KERALA AGRICULTURAL UNIVERSITY

DEPARTMENT OF PLANT BREEDING AND GENETICS

PRACTICAL MANUAL

Fundamentals of Genetics (2 + 1)
(Pbgn 1101)

BY
Dr. ARYA K.
Dr. GAYATHRI G.
Mrs. ANJU M. JOB
Ms. NIJI M. S.
Dr. BEENA THOMAS

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A PrActicAl MAnuAl
On
FUNDAMENTALS OF GENETICS (2 + 1)
(Pbgn 1101)

Prepared by
Dr. Arya K.
Dr. Gayathri G.
Mrs. Anju M. Job
Ms. Niji M. S.
Dr. Beena Thomas

Department of Plant Breeding and Genetics

College of Agriculture, Vellayani
Kerala Agricultural University

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 Preface
The present manual has been envisaged for

handling practical classes for the undergraduate students.

It is based on the new syllabus for the course

Fundamentals of Genetics. The exercises aim to instill in

the students an interest in basics of genetics related to

plant breeding. It will help them to understand gene

interactions and checking the ratios in practical situation.

Preparation of stains, studies on mitosis and meiosis are

also included to help them in labwork.

The manual has drawn heavily from several texts

books and manuals related to genetics and cytogenetics

to cover the syllabus in toto. The permission accorded

by the Dean, Faculty of Agriculture to publish this

manual is gratefully acknowledged. The authors extend

their appreciation to their colleagues for all the help

rendered in the preparation of the manuscript and to

family members for their love, encouragement and

support.

Authors

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 CONTENTS

Ex. No Title Page

1 Study of microscope 4-8
Preparation and use of fixatives and staining for light microscope
2 9-12
3-4 Preparation of micro slides and identification of various stages of mitosis
13-16

5-6 Preparation of micro slides and identification of various stages of meiosis 17-19

7 Probability and Chi-square analysis 20-24

8 Monohybrid ratio and its modifications 25-32
9 Dihybrid ratio and its modifications 33-36
10 Trihybrid ratio 37-38

11 Multiple alleles 39-40

Gene interactions
12 41-43

13 Complementary factor and epistasis 44-46

14 Supplementary factors and inhibitory factors 47-48

15 Duplicate factors and polymeric gene action 49-52
16 Determination of linkage - Two point test cross 53-58
17 Determination of linkage - Three point test cross 59-61
18 Study on models of DNA and RNA structures 62-68

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Ex. No: 1 Study of microscopy

The microscope is an optical system, which has a combination of lenses to enlarge the
image of a small object. It is a valuable instrument for studying objects which are too small to
be studied with naked eye. The naked eye is able to distinguish between two points 0.1 mm
apart. This is referred to as the resolution power of the eye. The resolution power of a good
light microscope is 0.2 microns (1 micron = 10-6 m). This is accomplished by the use of
magnifying glasses within the microscope.
Types of Microscope:-
1. Simple Microscope: A magnifying glass, an ordinary double convex lens having a
short focal length is a simple microscope. When an object is placed within its focal
length, an image is produced that is erect, virtual and larger than the original object
e.g. reading and hand lens. Microscopes invented by Leeuwenhoek are considered as
simple microscopes.
2. Compound Microscope: The compound microscope utilizes two lenses or lens
systems. The lower lens (nearest to the object) is called as objective and the upper
lens (nearest to the eye of the observer) is called as eyepiece. When and object is in
focus, a real, inverted image is formed by the objective at a point inside the principle
focus of the eye piece. One lens system focuses an enlarged image of the object and
the second magnifies the image focused by the first. A compound microscope has the
following parts:
1. Stage: a place where the specimen rests.
2. Clips: hold the specimen still on the stage
3. Micromanipulator: moves the specimen in controlled, small increments along
the X and Y axis.
4. Mirror: generally one side is level and the other concave. Concave side reflects
more light. Mirror is adjustable to reflect light on the specimen through diaphragm.
5. Condenser: lens system that aligns and focuses the light from the mirror on the
specimen.
6. Diaphragm: placed in the light path, it alters the amount of light that reaches the
condenser.
7. Revolving Nosepiece: three objective lenses are mounted on this structure.
8. Lower Power Objective Lens: provides 10x magnification and is used to view a
comparatively larger area of the specimen.
9. High Power Objective Lens: provides 45x magnification and is used to magnify
a smaller section of the specimen.
10. Oil immersion objective lens: provides 100x magnification. Oil is placed
between objective lens and specimen to prevent additional light refraction as it
enters from one medium to another.
11. Body Tube: holds the eyepiece at the proper distance from the objective lens and
blocks out stray light.
12. Coarse Focus Knob: used to bring the object into the focal plane of the objective
lens.

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 13. Fine Focus Knob: used to make fine adjustments to focus the image.
14. Arm: curved portion that holds all of the optical parts at a fixed distance and
aligns them and is always held in carrying the microscope.
15. Base: supports the weight of all of the microscope parts.
16. Eye Piece (ocular): it is the lens at the top of the microscope. It generally
magnifies 10x. Most eyepieces have a magnification between 8x and 12.5x.
All modern microscopes are parfocal, meaning the focus does not change appreciably
when objective lens is changed, and are parcentral, meaning the centre of the field does not
change when the objective is changed. Microscopes come in two basic configurations,
upright and inverted. Upright microscope has the illumination system below the stage and the
lens system above the stage. An inverted microscope has the illumination system above the
stage and the lens system below the stage. Inverted microscopes are better for looking
through thick specimens such as dishes of cultured cells because the lenses can get close to
the bottom of the dish where the cells grow.

Magnification
Magnification can be defined as the number of times a sample appears to be larger
under the microscope than it really is. The total magnification of the microscope is the
product of magnification powers of both eyepiece and objective lens. If the eyepiece has 10x
power and objective has 40x power, then the total magnification of the microscope will be 10
X 40. In other words, the image of the material is enlarged by 400 times of its original size.
The magnifying powers of eye piece and objective are engraved on their metal bodies.
Magnification = Objective (magnification) X Eyepiece (magnification)
Resolution
By using more lenses, microscopes can magnify upto a larger extent, but this does not
always mean that more details can be seen. The amount of details visible depends on the
resolving power of a microscope, which is the smallest distance by which two separate
objects can be distinguished. It is the ability to discriminate between two adjacent structural
components of the object, revealing them as distinct and as wide as possible.
The resolving power of a microscope is inversely proportional to the wavelength of
the light, which means shorter the wavelength more would be the resolving power of the
microscope. Magnification is different from resolving power. By increasing the magnification
power, clarity of the images declines but by increasing the resolving power, clarity is restored.
Numerical Aperture
The numerical aperture of a microscope objective lens is a measure of its ability to
gather light and resolve fine specimen details at a fixed object distance. The numerical
aperture of an optical system such as an objective lens is defined by
NA = n sin Ө
Where n is the index of refraction of the medium in which the lens is working (1.0 for air,
1.33 for pure water and upto 1.56 for oils) and Ө is the half-angle of the maximum cone of

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light that can enter or exit the lens. In microscopy, NA is important because it indicates the
resolving power of a lens.
1. Light Microscopy: This is the oldest, simplest and most widely used form of
microscopy. Specimens are illuminated with light, which is focused using glass lenses.
Specimens often need to be stained with a coloured dye to make them visible. Light
microscopy has a resolution which is good enough to see cells, but not the details of
cell organelles. Here are the various types of microscopy techniques:
(i)Bright Field: With a conventional bright field microscope, light from an
incandescent source is aimed towards a lens beneath the stage called condenser,
through the specimen, through an objective lens, and to the eye through a second
magnifying lens, the ocular or eye piece. We see objects in the light path because
natural pigmentation or stains absorb light differentially, or because they are thick
enough to absorb a significant amount of light despite being colourless.

(ii) Dark Field: It is based on the principle that light rays are scattered due to
difference in refractive index of two phases. With increasing contrast between object
and background, the degree of visibility enhances. The microscope is provided with
dark field condenser which does not allow direct light to pass into objective as the
object is obliquely illuminated.
(iii) Phase contrast: This technique is best for looking at living specimens such as
cultured cells.
(iv) Polarized microscopy: The polarized microscope is used to study the ultra-
structure of cells including mitotic spindle, structure of nerve fibers and chloroplast,
etc.
(v) Fluorescence Microscopy: This type of microscope uses high energy, short
wavelength light (usually ultraviolet) to excite electrons in certain molecules inside a
specimen, causing those electrons to shift to higher orbits. When they fall back to
their original energy levels, they emit lower energy, longer wavelength light (usually
in visible spectrum), which forms the image.
(vi) Stereo Microscopy: The stereo or dissecting microscope is designed
differentially from the light microscope and serves a different purpose. It uses two
separate optical paths with two objectives and two eyepieces to provide slightly
different viewing angles to the left and right eyes. In this way it produces a three
dimensional visualization of the sample being examined. The stereo microscope is
often used to study the surfaces of solid specimens or to carry out close work such as
sorting, dissection or microsurgery.
(vii) Interference Microscopy: Microscope that is capable of creating visible contrast
from differences in refractive index is the interference microscope, which is more
versatile than the phase contrast microscope but also more complex and expensive.
 Small detail not easily discernible by the phase contrast microscope can be
seen by interference microscope.

