Course: Fundamentals of Genetics

Class: - Ist Year, IInd Semester

Lecture No. XVIII
Title of topic: - Estimation of linkage
Prepared by- Vinod Kumar, Assistant Professor, (PB & G)
College of Agriculture, Powarkheda
Linkage
Sutton and Boveri proposed the chromosome theory of inheritance. According to
chromosome theory of inheritance, it is well established that many genes are located in
each chromosome in a linear fashion. It may therefore be expected that all genes
located in same chromosome would move to same pole during cell division. As a
consequence, such genes will fail to show independent segregation and would tend to
be inherited together. This tendency of genes to remain together in their original
combination during inheritance is called linkage. Mendel’s law of independent
assortment is applicable only when the genes are located in different chromosomes
while linkage refers to the genes located in the same chromosome.
The phenomenon of linkage was first reported by Bateson and Punnet in 1906. They
studied flower colour and pollen shape in sweet pea involving two varieties / races.
Plants with red flowers and long pollen grains were crossed to plants with white
flowers and short pollen grains. All the F1 plants had red flowers and long pollen grains,
indicating that the alleles for these two phenotypes were dominant. When the F1 plants were
self-fertilized, Bateson and Punnett observed a peculiar distribution of phenotypes among
the offspring. Instead of the 9:3:3:1 ratio expected for two independently assorting genes,
they obtained a ratio of 24.3:1.1:1:7.1. We can see the extent of the disagreement between
the observed results and the expected results at the bottom of Figure 7.2. Among the 803 F 2
plants that were examined, the classes that resembled the original parents (called the
parental classes) are significantly overrepresented and the two other (non parental) classes
are significantly under represented.
 The correct explanation for the lack of independent assortment in the data is that the
genes for flower color and pollen length are located on the same chromosome—that is, they
are linked.
Phases of linkage: In the two races experimented one parent has purple flowers with
long pollen grains. The other parent has red flowers and round pollen grains. The
character purple (P) is a simple monogenic dominant to red (p); while long (L) pollen is
dominant to round (l) pollen, when these two plants were crossed the F1 (PL/pl) was
purple flowered with long pollen. But in F2, the ratio of four types of plants deviated from
the normal di-hybrid ratio of 9:3:3:1 expected on the principle of independent assortment
of flower colour and pollen shape.
PARENTS Purple Long x Red Round
PL/PL pI/pI
F1 Purple Long
PL/pl
F2 Purple long Purple round Red long Red round Total
Actual numbers 4831 390 393 1338 6952
Expected numbers 3910.5 1303.5 1303.5 434.5 6952
Expected Ratio 9/16 3/16 3/16 1/16
The plants with the character combination as in the original parents are known as
parental forms or parental combinations, i.e., (i) Plants with purple flowers and long
pollen and (ii) Plants with red flowers and round pollen. Plants possessing one character
from one parent and another character from the second parent are known as
recombined types or recombinations or new combinations or cross-overs, i.e. (i) plants
with red flowers and long pollen and (ii) plants with purple flowers and round pollen.
From the above table the following features can be noted that in F2, there are
both parental forms and recombinations. The chief peculiarity of the results in the above
table was that parental forms are in far excess of the expected number while the
recombinations were fewer. This deviation from the expected ratio is due to linkage of
the character pairs, viz., 1. Purple flowers (P) and long pollen (L) are linked because
both the genes are located on the same chromosome.
Similarly red flowers (p) and round pollen (l) are linked together because the
genes are located in the same chromosome (which is the homologue of the previous
one). The recombinations are obtained due to crossing over between the two concerned
genes in some of the spore mother cells.
Another cross was made in sweet peas with the following combinations:
PARENTS Purple Round x Red Long
PI/PI pL/pL
Purple Long
Pl/pL
F2 Purple Purple Red Red Total
long round long round
Actual numbers 225 95 97 2 419
Expected numbers 235.7 78.5 78.5 26.2 419
Expected Ratio 9/16 3/16 3/16 1/16
 Here again the parental types are more while the recombinant types are less than
expected on the basis of independent assortment, viz., 9:3:3:1. This deviation is also
due to linkage.

