany and Research Centre,
Padmashri Vikhe Patil College of Arts, Science and Commerce,
Pravaranagar- 413 713
dranilwabale78@gmail.com

2. 2
August 21, 2020
CONTENTS
 Linkage- Definition and Types
 Crossing over: Definition and Types
 Construction of a linkage map
 Two point test cross
 Three point test cross
Dr. A. S. Wabale

3. 3
June 27, 2020 Dr. A. S. Wabale
Immediately after the rediscovery of Mendel's Laws of Inheritance, Bateson and Punnett
(1905) in sweet pea found that two pairs of alleles do not separate independently. Morgan
(1910) also observed the similar fact in Drosophilla and Hutchinson recorded a
comprehensible case of linkage in Maize.
Conclusion of all above findings was that the genes come into in groups rather than
individually. The trend of two or more genes to stay together in the same chromosome
during inheritance is referred to as linkage .
Main features of linkage are as follows:
1. Linkage occupy two or more genes that are placed on the same chromosome in a linear
fashion
2. It may involve either dominant or recessive genes or some dominant and some recessive
genes
3. Linkage involves those genes which are closely positioned
4. Linkage may involve either two or more desirable traits or all undesirable traits or some
desirable and some undesirable traits
5. The potency (effectiveness) of linkage depends on the distance between the linked genes.
Lesser the distance, higher the strength
6. If crossing over does not occur, all the genes situated on one chromosome are expected to
be inherited together. Thus the maximum number of linkage groups in an organism is
equal to its haploid chromosome number
7. Linkage can be broken by repeated intermating of aimlessly (at random) selected plants in
segregating populations for several generations
WHAT MEANS LINKAGE?

4. 4
June 27, 2020
WHAT MEANS LINKAGE?
Dr. A. S. Wabale

5. 5
June 27, 2020
WHAT ARE TYPES OF LINKAGE?
Dr. A. S. Wabale
1. Based on Crossing Over
i. Complete Linkage is a linkage in which crossing over does not occur. In other words, when only
parental types are obtained from the test cross progeny, it refers to absolute linkage E.g. Complete
linkage in Drosophila
Parents Gray Body Colour and Long Wings X Black Body Colour and Vestigial Wings
(GL/GL) (gl/gl)
Gametes GL gl
F1 Generation Gray Body Colour and Long Wings
GL/gl
Test Cross Gray Body Colour and Long Wings Black Body Colour and Vestigial Wings
(GL/gl) X (gl/gl)
F2 Generation
50% Gray Body Colour and Long Wings
50 % Black Body Colour and Vestigial Wings
Gametes gl gl
GL GL/gl GL/gl
gl gl/gl gl/gl
G G
L L
g g
l l
G
L
g
l
g
l
G
L
g
l
G
L
g
l
g
l
g
l
G
L
g
l
g
l

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June 27, 2020
WHAT ARE TYPES OF LINKAGE?
Dr. A. S. Wabale
ii. Incomplete Linkage is a linkage in which crossing over occurs between linked genes. As a result
along with parental combinations, recombination are also observed in the test cross progeny.
Parents Gray Body Colour and Long Wings X Black Body Colour and Vestigial Wings
b+ b+ vg+ vg+ bb vg vg
Gametes b+ vg+ b vg
F1 Generation Gray Body Colour and Long Wings
b+ b vg+ vg
Test Cross Gray Body Colour and Long Wings Black Body Colour and Vestigial Wings
b+ b vg+ vg X b b vg vg
Gametes b+ vg+ , b+ vg , b vg+ , b vg b vg
F2 Generation
b+ b
vg+ vg
b+ b
vg vg
b b
vg+ vg
b b
vg vg
Parental Types Recombinants
b+ b+
vg+ vg+
b b
vg vg
b+
vg+
b
vg
b+ b
vg+ vg
b+ b
vg+ vg
b b
vg vg

7. 7
WHAT ARE TYPES OF LINKAGE?
Dr. A. S. Wabale
2. Based on Genes Involved
i. Coupling Linkage: It refers to linkage either between dominant genes or between
recessive genes. This type of linkage is found in pea, maize and several other crops
ii. Repulsion Linkage: It refers to linkage of some dominant genes with some recessive genes.
This type of linkage is found in pea, maize and several other crops
3. Based on Chromosomes Involved
i. Autosomal Linkage: It refers to linkage of such genes which are located on other sex
chromosomes (Autosomes)
ii. X-Chromosomal Linkage: It refers to linkage of such genes which are located on sex
chromosomes
June 27, 2020