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  Quantitative measurements not possible with the phase contrast microscope
can be made by interference microscope
 By the use of interference microscope it is possible to measure the dry weight
of the object

2. Electron Microscopy: Electron Microscopes are scientific instruments that use a

beam of highly energetic electrons to examine objects on a very fine scale. Electron
microscopes (EMs) function exactly as their optical counterparts except that they use
a focused beam of electrons instead of light to “image” the specimen and gain
information as to its structure and composition.
The basic steps involved in all Ems:
(i) A stream of electrons is formed (by the electron source) and accelerated
towards the specimen using a positive electrical potential.
(ii) The stream is confined and focused using metal apertures and magnetic lenses
into a thin, focused, monochromatic beam
(iii) This beam is focused onto the sample using a magnetic lens.
(iv) Interactions occur inside the irradiated sample, affecting the electron beam.
These interactions and effects are detected and transformed into an image.
The electron microscopes are basically of two types:
a. Transmission Electron Microscope: The Transmission Electron Microscope
(TEM) was the first type of Electron Microscope to be developed and is modeled
exactly like light microscope except that a focused beam of electrons is used
instead of light to “see through” the specimen.
b. Scanning Electron Microscope: The Scanning Electron Microscope (SEM) is a
type of electron microscope that images the sample surface by scanning it with a
high-energy beam of electrons of random scan pattern. The electrons interact with
the atoms that make up the sample producing signals that contain information
about the sample’s surface topography, composition and other properties such as
electrical conductivity.

Comparison between Light and Electron Microscopes

Light Microscope Electron Microscope
Cheap to purchase Expensive
Cheap to operate Expensive to produce electron beam
Small and portable Large and requires special rooms
Simple and easy sample preparation Lengthy and complex sample preparation
Material rarely distorted by preparation Preparation distorts material
Vacuum is not required Vacuum is required

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Comparison between TEM and SEM
TEM SEM
The electrons are detected by beam It detects secondary electrons which are
transmission emitted from surface due to excitation by
primary electron beam
It is used for analyzing sections, so electrons It is used for surface analysis and electron
pass through thin sample beam scans over surface of sample
Specially prepared thin samples or particulate Sample can be of any thickness and is
material are supported on TEM grids mounted on aluminum stub
Image is shown on fluorescent screen Image is shown on TV monitor
Image is 2D projection of the sample It gives good representation of 3D structure
of sample
TEM resolution is better than SEM Its resolution is less than that of TEM
(about an order of magnitude)

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 Ex. No: 2 PREPARATION AND USE OF FIXATIVES AND STAINS
FOR LIGHT MICROSCOPY

A living cell, which consists of many component organelles, resists absorption of dyes,
posing problems for proper study of its vital parts on the basis of differential staining.
However, such a problem is not confronted with dead but fixed cells. This may be
accomplished by instantly stopping the life processes of each and every cell and tissue of the
organism by treating them with an appropriate fixative fluid. The cytological studies of
chromosomes in a cell can be carried out through the following steps:
1. Pre treatment
2. Fixation
3. Staining
Pre treatment
Pre treatment is done to treat the dividing tissue with an anti-mitotic agent that blocks
division at metaphase. Pre treatment leads to:
(a) Separation of the middle lamella causing softening of the tissue.
(b) Dissoulution of spindle fibers resulting in separation of chromosomes
(c) Clearing of cytoplasm
(d) Viscosity changes in cytoplasm
(e) Clarification of chromosomal constrictions
Treatment time varies and depends upon species to species. Care should be taken that
after treatment, the material is washed for 5-10 minutes under running tap water.
Pre treatment reagents
1. Colchicine: Colchicine, an alkaloid extracted from roots or corm of
Colchicumautumnale, arrests the development of spindle fibers resulting in separation
of chromosomes. It is water soluble. For its manifestation, range of concentration lies
between 0.001 to 1.0 per cent and the period of treatment varies from 10 minutes to 1
hour.
2. 8-hydroxy quinoline: It causes mitotic arrest and constrictions become conspicuous
under the influence of this chemical. Treatment of 0.0002M aqueous solution of 8-
hydroxy quinoline for 1-1.5 hours is given for chromosomal studies.
3. Chloral hydrate: It has the same effect as colchicine and the concentration required
for its treatment is also the same as that of colchicine.
4. α-bromo-naphthalene: The material is kept for one to two hours in saturated
solution of α-bromo naphthalene at 10-15oC temperature. This pre-fixative is most
commonly used for treating pollen mother cells.
5. p-dichloro-benzene: The material is kept in saturated solution of para-dichloro-
benzene for two to three hours at 12-16oC.

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Fixation
The instant killing of the material coupled with non-mutilation of its cells and tissues
is called fixation and is accomplished by a fluid known as a fixative. A good fixative should
have the capacity of rapid penetration so that the tissue is killed instantaneously, causing
arrest of the divisional configurations. Besides, it should be able to check autolysis of protein.
It should have antiseptic effect so that the material is preserved and no further decay occurs.
Fixation results in : coagulation of the constituents of the cell without disintegration or
disturbance of their internal structure; production of such surface conditions in the fixed
material that will enable them to hold the stain for making them visible and toughening the
material to resist embedding or squashing.
Preparation of fixing mixtures
These should be prepared fresh at the time of use. Fixation is done for 15 minutes to
24 hours in cold or at room temperature. The fluid is washed out with 70% to 90% alcohol.
1. Carnoy’s Fluid I (1886): It is also known as Farmer’s Fluid. It is used for fixing root
tips. It consists of :
Glacial acetic acid 1 part
Absolute ethyl alcohol 3 parts
2. Carnoy’s Fluid II (1886): It is used for fixing flower buds. Its composition is as
follows:
Glacial acetic acid 1 part
Chloroform 3 parts
Absolute ethyl alcohol 6 parts

Storage
After fixation, the material is transferred to 70% alcohol and can be stored in a cool place
preferably in a refrigerator for several weeks or months without any harm to the tissues.
Staining
Staining provides clear and distinct visibility of cell and tissue components
differentially coloured by specific dyes (stains). Staining may be viewed as vital or non-vital
staining. In vital staining, living cells and tissues can be studied under non-toxic dyes. For
example, Methylene blue may be used to study chromosomal division in growing cells.
However, vital staining is not very effective for chromosomal studies. In case of non-vital
staining, chemicals which are insoluble in the nucleoproteins of chromosomes impart
visibility of chromosomes in the fixed and killed tissues. The selection of dye or stain for a
particular material usually depends on its chemical nature, pH value of fixative used and
chemical reactivity of stain to the material. Stains may be acidic, basic or neutral in chemical
reaction.
1. Acidic stains: The ions of acidic stains are negatively charged which are effective in
staining of cytoplasmic proteins. Eg. Picric acid, Fuchsin acid, Aniline blue, Orange-
G, Eosin, Congored, Bismarck brown and Fluorescent dyes.
2. Basic stains:The stains with positively charged ions are used for staining of nucleic
acids. Eg. Bausch Fuchsin, Methyl green, Crystal Violet, Safranin and Haematoxylin.

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 3. Neutral stains: Some stains are amphoteric and have both properties of acidic and
basic nature. In some tissues and cellular components, the cytoplasm takes acidic
stains whereas nucleus takes basic stains. Neutral stains have a coloured cation and a
coloured anion; an example is Leishman’s stain.
Preparation of stains
1. Acetocarmine (C22H20O13): Carmine is obtained from dried female bodies of Coccus
cacti (an insect). The ingredients required for preparing 2% acetocarmine are:
Carmine powder 2g
Glacial acetic acid 45ml
Distilled water 55ml
Ferric acetate Few drops

Add distilled water to glacial acetic acid to form 45% acetic acid solution and heat
gently till the mixture boils. Remove it from the flame and immediately add 2gm
carmine powder slowly. Cool the solution and filter it through coarse filter paper. Add
few drops of ferric acetate solution until colour of the stain becomes dark wine red.
Then store at 4oC in the refrigerator for further use.