In the above two examples, it can be seen that in one cross the two dominant factors
(PL) are linked in one parent and two recessive factors (pl) are linked in the other.
Linkage in such crosses is said to be in coupling phase. In the second cross, dominant
allele of one character pair (P) and the recessive allele of another character pair (I) are
linked together in one parent, while in the second parent the other recessive (p) and
dominant alleles (L) are linked. Linkage in such crosses is said to be in repulsion phase.
Later, T H Morgan put forth the theory of linkage and concluded that coupling and
repulsion were two phases of single phenomenon, linkage.
Types of Linkage: Linkage is generally classified on the basis of three criteria viz., (i)
Crossing over, (ii) Genes involved and (iii) Chromosomes involved
(i) Based on crossing over: Linkage may be classified into (a) complete and (b)
incomplete / partial depending up on absence or presence of recombinant
phenotypes in test cross progeny.
(a) Complete linkage: It is known in case of males of Drosophila and females of
silkworms, where there is complete absence of recombinant types due to absence
of crossing over.
(b) Incomplete / partial linkage: If some frequency of crossing over also occurs
between the linked genes, it is known as incomplete / partial linkage. Recombinant
types are also observed besides parental combinations in the test cross progeny.
Incomplete linkage has been observed in maize, p ea, Drosophila female and
several other organisms.
(ii) Based on genes involved : Depending on whether all dominant or some dominant
and some recessive alleles are linked together, linkage can be categorized into (a)
Coupling phase and (b) Repulsion phase
(a) Coupling phase: All dominant alleles are present on the same chromosome or all
recessive alleles are present on same chromosome.
TR tr
----- --- Coupling phase
TR tr
(b) Repulsion phase: Dominant alleles of some genes are linked with recessive alleles
of other genes on same chromosome.
Tr tR
 ----- --- Repulsion phase
Tr tR
(iii) Based on chromosomes involved: Based on the location of genes on the
chromosomes, linkage can be categorized into (a) autosomal linkage and (b) X-
chromosomal linkage / allosomal linkage / sex linkage
(a) Autosomal linkage: It refers to linkage of those genes which are located in
autosomes (other than sex chromosomes).
(b) X-chromosomal linkage / allosomal linkage / sex linkage: It refers to linkage of
genes which are located in sex chromosomes i.e. either ‘X’ or ‘Y’ (generally ‘X’)
Characteristic features of Linkage:
1. Linkage involves two or more genes which are located in same chromosome in a
linear fashion.
2. Linkage reduces variability.
3. Linkage may involve either dominant or recessive alleles (coupling phase) or some
dominant and some recessive alleles (repulsion phase).
4. It may involve either all desirable traits or all undesirable traits or some desirable and
some undesirable traits.
5. It is observed for oligo-genic traits as well as polygenic traits.
6. Linkage usually involves those genes which are located close to each other.
7. The strength of linkage depends on the distance between the linked genes. Lesser
the distance, higher the strength and vice versa.
8. Presence of linkage leads to higher frequency of parental types than recombinants in
test cross. When two genes are linked the segregation ratio of dihybrid test cross
progeny deviates significantly from 1:1:1:1 ratio.
9. Linkage can be determined from test cross progeny data.
10. If crossing over does not occur, all genes located on one chromosome are expected
to be inherited together. Thus the maximum number of linkage groups possible in an
organism is equal to the haploid chromosome number.
Eg. Onion 2n = 16 n = 8 maximum linkage groups possible = 8 Maize 2n = 20 n = 10
maximum linkage groups possible = 10
11. Linkage can be broken by repeated intermating of randomly selected plants in
segregating population for several generations or by mutagenic treatment.
12. Besides pleiotropy, linkage is an important cause of genetic correlation between
various plant characters.
Linkage and pleiotropy: A close association between two or more characters may
result either due to linkage or pleiotropy or both. Pleiotropy refers to the control of two or
more characters by a single gene. A tight linkage between two loci can be often
confused with pleiotropy. The only way to distinguish between linkage and pleiotropy is
to find out a crossover product between linked characters. Intermating in segregating
populations may break a tight linkage, but a huge population has to be raised to find out
the crossover product. If a cross over product is not found in spite of repeated
intermatings, there seems to be the case of pleiotropy rather than linkage.
Linkage groups: Linkage group refers to a group of genes which are present in one
chromosome. In other words, all those genes which are located in one chromosome
constitute one linkage group. The number of linkage groups is limited in each individual.
The maximum number of linka ge groups is equal to the haploid chromosome number of
an organism. For example there are ten linkage groups in corn (2n = 20), seven in
garden pea (2n = 14), seven in barley (2n = 14), four in Drosophila melanogaster (2n =
8) and 23 in man (2n = 46).
Detection of linkage: Test cross is the most common method of detecting the linkage.