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WHAT IS CROSSING OVER?
Dr. A. S. Wabale
Crossing Over refers to the interchange of part between non-sister chromatids of homologous pair during
meiotic prophase (pachytene). It means that, the genetic material is exchanged between non-sister
chromatids by breakage and reunion at a specific point. The term ‘crossing over’ was first used by Morgan
and Cattell in 1912.
Main features of crossing over are as follows
1. Crossing over happens during meiotic prophase i.e. during pachytene. Each pair of homologous
chromosome has four chromatids at such time
2. Crossing over takes place between two non-sister chromatids. i.e. one chromatid from each of the
chromosome is involved in the process of crossing over.
3. It is universally accepted that crossing over occurs at four strand stage
4. Double or multiple crossing over may occupy all four, three or two of the four chromatids, which is very
rare
5. Crossing over directs to recombination or new combinations between linked genes i.e. It generally build
up two recombinant types or crossover types and two parental types or non-crossover types
6. It leads to exchange of equal segments or genes and recombination is always reciprocal. However, in
some cases unequal crossing over has also been reported
7. The frequency of recombinants can be found out from the test cross progeny as
Number of Recombinants
Total Progeny
X 100
Crossing over Frequency (%) =
June 27, 2020

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WHAT IS THE MECHANISM CROSSING OVER?
Dr. A. S. Wabale
Steps in crossing-over can be enlightened as under
1. Synapsis: In prophase-I of meiosis, the homologous chromosomes pair lengthwise due to a force of mutual
attraction. The pairing starts at one or more points and proceeds along the whole length in a zipper
manner. This process of pairing is termed as synapsis. Paired homologous chromosomes are called
bivalents. A synaptonemal complex (a protein structure) is formed between homologous chromosomes
(two pairs of sister chromatids) to carry on synapsis and recombination during meiosis-I in eukaryotes.
2. Duplication of chromosomes: It changes the bivalent nature of chromosome to four- stranded stage or
tetravalent occurs immediately after synapsis. Four stranded stage of chromatids occurs due to splitting of
homologous chromosomes into sister chromatids attached with un-splitted centromeres.
June 27, 2020
Longitudinal
Splitting
Sister
Chromatid
s
Non-sister
Chromatids
Synaptonemal
complex

10. 10
WHAT IS THE MECHANISM CROSSING OVER?
Dr. A. S. Wabale
3. Crossing-over: It occurs during pachytene stage. Non-sister chromatids of homologous pair twist over
each other due to action of enzyme endonuclease. The chromatids get connected with each other at points
known as chiasmata. It can take place at several points. According to Stem and Hota (1978) breaks or nick
appear in the chromatids due to endonuclease enzyme. A gap is developed between these nicks with the
help of enzyme exonuclease. The segments of chromatids separate in between two gaps with the help of an
enzyme called U-protein. These segments re-unite with the help of R-protein. The genes at distant loci
undergo crossing-over but closely placed genes do not undergo cross-over and exhibit the phenomenon of
linkage.
At diakinesis of prophase-I chiasmata move to the end of bivalent by a process termed as
terminalization as a result the chromatids move away from each other so that the homologous
chromosomes are separated completely. It is clear that one of the chromatids of each chromosome carries a
portion of chromatid from its homologous chromosome. At the end of meiosis, four types of gametes are
formed of which two are of parental types and two are of recombinant types.
June 27, 2020
Synapsis
HomologousPair
Sister Chrom. Sister Chrom.
Non-sister Chromatids
Cross over Terminalization Formation of
gametes
ParentalType
RecombinantType
Chiasmata

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Depending upon the number of chiasmata formed, crossing over may be classified into following three
types, viz.,
i. Single Crossing Over: In this condition a single chiasma is formed between two non-sister chromatids of
homologous chromosomes.
ii. Double Crossing Over: In this condition, two chiasmata are formed between non-sister chromatids of
homologous chromosomes. Double crossovers may involve either two strands or three or all the four
strands.
iii. Multiple Crossing Over: Presence of more than two crossovers between non-sister chromatids of
homologous chromosomes is referred to as multiple crossing over. Frequency of such type of crossing
over is extremely low.
WHAT ARE THE TYPES OF CROSSING OVER?
Dr. A. S. Wabale
June 27, 2020