2. Feulgen: It should be noted that all the glassware to be used for preparation of
Feulgen stain must be acid cleaned. The ingredients required for preparing this stain
are:
Leuco-basic Fuchsin 3g
1N HCl 90ml
K2S2O5 9g
Activated charcoal 3g
Distilled water 600ml

Dissolve Fuchsin in distilled water by boiling gently and repeated shaking. Cool to
50oC. Filter and add 1N HCl. Then add potassium meta-bisulphate (K2S2O5) and store
in dark at room temperature for 24 hours. Add charcoal powder to remove impurities.
Shake, filter and store in an amber bottle in a refrigerator at 4oC. Stain should be
colourless. Avoid direct inhalation of fumes.
3. Aceto-Orcein: The ingredients required for preparing this stain are:
Orcein 2g
Glacial acetic acid 45ml
Distilled water 55ml

Add distilled water to glacial acetic acid; boil and add powdered orcein stain slowly
by stirring the hot solution with a glass rod. Cool down the solution and filter.

Techniques for preparing slides for cytological studies

There are two techniques for preparing slides for cytological studies:

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 1) Smears: The cells are directly spread over a slide before fixation and in this process
no treatment is necessary to separate the cells. For example, slide preparation from
pollen mother cells of anthers.

2) Squashes: After fixation, 1N HCl at 60oC is added to dissolve the middle lamella
which separates the cells and the tissues are softened. The soft tissue is stained and
squashed on a slide by applying pressure over the cover slip. For example, slide
preparation for mitosis study from root tips of onion.

Preparation of permanent slides

For cytological studies, the temporary slide has to be made permanent. A clean and
dry slide is used to make a temporary slide as described above and it is then sealed with
paraffin wax to prevent drying. For preparation of permanent slide, anyone of the methods
given below can be followed after two to three hours of sealing the temporary slide.
Method I
Carefully remove paraffin wax without displacing cover slip. Invert the slide in acetic
acid : ethyl alcohol (1:1) solution and allow the cover slip to get detached. Then pass the slide
through solutions of absolute alcohol, absolute alcohol : xylene (1:1), and xylene series,
keeping for 10 minutes in each. Now the slide can be mounted in Euparal or Canada balsum.
Method II
Carefully remove paraffin wax without displacing cover slip. Invert the slide in a
solution of acetic acid : butyl alcohol (1:1) and the cover slip to get detached. Then pass the
slide through two containers of butyl alcohol containing different concentrations, keeping for
15-20 minutes in each. The slide can now be mounted in Euparal before the alcohol is
evaporated.

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Ex. No: 3-4 Preparation of microslides and identification of various
stages of mitosis

The process of reproduction or formations of new cells from pre-existing cells are
referred as cell division. Plant growth is the result of increase in cell number and cell size.
Cell division is the cause of increased cell number. This process take place in fast growing
tissues called meristematic tissues which are located mainly at stem apex and root apex.
There are two types of cell divisions viz., mitosis and meiosis.
Mitosis: (Greek mitos ‘thread’)
The term mitosis was coined by Flemming in 1882. Mitosis is the process by which
chromosomes replicate precisely and are distributed equally to two daughter nucleus such
that they have identical sets of chromosomes as that of the parental cell.
Cell cycle
It is the period in which one cycle of cell division is completed. It consists of two phases:
Interphase (phase of DNA synthesis) and mitotic phase (phase of nuclear division).

Cell cycle can be divided in to,

1. Inter phase: (inter (L) = between; Phase (Grk) = stage)
 G1, S and G2

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 2. M phase/ Mitotic phase (Period of cell division)
 Karyokinesis (nuclear division) and Cytokinesis (cytoplasmic division)

Interphase
Before the commencement of mitosis, the chromosomal material is in a special stage
known as interphase. It occupies the time between the end of telophase and the beginning of
next prophase. The interphase is the largest phase in the cell cycle. It is often not regarded as
a stage of cell division. The interphase is again divided in to 3 sub phases i.e. G1, S and G2.
 G1(Gap 1)Phase: Synthesis of RNA and protein (pre-DNA replication phase).
 S (synthesis)Phase: Synthesis of DNA (DNA replication phase)
 G2(Gap 2) Phase : Synthesis of mRNA and some fraction of RNA and proteins (post-
DNA replication phase)

During interphase, each DNA molecule replicates an exact copy of itself. This process
produces a chromosome with two identical functional strands called chromatids. The newly
formed chromatid will attached to a common point, centromere and function as a single
chromosome. As a result, chromosome number remains constant and definite in each species
at the end of mitosis.
Mitosis
Mitotic phase is studied in the meristematic cells of young root tips.
Material – Onion (Allium cepa)
Chromosome Number – 2n – 16
Dry scale leaves and roots are removed from the onion bulbs and soaked in water for 2 hours.
Then the bulbs are placed on the mouth of the test tubes filled with water in such a way that
basal stem portion is immersed in water. On the next day small white roots can be seen at the
basal part of portion of the bulbs. The root
Ordinarily cell division, mitosis, is similar in plants and animals. Mitosis occurs in
somatic cells and the chromosomes present in somatic cells are termed as somatic
chromosomes. The somatic chromosome number is represented by 2n. it comprises of
division of nucleus (karyokinesis) and division of cytoplasm (cytokinesis). For understanding,
mitosis is subdivided into 4 stages:
1. Prophase: Chromosome begins to appear as discrete units; each chromosome consists of
two chromatids; spindle formation begins.
2. Metaphase: Chromosomes arrange themselves at the equator of the spindle.
3. Anaphase: Centromere of each chromosome divides and the two chromatids (daughter
chromosomes) move to the opposite pole of the spindle.
4. Telophase: chromosomes are assembled at each pole and formation of daughter nuclei
starts.

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Cytokinesis: A cell wall is formed between the two newly formed nuclei and the cell divides
into two daughter cells.

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Ex. No: 5-6 Preparation of microslides and identification of various
stages of meiosis

All the sexually reproducing organisms undergo meiosis at the time of spore
formation and as a result, cells are formed having haploid (n) chromosome number. Meiosis
consists of one DNA replication followed by two successive nuclear divisions. First meiotic
division is accomplished by reduction of chromosome number while second division involves
separation of chromatids just like mitosis. First division (meiosis I) is also known as
reduction division since the chromosome number is reduced to half. Second division (meiosis
II) is known as equational division. The term meiosis was coined by Moore and Farmer
(1905).

Material: Rhoeo discolour

Chromosome number (2n) = 12
It is an ornamental plant belonging to the family Liliaceae. The somatic chromosome
number is 12 and the chromosomes are comparatively large in size which enables easy
identification of stages. The pollen mother cells are thin walled and comparatively very large
sized compared to the anther wall cells. Pollen grains are very closely arranged within the
anther lobes. To study the different stages of meiosis smear preparation of pollen mother cells
are done.
Meiosis: Before meiosis there is an interphase, which is same as that of mitosis (G1, S and
G2)
A. First meiotic division / meiosis I (reduction division): it consists of the following sub
stages
1. Prophase-I: chromosomes make distinct appearance and arrange themselves in
homologous pairs. During pairing the chromosomes undergo an exchange of
chromatid segments. This phenomenon is termed as crossing over, has important
genetic consequences. Prophase-I is again sub-divided into five as explained below
(Fig 4.1) :
a. Leptotene: Each chromosome appears as a single thread like structure.
Spindle formation starts.
b. Zygotene: Homologous chromosomes begin to pair.
c. Pachytene: Homologous chromosomes are paired; they reduce in
length and each chromosome split into two chromatids; thus, four
chromatids can be seen. Crossing over takes place at this stage.
d. Diplotene: Paired chromosomes (bivalents) contract and points of
crossing over (chiasmata) are visible.
e. Diakinesis: Bivalents contract very much and chiasmata
terminalization starts. A complete spindle is formed.

2. Metaphase-I: The paired homologous chromosomes (bivalents) arrange themselves

at the equator of the spindle; bivalents may be in the form of rings and rods.

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 Fig 4.1: Different stage of Prophase

3. Anaphase -I:The homologous chromosomes separate and move to the opposite

poles of the spindle; in this way, half of the chromosomes go tone pole and the
other half go to the opposite pole.
4. Telophase –I: The chromosomes assemble at each pole and start forming
daughter nuclei.
Cytokinesis:At the end of telophase-I, cytoplasm is divided into two halves; each of
the two halves stays together and this structure is called dyad.
B. Second meiotic division / meiosis II (equational division): Same as mitosis
1. Prophase -II:This stage is very short, chromosomes are visible in coiled form; at
the end of prophase-II, nucleolus and nuclear membrane disappear.

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 2. Metaphase-II:Spindle fibers are generally oriented at right angle to the first
division spindle fibers which connect the centromeres to the pole and
chromosomes are arranged at equatorial plate.
3. Anaphase -I:There is longitudinal division of centromere of each chromosome
and chromatids move towards opposite poles.
4. Telophase –I: Uncoiling of chromosomes takesplace and there is reappearance of
nucleolus and nuclear membrane.
Cytokinesis: By the end of telophase –II, the cytoplasm of each of the two cells of
dyad divides into two parts; as a result, one parent cell produces four haploid
daughter cells after completion of the two meiotic divisions; the four daughter
cells from a parent cell are present together and are known as tetrad.
A schematic representation of different stages of meiosis is depicted in Fig 4.2.