In this method, the F, heterozygous at two loci (AB/ab) is crossed to a double recessive
parent (ab/ab) and the phenotypic ratio of test cross progeny is examined. If the
phenotypic ratio of test cross progeny shows 1:1:1:1 ratio of parental and recombinant
genotypes, it indicates absence of linkage. If the frequency of parental types and
recombinant types deviate significantly from the normal dihybrid test cross ratio of
1:1:1:1, it reveals presence of linkage between two genes under study.
Another way to detect the presence or absence of linkage is to self pollinate the
individual heterozygous at two loci. If there is complete dominance at each locus and no
epistasis, the segregation ratio of the progeny will be 9:3:3:1. Presence of linkage either
in coupling or repulsion phase will lead to significant deviation from 9:3:3:1 ratio. The
deviation of observed values from the expected ratio is tested with the help of x2 test.
Significance of Linkage in Plant Breeding :
1. Linkage limits the variability among the individuals.
2. Linkage between two or more loci controlling different desirable characters is
advantageous for a plant breeder. A linkage between genes controlling two different
desirable characters will help in simultaneous improvement of both the characters.
3. Linkage is undesirable when desirable and undesirable genes are linked together.
4. The estimates of genetic variances for quantitative characters are greatly influenced
by the presence of linkage.
Lecture No.: XIX
Crossing Over
The term crossing over was first used by Morgan and Cattell in 1912. “The exchange of
precisely homologous segments between non-sister chromatids of homologous
chromosomes is called crossing over.”
Mechanism of crossing over: It is responsible for recombination between linked genes
and takes place during pachytene stage of meiosis i.e. after the homologous
chromosomes have undergone pairing and before they begin to separate. It occurs
through the process of breakage and reunion of chromatids.
 During pachytene, each chromosome of a bivalent (chromosome pair) has two
chromatids so that each bivalent has four chromatids or strands (four-strand stage).
Generally one chromatid from each of the two homologues of a bivalent is involved in
crossing over. In this process, a segment of one of the chromatids becomes attached in
place of the homologous segment of the non sister chromatid and vice-versa. It is
assumed that breaks occur at precisely homologous points in the two non sister
chromatids involved in crossing over; this is followed by reunion of the acentric
segments. This produces a cross (x) like figure at the point of exchange of the chromatid
segments. This figure is called chiasma (which is seen in diplotene stage of meiosis)
(plural-chiasmata).
Obviously, each event of crossing over produces two recombinant chromatids (involved
in the crossing over) called cross over chromatids and two original chromatids (not
involved in crossing over) referred to as non crossover
chromatids. The crossover chromatids will have new combinations of the linked genes,
i.e. will be recombinant; gametes carrying them will produce the recombinant
phenotypes in test-crosses, which are called crossover types.
Similarly, the noncrossover chromatids will contain the parental gene combinations and
the gametes carrying them will give rise to the parental phenotypes or noncrossover
types. Therefore the frequency of crossing over between two genes can be estimated as
the frequency of recombinant progeny from a test-cross for these genes. This frequency
is usually expressed as percent.
Thus, the frequency of crossing over (%) can be calculated using the formula;
No. of recombinant progeny from a test cross
Frequency of crossing over(%) = ------------------------------------------------------------------- x 100
Total number of progeny
Types of crossing over: Depending upon the number of chiasmata involved, crossing
over is of three types.
1. Single crossing over: It refers to the formation of single chiasma between non-sister
chromatids of homologous chromosomes. It involves two linked genes (Two point test
cross).
2. Double crossing over: It refers to the formation of two chiasmata between non-sister
chromatids of homologous chromosomes. It involves three linked genes (Three point
test cross).
3. Multiple crossing over: Occurrence of more than two crossing overs between non-
sister chromatids of homologous chromosomes is known as multiple crossing over.
However, the frequency of such type of crossing over is extremely low.
Factors affecting crossing over: The frequency of crossing over between the linked
genes is affected by several factors.
1. Distance between the genes: The frequency of crossing over between the two
genes is positively associated with the distance between their location in the
chromosome. Crossing over between the two genes would increase with an increase in
distance between them.
2. Sex: The frequency of recombination is markedly influenced by the sex of
heterozygotes for linked genes. In general, the heterogametic sex shows relatively lower
recombination frequencies than the homogametic sex of the same species. Eg: No
crossing over occurs between linked genes in Drosophila males and females of
silkworm.
3. Age of female: The frequency of crossing over shows a progressive decline with the
advancing age of Drosophila females.