12. 12
The frequency of crossing over is affected by several factors as discussed below:
i. Distance: The distance between genes affects the frequency of crossing over. Greater the
distance between genes higher is the chance of crossing over and vice versa.
ii. Age: Generally crossing over decreases with advancement in the age in the female
Drosophila.
iii. Temperature: The rate of crossing over in Drosophila increases above and below the
temperature of 22°C.
iv. Sex: The rate of crossing over also differs according to sex. There is lack of crossing over in
Drosophila male and female silk moth.
v. Nutrition: Presence of metallic ions like calcium and magnesium in the food caused reduction
in recombination in Drosophila. However, removal of such chemicals from the diet increased
the rate of crossing over.
vi. Chemicals: Treatment with mutagenic chemicals was found to increase the frequency of
crossing over in Drosophila female.
vii. Irradiation: Irradiation with X-rays and gamma rays was found to enhance the frequency of
crossing over in Drosophila females.
viii. Structural Changes: Structural chromosomal changes especially inversions and
translocations reduce the frequency of crossing over in the chromosomes where such changes
are involved.
ix. Centromere Effect: Generally genes that are located adjacent to the centromere show
reduced frequency of crossing over
WHAT ARE THE FACTORS AFFECTING CROSSING OVER?
Dr. A. S. Wabale
June 27, 2020

13. 13
Crossing over is useful in three principal ways, viz:
i. Creation of Variability:
Crossing over leads to recombination or new combination and thus is a potential
genetic mechanism for creating variability which is essential for improvement of genotypes
through selection.
ii. Locating Genes:
Crossing over is a useful tool for locating genes in the chromosomes.
iii. Linkage Maps:
Crossing over plays an important role in the preparation of chromosome maps or
linkage maps. It provides information about frequency of recombination’s and sequence of
genes which are required for preparation of linkage maps.
WHAT IS THE SIGNIFICANCE OF CROSSING OVER?
Dr. A. S. Wabale
June 27, 2020

14. 14
Gene mapping is the process of determining the genes and their location along the length of
chromosome.
T. D Morgan laid the foundation of gene map by identifying gene for white eye Drosophila on X-
chromosome of mutant.
The procedure of gene mapping was developed by Alfred H Sturtevent. His procedure is based
on the principle of linkage. The gene located on same chromosome inherits together known as
linked gene. However, some gene on same chromosome could separate during meiosis and new
combination of genes are formed. The phenomenon of recombination is due to crossover and
chiasmata formation during meiosis.
Gene map is by counting the number of crossovers that occur during meiosis. However, because
the actual crossover events cannot be seen, they cannot count them directly. So, recombination
frequency is calculated to estimate the crossover.
Chiasmata are counted through cytological analysis, whereas recombinant chromosomes are
counted through genetic analysis.
Map distance is the distance between genes.
The unit of map distance is written as centimorgan or map unit
WHAT IS GENE MAPPING?
Dr. A. S. Wabale
June 27, 2020

15. 15
June 27, 2020
WHAT IS TWO POINT TEST CROSS?
Dr. A. S. Wabale
A test cross discovered by Mendel generally involves crossing of Heterozygote or F1 hybrid
individual with a homozygous recessive individual to determine the recombinant frequency and
zygosity.
A two-point test cross is done to determine the recombinant frequency between 2 linked genes.
The Mendelian genotypic dihybrid ratio (round yellow and green wrinkled seeds) is 1:1:1:1, which
shows that genes are not linked.
The distance between those two genes determines the recombinant frequency and chances of
crossover during meiotic division. Three situations can be considered for these two genes locuses R
and Y:
1. If R and Y genes are very close: crossover occurs outside the loci and inherited as linked genes;
therefore, no recombinant classes and 100% parental.
2. If R and Y genes are very far: crossover occurs and 50% will be parental and 50% recombinant.
3. If R and Y genes are at an intermediate distance: crossover can occur and can generate 0–50%
of recombinant forms and 50–100% parental.
Thus linkage mapping or map distance can be developed using a two-point testcross based on the
recombinant frequency.