Fig 4.2: Different stages of meiosis

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 Ex. No: 7 Probability and chi-square analysis

Probability
Probability represents the likelihood of the occurrence of a particular event. It is
expressed by the number of times a particular event occurs divided by number of all possible
outcomes. In genetics it is used to express the chances of obtaining a particular genotype
from a specified cross under described conditions. Addition rule and multiplication rule are
the two rules of probability used to predict the ratio of progeny expected from a cross.
 Addition rule: It is used to predict the probability of two mutually exclusive events.
Mutually exclusive events are those in which occurrence of any one of the events
excludes the occurrence of other event. Here the probability of occurrence of two
events is the sum of their individual probabilities.
 Multiplication rule: It is used to predict the probability of two or more independent
events occurring together. Two or more events are said to be independent if the
occurrence of one event independent of occurrence or non-occurrence of other event.
Consider two independent event occurs with the probabilities p and q respectively,
then the probability of their joint occurrence will be (pxq) ie., combined probability is
the product of their individual probabilities.

Application of probability in genetics

Consider a cross between two plants which are heterozygous for high determining
locus, T and t (Tt x Tt). Here each parent will produce two types of gametes in which half
will be having T allele and half will be with t allele. In progenies these gametes from the two
parents will combine in 4 different ways. Since probability of receiving gametes from two
parents is independent of each other, multiplication rule should be used to determine the
progenies. The four types of progenies from the cross and corresponding probabilities areas
follow.
TT = ½ x ½= ¼ Tall
Tt = ½ x ½ = ¼ Tall
tT = ½ x ½ = ¼ Tall
tt = ½ x ½ = ¼ Dwaff
The heterozygous progenies are formed in two different ways i.e, they can receive a T
allele form first parent and t allele from second parent and vice versa.
To determine overall phenotypic ration of progenies, addition rule is used. Probability
of obtaining tall progeny is ¼ + ¼ + ¼ = ¾ , since due to dominance TT, Tt and tT will have
tall phenotypic expression. Probability of dwarf phenotype is ¼ since it is a recessive trait.
Chi-square and Goodness of fit
When we cross two individuals of known genotype, we expect certain genotypic and
phenotypic ratios base on the Mendalian principles of genetics. But in practical situation it

20
may not strictly divide the progeny population in to 3:1, 1:1 or 9:3:3:1proportion and we may
experience deviations. But these deviations may be due to chance or may be due to some
complicated inheritance behaviour of the character. In such situations we need some means of
evaluating hoe likely that chance is responsible for the deviation between the observed and
the expected numbers. Chi-square is a valuable statistical tool to determine the goodness of
fit. Chi-square(χ2) analysis test whether a particular ratio obtained during observations is
good enough or fit enough to be accepted as compared to an expected ratio. Chi- square
method is one of the most versatile in the statistical theory. In this method we can test
whether the observed frequencies in a distribution differ significantly from the frequencies
which can be expected according to some hypothesis.
Conditions for application of Chi-square test
 There should be sufficiently large number of observations i.e., at leat 50 number of
observations
 In each class the number of observations should not be two low i.e., at least 5
 It is applicable to numerical data only

Chi-square (χ2) is calculated using the following formula

χ2 = ∑ Xo – Xe
Xe
Where ∑ = Sum of observations
Xo = Observed numbers
Xe = Expected numbers
Procedure for Chi-square test
1. Formulation of null hypothesis
It is the first step in a Chi-square test.it states that the observed data are in agreement
with the expected ratio or the deviation observed is due to chance only.
2. Computation of Chi-square value from the experimental data
The procedure for calculatingχ2 value is as follows.
The observed frequencies (O) for each class in the data are sequentially arranged.
Expected frequencies (E) for each class are computed on the basis of expected ratios for that
particular cross (Eg: Monohybrid 3:1, monohybrid test cross 1:1, dihybrid 9:3:3:1 etc.) and
total number of observations.
3. Computation of deviations for each classes (O-E)
4. Square the deviations for each class to avoid sign ((O –E)2)
5. Divide the deviation square by the expected frequencies ((O-E)/E)
6. Summation of the above value for all the classes will give χ2 value for that particular cross.

21
7. Compare the calculated χ2 value with table χ2 value at degree of freedom at 5% and 1%
level of significance. Degree of freedom is one less than the total number of classes in the
data.
df = n – 1, n = Number of classes
Degree of freedom Probability of larger value of χ2 (P) 0.05
1 3.841
2 5.991
3 7.815
4 9.488
5 11.070

8. Drawing conclusion
After comparing the calculated value and table value at corresponding degree of freedom, if
the calculated value is less than the table value, then the null hypothesis is accepted. It means
that the deviation is not significant and it is due to chance only. If the calculated value is
equal or greater than the table value, then the null hypothesis is rejected. It means the
deviation is real and not due to chance.
Note: In most of the biological experiments 0.05 probability (5% level) is accepted as the
standard probability level for decision making.
Example I
In F2 generation Mendel got 1064 plants in which 787 plants were tall and 277 were dwarf.
Test for goodness of fit.
Null hypothesis (H0): There is no significant deviation from 3:1 segregation ratio.
Number of classes (n) = 2
Degree of freedom (df) = (n-1) = 1
Chi-square analysis
If the plants are segregating in 3:1 ratio according to the expectation, then there should be
798 tall and 266 dwarf plants (1064 x ¾, 1064 x ¼ respectively).
Observed Expected
Classes number number O–E (O – E)2 χ2 = (O - E)/E
(O) (E)
¾ x 1064 =
Tall 787 11 121 121/798= 0.15
798
¼ x 1064 =
Dwarf 277 11 121 121/266= 0.45
266
Total 1064 0.60

Total χ2 value is tested against 1 df.

Table value of χ2 at 0.05 level of significance = 3.84

22
Since the calculated value of χ2 is less than the table value at 5% level of significant, the null
hypothesis is accepted, i.e., there is no significant difference in observed value from the
expected 3:1 segregation and the deviation is due to chance.
Example 2
Mendel did a dihybrid cross involving the characters, shape of seed and colour of pods. he
observed the following data from F2.
Round yellow – 315, Round green – 108, Wrinkled yellow – 101 and wrinkled green – 32
Test for goodness of fit.
Null hypothesis (H0): There is no significant deviation from 9:3:3:1 ratio.
Number of classes (n) = 4
Degree of freedom (df) = (n-1) = 3
Chi-square analysis
The expected F2 ration is 9:3:3:1
Observed Expected χ2 =
Classes number number O-E (O – E)2 (O - E)/E
(O) (E)
Round yellow 315 312.75 2.25 5.0625 0.01
Round green 108 104.25 3.75 14.0625 0.12
Wrinkled yellow 101 104.25 3.25 10.5625 0.12
Wrinkled green 32 34.75 2.75 7.5625 0.21
Total 556 0.48

Total χ2 value is tested against 3 df.

Table value of χ2 at 0.05 level of significance = 7.815
χ2 3 (0.05) = 7.815> 0.48
Since the calculated value of χ2 is less than the table value at 5% level of significant, the null
hypothesis is accepted, i.e., there is no significant difference in observed value from the
expected 9:3:3:1 segregation and the deviation is due to chance.
Problems
1. The flowers of four o’ clock plant may be red, pink or white. When red crossed to white
only pink offsprings were produced. When pink were crossed to pink, 113 red, 129 white
and 242 pink offsprings were produced. The inheritance of flower colour in four o’ clock
plant is co-dominant in nature. It is thought that the genes segregate in 1:2:1 ratio. Test
whether the hypothesis is acceptable on the basis of chi-square test.
2. In summer squash white fruit flower (W) is dominant over yellow (w). Disc fruit (D) is
dominant over sphere (d). In a cross between homozygous white disc and yellow sphere
the F2 segregated as follows white disc (50), white sphere (18), yellow disc(16) and yellow
sphere (6). Test whether the data show independent assortment using chi square test.

23
3. In a test cross of F1 hybrid in the above problem, the following results were obtained. Test
whether there is independent assortment. White disc (25), white sphere (27), yellow disc
(20) and yellow sphere (28).
4. Let purple and green are the two types of stem colour in pea plant and it is controlled by A
and a respectively. A true breeding purple plant is crossed with a green plant and all the
progenies were of purple colour. When the F1 was crossed to another green plant, the
progeny segregated as 482 purple and 526 green. Test whether the observed frequencies
are in agreement with the expected frequencies.
5. In sweet pea, gene P is showing its effect only along with the colour gene C. C and P
together produce purple colour. P has no effect of its own and C and P independently
produced white colour. When two homozygous white plants were crossed, the F1
produced purple and the F2 segregated as 500 purple and 380 white. Test whether the
deviation observed can be considered as due to sampling error.
6. In summer squash gene for white fruit colour (W) is epistatic over those for yellow and
green. Consider ‘Y’ for yellow and ‘y’ for green. When WWYY plant is crossed with
wwyy, the F2 produced 255 white, 98 yellow and 31 green. Test whether there is epistatic
gene interaction using chi-square test.