4. Temperature: In Drosophila, the lowest frequency of crossing over is observed when
females are cultured at 220C. The frequency of recombination tends to increase both at
the lower and higher temperatures than 220C.
5. Nutrition: The frequency of crossing over in Drosophila is affected by the presence of
metallic ions Eg: Ca+2 and Mg+2 in its food. Higher the amount, lower will be the crossing
over frequenc y and vice-versa.
6. Chemicals: Treatment of Drosophila females with certain antibiotics like mitomycin D
and actinomycin D and certain alkylating agents such as ethylmethane sulphonate
promotes crossing over.
7. Radiations: An increase in frequency of crossing over is observed when Drosophila
females are irradiated with x-rays and g-rays.
8. Plasmagenes: In some species, plasma genes reduce the frequency of crossing
over. Eg: The Tifton male sterile cytoplasm reduces the frequency of crossing over in
bajra.
9. Genotype: Many genes are known to affect the occurrence as well as the rate of
crossing over. For example C3G gene of Drosophila located in chromosome 3 prevents
crossing over when present in homozygous state while it promotes crossing over in the
heterozygous state.
10.Chromosomal aberrations: In Drosophila, some chromosomal aberrations Eg:
paracentric inversions, reduce recombination between the genes located within the
inverted segment.
11.Distance from centromere: Centromere tends to suppress recombination.
Therefore genes located in the vicinity of centromeres show a relatively lower frequency
of crossing over than those located away from them.
Significance of crossing over in Plant Breeding:
1. It increases variability
2. It helps to break linkages
3. It makes possible to construct chromosome maps
Cytological proof of crossing over: The first cytological evidence in support of genetic
crossing over was provided by Curt stern in 1931 on the basis of his experiments with
Drosophila by using cytological markers. He used a Drosophila female fly in which one
X-chromosome was broken into two segments and out of these two segments, one
behaved as X-chromosome. This chromosome had one recessive mutant allele car
(carnation) for eye colour and another dominant allele B (Bar) for eye shape. The other
X-chromosome had small portion of Y chromosome attached to its one end. This
chromosome had the dominant allele + (wild type allele of car) producing dull red eye
colour and a recessive allele + (wild type allele of B) producing normal ovate eye shape.
Thus the phenotype of female is barred (since B is dominant to +) with normal eye
colour (since car is recessive to +) and both the X-chromosomes in the female had
distinct morphology and could be easily identified under microscope. Such females were
crossed with male flies having recessive alleles for both genes (car +). As a result of
crossing over female flies produce four types of gametes viz., two parental types or non
crossover types (car B and + +) and two recombinant types or cross over types (car +
and + B). The male flies produce only two types of gametes (car + and Y), because
crossing over does not occur in Drosophila male. A random union of two types of male
gametes with four types of female gametes will produce males and females in equal
number of four each.
Stern cytologically examined the chromosomes of recombinant types i. e.
carnation with normal eye shape and barred with normal eye colour. It was found that
carnation flies did not have any fragmented X-chromosome but rather had normal X-
chromosome. On the other hand barred flies had a fragmented X-chromosome with a
segment of Y-chromosome attached to one of the two fragments of X-chromosome.
Such chromosome combination in barred is possible only through exchange of
segments between non-sister chromatids of homologous chromosomes. This has
proved that genetic crossing over was accompanied with an actual exchange of
chromosome segments.
Similar proof of cytological crossing over was provided by Creighton and McClintock in
maize.
Coincidence: It refers to the occurrence of two or more distinct crossing overs at the
same time in the same region of a pair of homologous chromosomes and as a result, a
double cross over product is obtained. Coefficient of coincidence is estimated b y using
the formula:
Observed frequency of double cross over
Coefficient of coincidence = -------------------------------------------------------
Expected frequency of double cross over
(The ratio between the observed and the expected frequencies of double crossovers is
called coefficient of coincidence)
Interference: The occurrence of crossing over in one region of a chromosome interferes
with its occurrence in the neighbouring segment. This is known as interference. The term
interference was coined by Muller. It may also be defined as the tendency of one
crossing over to prevent another crossing over from occurring in its vicinity. This is called
positive interference. Sometimes, one crossing over enhances the chance of another
crossing over in the adjacent region. This is termed as negative interference. Eg:
Aspergillus, bacteriophages.
The effect of interference reduces as the distance from the first crossing over
increases. The intensity of interference may be estimated as coefficient of interference.
Coefficient of interference = 1 - coefficient of coincidence
Differences between crossing over and linkage
Crossing over Linkage