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June 27, 2020
EXAMPLE TO STUDY TWO POINT TEST CROSS
Dr. A. S. Wabale
Example: A two-point testcross in Drosophila with following character can be studied as an
example:
• Eye color: purple (pr) or red (pr+) and Wing length: vestigial (vg) or normal (vg+)
Gametes prvg prvg prvg prvg
pr+vg+ pr+vg+prvg pr+vg+prvg pr+vg+prvg pr+vg+prvg
pr+vg pr+vgprvg pr+vgprvg pr+vgprvg pr+vgprvg
prvg+ prvg+prvg prvg+prvg prvg+prvg prvg+prvg
prvg prvgprvg prvgprvg prvgprvg prvgprvg
Parents Red Eye and Normal Wings X Purple Eye and Vestigial Wings
(pr+vg+/pr+vg+) (pr vg/pr vg)
Gametes pr+vg+ pr vg
F1 Generation Red Eye and Normal Wings
pr+pg vg+vg
Test Cross Red Eye and Normal Wings Purple Eye and Vestigial Wings
pr+vg+/pgvg prvg/prvg
If Crossing over occurs following types of gametes will be produced
pr+ pr+
vg+ vg+
pr pr
vg vg
pr+
vg+
pr
vg
pr+
vg+
pr
vg
pr+
vg+
pr
vg
pr pr
vg vg

17. 17
F2 Generation: Ratio deviating from 1:1:1:1 showing that alleles are on same chromosomes and are linked
together.
pr+
vg+
pr
vg
pr+
vg
pr
vg
pr
vg+
pr
vg
pr
vg
pr
vg
Parental Type Parental Type
Recombinant Type Recombinant Type
Red Eye and
Normal Wings
1339
Purple Eye and
Vestigial Wings
1195
Red Eye and
Vestigial Wings
151
Purple Eye and
Normal Wings
154
Parental Types: Highest number of off springs
Recombinant types: Lowest number of off springs
Percent Recombination Frequency = Total Number of Recombinants
Total Number of off springs
X 100
= 151 + 154
1339 + 1195
X 100
= 305
2534
X 100
Percent Recombination Frequency = 12.03
Percent Recombination Frequency α map distance
Map Distance between two genes pr and vg is 12.03 cM or Map unit
EXAMPLE TO STUDY TWO POINT TEST CROSS
June 27, 2020
pr
vg
12.03 cM
Dr. A. S. Wabale

18. 18
June 27, 2020
WHAT IS THREE POINT TEST CROSS?
Dr. A. S. Wabale
A three-point test cross involves three linked genes. It is done
1. To determine the recombinant frequency between 2 linked genes.
2. To determine the linear order in which the genes are arranged on
the chromosome
3. To determine the relative distances between the linked genes
According to Morgan, recombination percentage is equal to map distance which can
be expressed as Mu (Map units, Morgan Units or centimorgan)
In a test cross involving three linked genes, the parental types are expected to be
more frequent and double cross over to be the least frequent.
Percent Recombination Frequency = Total Number of Recombinants
Total Number of off springs
X 100

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June 27, 2020
EXAMPLE TO STUDY THREE POINT TEST CROSS
Dr. A. S. Wabale
Example: A trihybrid test cross in Drosophila melanogaster involving three linked genes Scoute
(SC), Echinus (EC) and Cross Vein less (CV), the following progeny has been obtained
Echinus (Ec + +) 8576
Scoute and Cross vein less (Sc Cv +) 8808
Scoute and Echinus (Sc Ec +) 681
Cross vein less (Cv + +) 716
Echinus and Cross vein less (Ec Cv +) 1002
Scoute (Sc + +) 997
Scoute, echinus and Cross vein less (Sc Ec Cv) 4
Wild type (+ + +) 1
From the given data find out
a) Parental types
b) Linear order of the genes
c) Distance between each of the genes
d) Construct a genetic map

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June 27, 2020
EXAMPLE TO STUDY THREE POINT TEST CROSS
Dr. A. S. Wabale
Solution:
Step-1: Identify Parental and Double Cross Over Types
Parental types are the highest number of offspring's. i.e., Ec + + = 8576 and Sc Cv + =8808
Double Cross Over are the lowest number of offspring's i.e. Ec Sc Cv = 4 and + + + = 1
Step-2: What is actual gene order?
Ec Sc Cv / Ec Cv Sc / Sc Ec Cv / Sc Cv Ec
Step-3: Compare parental and double cross over types to get gene order
Parental types = Sc Cv + and Ec + +
Double Cross Over = Ec Sc Cv and + + +
Ec is not matching between parental and double cross over types. It means that gene Ec is in
the middle of other two genes. So the proper gene sequence is Sc---Ec---Cv