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 Ex. No: 8 Monohybrid ratio and its modifications

BASIC CONCEPTS OF GENETICS

Genetics : Study of inheritance (heredity and variation).

Heredity : Resemblance among individuals related by descent.
Variation : Differences among individuals of the same species.
Gene : A unit of inheritance which controls a character and having a fixed locus in the
chromosome.
Allele : Alternative forms of a gene, also known as Allelomorph.
character : Any heritable attribute of an organism. Controlled by genes.
Trait : Alternative forms of a character. Controlled by alleles of the gene.
True breeding : Individual which produce offsprings of same kind.
Homozygote :Individuals having identical alleles at corresponding loci of homologous
chromosomes. They are true breeding eg. TT, tt.
Heterozygote :Individuals with dissimilar alleles at corresponding loci of homologous
chromosomes. Eg. Tt.
F1(First filial : It is the progeny of a cross between two true breeding individuals having two
generation) contrasting traits of a character. It is the hybrid generation.
F2 (Second filial : It is obtained by selfing or inter-mating of F1 individuals.
generation)
Hybridization : Crossing of genetically different individuals.
Hybrid : The progeny obtained by crossing two genetically different pure breeding parents.
Eg. TT x tt , this cross give rise to Tt individual (hybrids).
Monohybrid : Individuals heterozygous for a single gene.
Di hybrid : Individuals heterozygous for two genes.
Tri hybrid : Individuals heterozygous for three genes.
Phenotype :The external expression of an individual in a given environment. It is the sum of
genotype and environmental interaction.
Genotype : The genetic makeup of an organism.
Dominance : It is an intra-allele interaction where one allele masks the effect of the other allele
in the F1 progeny is called dominant character.

25
Dominant allele : The allele which express itself in heterozygous condition under complete
dominance.
Recessive allele : Allele whose expression is masked in heterozygous condition.
Back cross : A cross between F1/hybrid with either of its parents.
Test cross : A cross between F1or hybrid corresponding recessive parent/individual. It is done
to test the genetic makeup of the individual (homozygous or heterozygous) as well
as for linkage.

Homozygous individual will produce only one type of gamete.

A heterozygous (at one locus) individual will produce two types of gametes in equal
proportion. Eg. Individual with Tt genotype will produce gamete carrying T and t
allele in equal proportion (1:1).
In test cross the parent should be a recessive individual

Genetics is the study of heredity and variation. The transmission of parental

characters to the progeny is called heredity. In other words, it is the tendency of individuals to
resemble their ancestors. However, though the offsprings resemble their parents very closely,
they never resemble them exactly. The differences between organisms that are related to one
another by descent are termed as variations.
Mendelism
Principles of heredity and variation were first formulated by Gregor Johann Mendel
(1822 – 1884) in 1866. Mendel is recognized as the “Father of Genetics” and his work is
known as “Mendelism”. He formulated the laws of Segregation and Independent Assortment.
In early 1900s, his laws were independently rediscovered by three scientists; Hugo de Vries
(Holland), Carl Correns (Germany), and Erich von Tschemark (Austria). Mendel used the
term Factors for genes and it was Johannsen (1909) gave the term allele for the alternative
forms of the factor.
Mendel studied seven independent characters in garden pea, Pisum sativum, each of
which had two alternative expressions or traits of which one has dominant and the other
recessive.
Sl. no Alternating Traits
Character
Dominant Recessive
1 Shape of seed Round Wrinkled
2 Seed coat colours Grey White
3 Cotyledon colour Yellow Green
4 Shape of the fruit/pod Inflated or full Constricted
5 Colour of fruit/pod Green yellow
6 Position of flower Axial Terminal
7 Length of stem Tall / long Dwarf / short

26
 He obtained purelines for each alternating expressions of the seven characters he
chose to study by self-fertilizing plants of each lines for several generations. Then he
conducted monohybrid crosses for the seven characters and studied each character one at a
time. For making crosses, he selected male and female parents having alternative traits of a
single character. Then he removed the male reproductive organ from the female parent. For
example, in the case of stem length, he crossed tall and dwarf true breeding plants (TT and tt
respectively) to each other by removing the stamens from some of the flowers from each
plant (emasculation) before they become mature and then dusting their stigma with pollen
obtained from the other plant. He also made reciprocal crosses by using both types as male
and female parents (TT x tt, tt x TT). The first-generation progeny of F1seeds were then
raised and each of the F1 plants were then self-fertilized to obtain the second filial generation
for F2 and then later in the same manner F3.

Parent Tall X Dwarf

F1 All Tall (Selfed)

F2 Tall Tall Tall Dwarf

(Selfed) (Selfed) (Selfed) (Selfed)

All Tall Tall: Dwarf Tall: Dwarf All Dwarf

When parents were tall and dwarf, allF1 progenies were tall. F2 segregated as 3 tall
and 1 dwarf. When F2 plants were selfed to get F3, all progenies from dwarf F2 yield dwarf
plants only. But of the tall F2 plants, 1/3 of them producer all tall progeny while 2/3 of them
produced tall and dwarf progenies in the ratio of 3:1.
For all the seven character tested, the results fitted in the same pattern. Mendel found
that:
1. F1 hybrid derived from a cross between two different parents showed only one of the
alternating traits of a character.
2. No reciprocal differences.
3. The trait that was hidden in the F1 reappeared in the F2 with a frequency of 25%.

Mendel visualized the determinants of each of the alternating trait as a

“factor”. According to him each parent had two of such factors and each parent

27
 passed only one factor to each of its progeny. He predicted that each factor retained its
individuality fromgeneration to generation i.e., there is no blending. The factors
contributed by the parents united at random in progeny. This is the Law of
segregation of factors.

When symbols are used, a monohybrid cross can be represented as follows.

Parent Tall X Dwarf
(TT) (tt)

Gametes T t

F1 Tall (Tt)
Gametes (Selfing)

T t T t

T t

T TT (Tall) Tt (Tall)

t Tt (Tall) tt (Dwarf)

F2 Monohybrid phenotypic ratio = 3 Tall: 1 Dwarf

F2 Monohybrid genotypic ratio = 1 TT: 2 Tt: 1 tt
F2 genotypes are selfed to get the F3.

F2 genotypes – TT Tt tt

selfed

F3 - All Tall 3 Tall : 1 Dwarf All Dwarf

From this results, it is clear that the tall F2 plants producing both tall and dwarf
progenies must contain both factors ie. T and t and are thus hybrids.
Testing genotypes:

28
 Mendel used two types of tests to distinguish between pure breeding homozygote and
hybrid heterozygote – selfing and test crossing.
In selfing, the plant to be tested is sefed and if all progenies show the same parental
phenotype, then the plant tested is pure or homozygote. On the other hand if the selfed
progenies segregate in to dominant and recessive phenotypes in a ratio approximating 3:1,
then the plant tested is hybrid or heterozygote.
Thus
 Tall plant All progenies tall, tested plant is homozygous for tall, TT genotype.
 Tall plant Progeny segregate into 3 Tall:1 Dwarf, tested plant is heterozygous for tall,
Tt.
In test crossing, the plant to be tested is crossed back to its recessive parent or any
other available recessive.
Thus if a test cross is done in order to identify the genotype of an individual, based on
the observations on progeny as follows;
 Tall plant x tt recessive parent All progenies are tall, the we can conclude that the
tested individual is homozygous ie., TT.
 Tall plant x tt recessive parent 1 Tall: 1 Dwarf, then we can conclude that the
tested individual is heterozygous ie., Tt.

The test cross is a back cross when it is involves crossing back to its recessive parent.
But all back crosses are not test crosses.
Dominance relationship, lethality and modification of monohybridratio
The 3:1 monohybridF2phenotypic ratio occurs only when a gene has two alternative
allels of which one is completely dominant over other recessive allele. When this relationship
is changed, the 3:1 ratio become modified and such interactions which operates within a gene
is known as intra-genic interactions/ inter-allelic interactions (between alleles of a gene)
Incomplete dominance (Partial dominance)
It is an intra-genic interaction, in which in a heterozygote one allele is unable to mask
the expression of the other allele. Hence, the heterozygote will be having a phenotype
intermediate between the two homozygous parents. Here the heterozygote can be easily
identified by its phenotype. So the phenotypic and genotypic ratios are the same ie, 1:2:1.
Eg: When a homozygous red flowered Mirabilis plant (RR) when crossed to a recessive
white flowered one (rr), the F1 will be heterozygote (Rr) with pink flowers. In the F2 the ratio
is 1 red: 2 pink: 1 white.