1. It leads to separation of linked genes 1. It keeps the genes together

2. It involves exchange of segments 2. It involves individual chromosomes
between non-sister chromatids of
homologous chromosomes
3. The frequency of crossing over can 3. The number of linkage groups can never
exceed 50 % never be more than haploid
chromosome number
4. It increases variability by forming new 4. It reduces variability
gene combinations
5. It provides equal frequency of parental 5. It produces higher frequency of parental
and recombinant types in test cross types than recombinant types in test cross
progeny progeny

Chromosome Maps
Chromosome maps can be prepared by genetical or cytological methods
1. Genetical method: This is the general method and is based upon cross over data.
The resulting map is the linkage map. Linkage map (cross over map or genetical map)
map be defined as a line on which the relative positions of genes proportional to the
amount of crossing over between them is represented.
A rule widely followed in plotting genes is that if genes A and B are known to be linked
and if a particular gene is found by experiment to be linked with gene A it must also be
linked with gene B. This principle follows from the fact that two linked genes are on the
same chromosome. The genes, which are linked together on the same chromosomes
are called syntenic genes.
Genetic mapping of chromosomes is based on the following assumptions:
a) The genes are arranged in a linear order.
b) Crossing over is due to breaks in the chromatids
c) Crossing over occurs by chance and is at random
d) The percentage of crossing over between the genes is an index of their distance
apart.
Map distance: Recombination frequencies between the linked genes are determined
from appropriate testcrosses. These percent frequencies are used as map units for
preparing linkage maps. A map unit is that distance in a chromosome, which permits one
percent recombination (crossing over) between two linked genes. A map unit is also
called a centri-Morgan, after the name of the scientist Morgan, who first constructed the
linkage map in Drosophila. Thus 5 % crossing over between genes A and B is taken to
mean that they are situated 5 map units of distance apart on the same chromosome. If a
third gene C with 7 % crossing over between A and C is included the relationship of
linkage between the three genes A, B and C is indicated as below:

B 5 cM A 7 cM C
12 cM
Or

A 5 cM B 7 cM C
7cM

To choose the correct one between these two alternatives, one more information
i.e. either the order of arrangement of the three genes or the cross over value between B
and C is required. Eg: If the crossover value between B and C is found to be 2 % by
actual experiments, the second arrangement is the correct one. Therefore, for preparing
a chromosome map of three genes either the map distances (cross over frequencies)
between all three gene pairs must be known or the cross over frequencies between any
two gene pairs plus the order or sequence of these three genes in the chromosome
must be known. In obtaining cross over value care should be taken about the
occurrence of double crossing over between the concerned genes. If two genes A and B
are rather far apart in a chromosome and if two crossing overs (i.e. double cross over)
occur between A and B, the chromatids involved do not show recombination of marker
genes. If double crossing over occurs frequently, the recombination value will be less
and gives a false impression that the distance between the concerned two genes is less.
To overcome this difficulty, data for chromosome mapping should be taken from linked
gene pairs that are quite close together. Usually double crossing over does not occur
within distances less than 5 map units or for certain chromosome segments within
distances upto 15 or 20 map units.
2. Cytological maps: By cytological studies of chromosomal aberrations and by their
behaviour in genetical experiments, it is possible to construct map of chromosomes
showing the actual physical location of gene in a chromosome. Such maps are called
cytological maps of chromosomes. The work on cytological maps also confirm the theory
of linear arrangement of genes in chromosomes.
Comparison between linkage maps and cytological maps: The relative distances
between the genes on linkage map and cytological map do not always correspond. The
discrepancies are greatest in the vicinity of the centromere where one cross over unit
corresponds to a relatively much greater physical distance on the chromosome than in
other regions of the same chromosome. These discrepancies may be explained on the
basis that different chromosomes and various regions in the same chromosome may
also show variations in frequency of crossing over. Eg: In Drosophila, frequency of
crossing over seems to be affected by temperature of the mother flies and by
environmental factors.
Importance of linkage and chromosome maps in plant breeding:
1. They give an idea whether particular genes are linked or segregate independently.
2. Linkage intensities can be known and the probability of obtaining a given combination
of genes can be assessed. If linkage between two genes is close, it is difficult to
obtain recombination. In such cases, linkage can be broken artificially by irradiating
with x-rays etc. and the desired combinations may be obtained. However, close
linkage is useful to preserve desirable gene combinations.
3. Help the geneticist to plan how large the experimental population should be to obtain
plants with the desired gene combination.
4. If an easily identifiable qualitative character is found to be linked with the quantitative
character, the qualitative character can be used to easily identify the recombinants.
This means that when a particular qualitative character is observed in a recombinant
plant, it can be understood that the associated linked quantitative character is also
present. Eg: Anthocyanin pigment and yield in rice
5. Linkage limits the variability among the individuals.