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June 27, 2020
EXAMPLE TO STUDY THREE POINT TEST CROSS
Dr. A. S. Wabale
+ EC
SC CV
+
+
No Crossing Over
(Parental Types)
A single crossing over occurring between the loci SC and EC will result in the formation of
recombinants i.e. Scoute and Echinus (681) and Cross Vein Less (716)
+ EC
SC CV
+
+
Single Cross Over
(Recombinant Types)
A single crossing over occurring between the loci EC and CV will result in the formation of
recombinants i.e. Echinus and Cross Vein Less (1002) and Scoute (997)
+ EC
SC CV
+
+
Single Cross Over
(Recombinant Types)
Step-4: Determine the single and double cross over’s
The most frequent phenotypes among the offspring will be the parents (Parental ditypes or non-crossovers).
So the progeny Echinus with 8576 and Scoute, Cross Vein Less with 8808 will be the parental types
+
SC
CV
EC +
+
+
+
SC
CV
+
EC

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June 27, 2020
EXAMPLE TO STUDY THREE POINT TEST CROSS
Dr. A. S. Wabale
A double cross over occurring between the loci SC and CV will result in formation of
recombinants i.e. Scoute, Echinus and Cross Vein Less (4) and wild types (1)
+ EC
SC CV
+
+
Double Cross Over
(Recombinant Types)
From the given test cross data let us find out the total progeny.
It is 8576 + 8808 + 681 + 716 + 1002 + 997 + 4 + 1 = 20785
Out of these 17384 (8576 + 8808) are parental types and 3401 (681 + 716 + 1002 + 997 + 4 + 1)
are the recombinants
+
SC CV
+
EC
+

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June 27, 2020 Dr. A. S. Wabale
2. % recombination between the loci EC-CV = No. of recombinants produced as a result
of cross over between the Loci EC & CV
Total number of progeny
= 1999
20785
= 9.61 %
X 100
X 100
3. % recombination between the loci SC-CV = No. of recombinants produced as a result
of cross over between the Loci SC & CV
Total number of progeny
= 5
20785
= 0.02 %
X 100
X 100
EXAMPLE TO STUDY THREE POINT TEST CROSS
Now let us find out the percent recombination frequency
1. % recombination between the loci SC-EC = No. of recombinants produced as a result
of cross over between the Loci SC & EC
Total number of progeny
= 1397
20785
= 6.72 %
X 100
X 100

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June 27, 2020 Dr. A. S. Wabale
Step-5: To find out the distance between genes SC and CV, we must count all cross over's (both
single and double) that occur in the region
Total recombination percentage between Loci Sc and Ec = 6.72 + 0.01 = 6.73%
Total recombination percentage between Loci Ec and Cv = 9.61 + 0.01 = 9.62%
Total recombination percentage between Loci Sc and Cv = 6.72 + 9.61 + 0.02 = 16.35%
According to Morgan, 1 % of recombination is equal to 1Mu (map unit or centi morgan). If we
depict these values on a imaginary line, the genetic map thus obtained will be as follows:
SC
EC
6.73 Mu
9.62 Mu
CV
16.35 Mu
EXAMPLE TO STUDY THREE POINT TEST CROSS

25. 25
WHAT IS THE DIFFERENCE BETWEEN LINKAGE
AND CROSSING OVER ?
Dr. A. S. Wabale
June 27, 2020
Sr. No. Linkage Crossing Over
1 Keeps genes together Leads to separation of linked genes
2 Involves individual chromosome Involves non-sister chromatids of the
homologous pair
3 Linkage groups can never be more
than haploid chromosome
number
Frequency of crossing over can never
exceed 50%
4 It reduces variability It increases variability by forming new
gene combinations
5 Provides higher frequency of
parental types than recombinant
types in test cross progeny
Provides equal frequency of parental and
recombinant types in test cross progeny

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1. Define linkage and write main features of linkage.
2. Explain complete linkage with suitable example.
3. Explain incomplete linkage with suitable example.
4. Explain linkage based on genes involved.
5. Explain linkage based on chromosomes involved.
6. Define crossing over and write main features of crossing over.
7. Explain in detail the mechanism of crossing over.
8. Comment on the types of crossing over
9. Comment on the factors affecting crossing over.
10. Give significance of crossing over.
11. Write a note on gene mapping.
12. Explain two point test cross with suitable example.
13. Explain three point test cross with suitable example.
14. Give difference between linkage and crossing over.
15. With help of given data find out the sequence of linked genes, distance between genes and construct a
gene map
June 27, 2020
ASSIGNMENT
Dr. A. S. Wabale