Co-dominance
In co-dominance, the alleles of a gene lack dominance recessive relationship and both
the alleles will express the effect simultaneously and independently in the heterozygous
condition. Hence, the heterozygote is having a phenotype different from both the
homozygous parents and can be easily distinguished. Here also the phenotypic and genotypic
ratios are same, 1: 2: 1.
Eg: Coat colorin short horn cattle is a classic example of co-dominance. The allele R for red
color is not dominant over R1 for white. The heterozygous (RR1) condition produces roan.

29
Parents Red x White
(RR) (R1R1)
Gametes R R1

Roan
F1 RR1

Roan Roan
RR1 RR1
Gametes R R1 X R R1

F2 Genotype 1 RR 2 RR1 1 R1R1

Phenotype 1 Red 2 Roan 1 White

Over dominance
When the heterozygote exceeds the phenotypic measurements of both the
homozygous parent, such condition is referred as over dominance (super dominance).This is
related to quantitative characters.

P1 20 cm X P2 10 cm

F1 25 cm

F2 1: 2: 1
20cm 25cm 10cm

Lethality
The term “lethality” is applied to those changes in an organism which causes its death
prematurely. When the dominant allele kills the carrier individual in the homozygous
condition, it is termed dominant lethality ie. Two doses of the alleles being needed to cause
death. If the allele can kill in the heterozygous condition ie. In a single dose, then it is also
called dominant lethality.

30
 In mice coat color, the dominant “Y” yellow allele cause dominant lethality, killing
the homozygous YY dominant genotype. The F2 ratio is modified to 2 yellow : 1 non-yellow.
In recessive lethality, individuals with recessive genotypes will not survive. The
recessive lethal gene remains unnoticed in the population in the heterozygous state. They may
be transmitted through heterozygous carriers for many generations without being noticed.
In mouse, hydrocephaly is due to a recessive lethal gene. During embryonic
development there is abnormal growth of cartilage. This leads to irregularities in formation of
skull and brain and excessive accumulation of cerebrospinal fluid. Embryos carrying the
homozygous gene do not survive. The heterozygotes are phenotypically normal.
Problems:
1. Yellow (Y) is dominant over the green (y) colour of the pea seeds. In the following
crosses find out the genotype of parents.
PROGENY
SI.NO CROSSES Yellow Green
1 Yellow X Green 78 80
2 Yellow X Yellow 122 42
3 Yellow X Green 74 0
4 Green X Green 0 50

2. In cattle, “polled” condition governed by P and “horned” condition governed by p. A

polled bull is crossed with 3 cows, with cow A which is horned, a polled calf is
produced. With cow B, which is horned, a horned calf is produced. With cow C,
which is polled, a horned calf is produced. Determine the genotype of the bull, cow
and calves?

3. In four O’clock plants, the allele for red (R1) flower colour has an effect that it is
incompletely dominant over the effect of the white (R2) colour allele. If a cross between
two plants produced 18 red, 32 pink and 15 white plants, what are the phenotypes of the
parents? What ratio of flower colour in four O’clock plant would you expect among the
off spring of the following crosses?
a. Red x Red b. Red x Pink
c. White x Pink d. Pink x pink

4. A pair of co-dominant alleles is known to govern cotyledon leaf colour in soybeans.

Homozygous genotype CGCG produces dark green; the heterozygous genotype CGCY
produces light green, and the other homozygous genotype CYCY produces yellow leaves
which are lethal. If dark green plants are pollinated only by light green plants and the F1
crosses are made at random to produce an F2, what phenotypic and genotypic ratios
would be expected in the mature F2 plants?

31
5. Yellow coat colour in guinea pigs are produced by homozygous genotype CYCY, cream
colour by the heterozygous genotype CYCW and white by the homozygous genotype
CWCW. what genotypic and phenotypic ratios are expected to produce in a mating
between cream coloured individuals?

6. The shape of radish may be long (SLSL), round (SLSR) or oval (SR SR). if long radishes
are crossed to oval radishes and the F1 then allowed to cross at random among
themselves, what phenotypic and genotypic ratio is expected in the F2?

7. Chicken with shortened wings and legs are called creepers. When creepers are mated
to normal birds, the produced creepers and normal in equal frequencies. When
creepers are mated to creepers, they produced two creepers to one normal. Cross
between normal birds produced only normal progeny. How can be these results are
explained?

32
 Ex. No: 9 Dihybrid ratio and its modifications

The law of independent assortment

Mendel’s study of monohybrid crosses (F2) led to the discovery of the principle of
segregation of factors. He then continued his experiments to study the pattern of segregation
in individuals differing in two characters (dihybrid crosses).
Dihybrid cross is a cross between two genetically dissimilar homozygous individuals
involving two gene pairs controlling two different characters simultaneously. Their F2is
9:3:3:1 in case of complete dominance. Mendel proposed law of Independent assortment
based on his observations on F2 dihybrid ratio. According to this law, the segregation of one
pair of allele is independent of the segregation of other pair.
He crossed smooth and yellow seeded plant to a wrinkled and green seeded one.

P: Round Yellow X Wrinkled Green

F1: Round Yellow

F2: Round Yellow Round Green Wrinkled Yellow Wrinkled Green

304 102 95 35

This result can be reclassified as follows:

If each character is analysed individually, then
Smooth x Wrinkled Yellow x Green
P1:

F1: Smooth Yellow

F2: ¾ Smooth : ¼ Wrinkled ¾ Yellow : ¼ Green

The F2 ratios therefore fit the monohybrid ratios for each character so each character
act independently of the other. Mathematically, this relationship can be expressed by
multiplying the probability of each character to get the probability of their appearance
together. There if a seed has the probability of ¾ to be smooth and ¾ to be yellow, its chance
to be smooth as well as yellow would be ¾ x ¾ = 9/16. Thus,

¾ Smooth X ¾ Yellow = 9/16 Smooth Yellow

¾ Smooth X ¼ Green = 3/16 Smooth Green

33
 ¼ Wrinkled X ¾ Yellow = 3/16 Wrinkled Yellow
¼ Wrinkled X ¼ Green = 1/16 Wrinkled Green

ie. a ratio of 9:3:3:1 which is the product of two 3:1 ratios multiplied ((3:1) x (3:1)) =
9:3:3:1.
Testing dihybrid F1
A dihybrid smooth yellow SsYy, when crossed to ssyy, will give a typical test cross
ratio of 1 Smooth Yellow: 1 Smooth Green: 1 Wrinkle Yellow: 1 Wrinkle Green ie. 1 SsYy:
1 Ssyy: 1 ssYy: 1 ssyy. This is the product of two monohybrid test crosses, ie. (1:1) (1:1) =
1:1:1:1.
Forked line method/ Branching method

P: Round Yellow (RRYY) X Wrinkled Green (rryy)

F1: Round Yellow (RrYy)

If we consider each character individually, then there will be 1RR, 2Rr, 1 rr and
1YY, 2Yy, 1yy type of gametes.

1YY 1RRYY 1 Round Yellow

1RR 2Yy 2RRYy 2 Round Yellow
1yy 1RRyy 1 Round Green

1YY 2RrYY 2 Round Yellow

2Rr 2Yy 4RrYy 4 Round Yellow
1yy 2Rryy 2 Round Green

1YY rrYY 1 Wrinkled Yellow

1rr 2Yy 2rrYy 2 Wrinkled Yellow
1yy 1rryy 1 Round Green

34
To obtain phenotypic ratios:
3 Yellow 9 Round Yellow
3 Round
1 Green 3 Round Green

3 Yellow 3 Wrinkled Yellow

1 Wrinkled
1 Green 1 Wrinkled Green

Problems

1. A tall (TT) pea plant with smooth seeds (SS) was crossed with plants with dwarf (tt)
and wrinkled seeds (ss). Find out the following,
a) Genotype and phenotype in F1
b) Genotype and phenotype in a cross between F1 plant and tall (TT) and smooth (SS)
seeded plant.
c) Genotype and phenotype in a cross between F1 plant and dwarf (tt) and wrinkled (ss)
seeded plant.
d) Genotype and phenotype in F2

2. Work out genotype of the progenies in the following crosses

Note: seed coat color grey (G) is dominant over white (g). Seed shape round (R) is
dominant over wrinkled (r):

a) GGRR x GgRr b) GGRR x GGrr

c) GgRr X ggrr d) Ggrr x Ggrr
d) GgRR x ggRr

3. In summer squash, white (W) fruit color is dominant over yellow (w) and disc (D) is
dominant over sphere (r). what will be the color and shape of fruit in F1? What will be
the genotypic and phenotypic segregation in F2. Indicate the F3 phenotypic behavior
of the F3 genotypes?

4. In ground nut semi-spreading habit (B) is dominant over bunch type (b), red seed
color (R) is incompletely dominant over white (r). The heterozygous genotype
35
 produces rose seed color. In a cross between a semi-spreading red with bunch rose,
the segregation was as follows, ¼ semi-spreading red, ¼ semi-spreading rose, ¼
bunch red and ¼ bunch rose. Determine the genotypes of parents and progeny.

5. The shape of radishes may be long (LL), round (L1 L1) or oval (LL1). Color may be
red (RR), white (R1R1) or purple (RR1). If a long white strain is crossed with a round,
red strain, what phenotypic proportions are expected in the F1 and F2.

6. In man, assume that eye colour brown (B) is dominant over blue eye (b) and right
handedness (L) is dominant over left handedness (l). when a brown eyed right-handed
man married to a blue-eyed left-handed woman, they had their first child with blue
eyed and left-handed. What will be the genotype of parents and the child?

36
 Ex. No:10 Trihybrid ratio

The principle of independent assortment can be verified for three pairs of

alleles. A cross involving three independently assorting character pairs are called
trihybrid cross. A trihybrid cross is between two individuals that are heterozygous for
three different traits. There are 8 phenotypic classes in the F2 of a trihybrid cross with a
general rule that there are 2n phenotypic classes in the F2, where n is the number of
independently assorting heterozygous gene pairs. There are 64 possible combinations of
the eight different gamete types contributed by each parent, creating 27 different
genotypes. There will be 8 different genotypes, two being parental types and six
recombinants in a predicted ratio of 27:9:9:9:3:3:3:1. It is obtained as a product of three
monohybrid F2 ratios ie. (3:1) x (3:1) x (3:1).
Likewise, a trihybrid test cross ratio can be obtained by multiplying three
monohybrid test cross ratios ie. (1:1) x (1:1) x (1:1) = 1:1:1:1:1:1:1:1.

Problems

1. A wheat variety with colored seeds is crossed to a colorless strain producing all
colored F1. In F2, 1/64 of the progeny has colorless seeds.
a) How many pairs of genes are controlling seed color?
b) What are the genotypes of parents and the F1 (use your own symbols)

2. In tomato red (R) fruit color is dominant over yellow (r).Two loculed (L) fruit is
dominant over many loculed (l). Tall (T) is dominant over dwarf (t). A tall plant with
yellow two loculed fruit when crossed with a dwarf red two loculed fruited plant,
progenies produced were in the following proportions. 3/16 tall red two loculed, 3/16
dwarf red two loculed, 3/16 tall yellow two loculed, 3/16 dwarf yellow two loculed,
1/16 tall red many loculed, 1/16 tall yellow many loculed, 1/16 dwarf red many
loculed and 1/16 dwarf yellow many loculed. Determine the genotype of parents and
progeny.

3. In peas tall (T) habit is dominant over dwarf (t), green (G) pod color is dominant over
yellow (g). Round (R) seed shape is dominant over wrinkled (r). a homozygous dwarf
green round plant is crossed with a tall yellow wrinkled plant. What will be the
appearance of F1? What type of gametes will be produced by the F1?

37
4. In man brown eye (B) is dominant over blue (b). Dark hair (D) is dominant over red
hair (d), and right handedness (R) is dominant over left handedness (r). What would
be the outcome of marriages between persons of the following genotypes?

a) BBDdRr X BbDdRR
b) bbDdRr X BBDDRR
c) BbDDRr X bbddrr
5. Consider a cross involving three independently segregating gene pairs as follow,
AABBCC x aabbcc, and the F1 progeny is self-fertilized. Answerer the following
questions.

Note: All the capital letters represent alleles with dominant phenotype.
a. How many types of genotypes are possible in F2?
b. How many F2 progenies will be phenotypically recessive for all
the three genes?
c. Number of progenies in F2 having homozygous condition for the
three dominant genes?
d. What kind of difference will be there in F2 data by considering the
cross AAbbCC x aaBBcc instead of AABBCC x aabbcc?

38
 Ex. No:11
Multiple alleles and Factor (gene) interaction

Usually a gene has two alternative forms or alleles. But there are some genes which
have more than two alternative forms such a condition is known as multiple allelism. It is
believed thatthese alleles have originated through mutations. The entire series of alleles
in a multipole allelic system are represented by the same basic letter. The different forms
are represented by superscript or subscripts (IA, IB and IO). All these alleles are arranged
according to their order od dominance gradation. But in one diploid, there will be only
two of these alleles, one at a given locus on each homologous chromosome. They control
the same character. A common example for a multiple allelic system is the blood group
pattern of man which is controlled by three multiple alleles. Person with IA has antigen A
and antibody against B. person with IB has antigen B and antibody against A. person with
IA and IB alleles have both antigen and no antibodies while, person with alleles IOIO has
no antigens and have antibodies against both A and B antigens. Hence O blood group can
be given to all and is called the universal donor. AB blood group can receive all blood
types and hence is called universal recipient.

Problems:

1. Blood groups and genotypes of parents are given. Find the probable blood groups of
offsprings.

Cross 1 OxO ii, ii

Cross 2 OxA ii, IAIA
Cross 3 OxA ii, IAi

2. Find impossible blood groups in the following combination of parents

1. A x B
2. A x Heterozygous B
3. Heterozygous A x Heterozygous B
4. AB x AB
5. O x Heterozygous B
6. O x AB

3. The dominant allele IA produces A group, recessive allele i alone produce O group.
In the case of a disputed parentage of child with O blood group, mother and
suspected father had A group. Substantiate your answer.

4. In rabbit there are four alleles at C locus – C, Cch, Ch and c. order of dominance is
C > Cch> Ch> c . C- Wild type full color, Cch - Chinchilla,Ch - Himalayan and c-

39
 Albio. A mating of a full colored with a himalayan gave a set of 8 offsprings with 4
full color, 2 himalyan and 2 albino. Find genotype of parents and offsprings?

5. One Himalayan and 1 chinchilla were crossed with albino males. In the progeny of
each cross 50% albino were produced. Find out genotype of parents and progenies.

6. In guinea pigs, one of the genes that affect coat color has a number of different
alleles. In a certain strains of guinea pig homozygous combinations of these alleles
produce the following phenotypes
CC = Black CdCd = Cream
CkCk = Sepia CaCa = Albino

Assuming that these alleles show complete dominance in the order C > Ck> Cd> Ca, what
will be the phenotype and proportions of progenies in the following crosses.
a) Homozygous Black x Homozygous Sepia
b) Homozygous Black x Homozygous Cream
c) Homozygous Black x Homozygous Albino
d) Homozygous Sepia x Homozygous Cream
e) The F1 of (a) x F1 of (c)
f) The F1 of (b) x F1 of (d)

7. What phenotypes and ratios would you expect among the progenies from the
following crosses?
a) IAIA x ii b) IAIA x IAIB
c) IAIA x IBi d) IAIA x IAi
e) IAi x IAi f) IAi x IAIB
g) IAi x ii

40
Ex. No: 12 Gene Interactions

Simple Interactions
William Bateson and R. C. Punnet in 1907 explained gene interaction based on their
studies on comb shape in chicken. There was visible difference in the comb shape in
domestic breeds of chicken. Wyandottes, Brahmaas and Leghorns are the three different
types of chicken breeds and have rose, pea and single combs respectively. Each of these can
breed true. A cross between pure breeding rose comb and single comb produced rose combed
progenies revealing the dominance of rose over single comb and F2 segregated in to rose and
single comb in 3:1 proportion. In a cross between pure breeding pea and single combed breed
produced pea combed progenies in F1, indicating the dominant action of pea over single.
Corresponding selfing of this F1 produced pea and single combed progenies in 3:1 proportion.
However, when a pure breeding rose and pea comb were crossed, an entirely new phenotype
was observed in F1 generation, walnut comb. Upon intermating of F1 produced walnut, rose,
pea and single comb in 9:3:3:1 proportion. The single comb and walnut were not expressed in
parental lines. The F1 resembles neither of their parents and new characters were appeared in
the F2 (here, walnut and single comb). Based on these two deviations from normal dihybrid
cross, let to the conclusion that walnut results from an interaction between two independent
dominant gene and the double recessive produced single comb.
Rose comb (R-pp) - 3 Pea comb (rrP-) -3
Walnut comb (R-P-) -9 Single comb (rrpp) -1

41
 The most important contribution of Mendel was the identification of unit nature of
inheritance. All those experiments conducted by him was having single gene with two
alternative forms for a specific character. All those ratios show this precise segregation of
two alleles of a single gene. In all such situations, one allele was dominant and other was
recessive. Later experiments in the same field by different scientist experienced a deviation in
this expected Mendalian ratios. According to Mendel’s experiments F1 from two genetically
different parents expressed phenotype of dominant parent. But in later experiments novel
phenotypes were produced which could not be explained in Mendelian pattern. They are
collectively grouped as factor interaction or gene interactions.
The phenomenon in which two or more genes governing the expression of a single
character is known as gene interaction (epistasis (Greek = stoppage)). It can be further
explained as an interaction between non allelic genes present in separate loci in which one
gene mask, inhibits or supresses the expression of other gene.

42
Problems
1. In poultry, gene for Rose comb (R) and Pea comb (P) interact and produce walnut
comb (R- P-). Recessive alleles of both together produce single comb (rrpp). When a
rose combed bird was crossed with a walnut, 3/8 walnut, 3/8 rose, 1/8 pea and 1/8
single were produced. Determine the genotype of parents and progeny.
2. Give four possible genotypes of walnut, if a pure walnut is crossed to a single and if
F1 is interbred. What will be the proportion of walnuts of four possible genotypes?
3. Four walnut hens were crossed to a single. In the first cross, progeny produced were
only walnut type. In second cross, walnuts and peas were produced. In the 3rd cross,
walnut and rose were produced. In the 4th cross, all types were produced. Find out
genotype of walnut parent in each cross?
4. In poultry, feathered shank (F) is dominant over clean shank. Inheritance of comb is
as in the above problem. A feathered shank rose combed bird crossed with a clean
shank pea combed bird produced offsprings in the following proportions, ¼ feathered
walnut, ¼ feathered pea, ¼ feathered rose and ¼ feathered single. Find out the
genotype of parents and offsprings?

43
 Ex. No:13 Complimentary and Masking gene action

Complimentary gene action (Duplicate Recessive gene action): 9:7

When Bateson and Punnett crossed two white flowered varieties of sweet pea, they
got a red or purple flowered hybrid. Up on selfing or inter-crossing of this F1 produced 9
coloured and 7 white flowered progenies. This is explained on the basis of an hypothesis that,
there are two pairs of factors (C/c and P/p) in determining flower colour in sweet pea and
they interact in the following way to produce coloured flowers.
Allele A Allele B
Colourless Colourless Purple/Red
precursor 1 precursor 1 pigment
Pigment change Pigment change
catalysed completed

Both the genes control the same character and produce identical phenotype in
dominant condition alone and in double recessive condition (C-pp, ccP-, ccpp). But when
both the genes together in dominant condition at least for a single dose will complement each
other and produce a new phenotype (C-P-).
P1 white flower X P2 white flower
(CCpp) (ccPP)

F1 generation All purple

(CcPp)
Self fertilization

CP Cp cP cp
CP CCPP (Purple) CCPp (Purple) CcPP (Purple) CcPp (Purple)
Cp CCPp (Purple) CCpp (White) CcPp (Purple) Ccpp (White)
cP CcPP (Purple) CcPp (Purple) ccPP (White) ccPp (White)
cp CcPp (Purple) Ccpp (White) ccPp (White) ccpp (White)

9 C-P- : 3 C-pp : 3 ccP- : 1 ccpp

Purple White

44
 In complementary gene action, to produce one of the two phenotypes require the presence
of both genes. When any one of the two or both the genes are in homozygous recessive state,
the contrasting phenotype is produced.
Problems
1. In sweet pea, gene C or P alone produces white coloured flowers. But when both the
genes are present together in dominant condition produces purple flowers. When a
white flowered plant was crossed with a purple one, 3/8 of the progenies were of
purple and 5/8 were white. Determine the genotype of parents and progeny with
explanation.
2. Two white flowered pea plants when inter-crossed produced white and purple
flowered plants in 3:1 proportion. Determine the genotype of parent and offspring.
3. In Maize, two genes C and R are both necessary for the production of red aleurone
colour. The absence of either or both of them produce white aleurone. If a third gene
P is present in addition to C and R, then the aleurone colour will be purple. But P has
no effect in the absence of C or R or both. Determine the aleurone colour of the
progenies in the following crosses

a. Cross 1: CcRrpp x ccRrPp

b. Cross 2: CCRrPp x CcRrpp

Masking gene action (Dominant Epistasis): 12:3:1

Dominant allele of the two genes affecting the same character produce distinct
phenotype when they are with homozygous recessive state of the other gene. But
when dominant allele of both genes are present together, the expression of one gene
mask that of the other. When both the genes are present in recessive state, a different
phenotype is produced.

12 (9) A-B- White

(3) aaB-

3 (3) A-bb Yellow

45
 1 (1) aabb Green

Problems

1. In summer squash, gene for white fruit colour (W) is epistatic over yellow (Y) and
green (y). what will be the colour of fruits and genotype of offsprings in the
following cross.

 WwYy x Wwyy

2. If a dihybrid white in the above problem is crossed with a green, then what
phenotypic and genotypic ratio can be expected in the progeny?

46
 Ex. No:14 Supplementary Factor and Inhibitory Factor

Supplementary gene interaction (Recessive Epistasis): 9:3:4

The dominant allele of one of the two genes governing a character produces a
phenotypic effect, however the dominant allele of the other gene dose not produce any
phenotypic effect of its own, but when present along with the dominant allele of first
gene, it modifies the phenotypic effect produced by that gene.

Example: suppose there are two gene P and R governing aleurone colour in maize.
P- Red colour
p- white colour and R- supplementary gene
Then the phenotypic ratio will be modified as

9 P-R- Purple

3 P-rr Red

ppR-
4 pprr White

Problems

1. In tomatoes, brown coloured stem is produced by a dominant gene. Gene P in

dominant condition along with C produces purple stem. Double recessive
being green (ccpp). Genotype possessing P alone in dominant condition also
produce green coloured stem. In a cross between true breeding purple plant
and true breeding green plant/ double recessive, what will be the appearance
of F1? Determine phenotypic and genotypic segregation in F2.
2. Find out genotypic and phenotypic ratio in the following cross of purple and
green
CcPp x ccpp

47
Inhibitory gene action (Dominant and Recessive Interaction): 13:3

One of the two completely dominant genes produce the concerned phenotype, while
its recessive allele produce the contrasting phenotype. The second dominant gene called
inhibitory gene has no effects of its own on the character, but it can stop the expression
of the dominant allele of the first gene.

Example:
In rice stem pigmentation is controlled by two genes, R and I. R produce purple
pigmentation, r ptoduce green pigmentation. Gene I is an inhibitory gene with two
alleles I and i.
R-I- genotypes produce green pigmentation. Since I inhibit the expression of R gene.
R-ii produce purple colour. rrI- produce green colour since I act only with R allele. rrii
will also produce green colour. Hence the phenotypic ratio will be as flows;

R-I- 9
13 Green
rrI- 3

rrii 1

R-ii 3 3 Purple

Problems:
1. In poultry, gene C produces coloured feathers and c produces white
feathers. Gene I inhibit the action of C and produces white. What will be
the feather colour of poultry in the following cross?
IiCc x iiCc
2. Find out the phenotypic segregation ratios of the following crosses of
white parents
a. IiCc x Iicc
b. IiCc x IiCc
3. When two homozygous white feathered birds were crossed, F1 was white
feathered. When test crossed, 3 white and 1 feathered were produced.
What will be the genotype of parents and progeny?

48
Ex. No:15 Duplicate gene action and Polymeric gene action

Duplicate gene action (Duplicate Dominant gene action): 15:1

In duplicate gene action, two completely dominant genes which produce the same
phenotypic effect whether they are alone or together. The contrasting phenotype is
produced when both the genes are in homozygous recessive state.
Example:
In rice awned character is controlled by two genes A1 and A2. Both these
genes are having duplicate gene action ie., A1, A2 alone or together will produce
awned phenotype and their double recessive genotype will produce awnless
phenotype. The phenotypic ratio is modified as follows;

9 A1- A2-
Awned (15)
3 A1- a2a2

3 a1 a1 A2-

1 a1 a1 a2 a2 Awnless (1)

Problems:

1. In rice, awn character is governed by 2 duplicate genes A1 and A2. A cross

between two awned plants produced 7/8 awned and 1/8 awnless. Determine the
genotype of parents and progeny.
2. Upon selfing an awned plant produced 15 awned and 1 awnless progeny. What
will be the genotype of parents and progeny?

Polymeric gene action (Duplicate gene with cumulative effect) : 9:6:1

This is also known as additive gene action. Additive genes are two
independent genes affecting the same character and producing the same
phenotype when either of them is present in dominant condition. When both are
present together in dominant condition, they add to each others effect and
produce a new phenotype.

Example
In barley, two completely dominant genes A and B affect the length of awns.
Dominant allele of the gene A or B alone (A-bb, aaB-) gives rise to awns of

49
 medium length. But when dominant alleles of both the genes are present
together (A-B-), they produce long awns. Individuals homozygous recessive for
both the genes (aabb) are awnless.

9 A-B- Long awns

6 A-bb Medium awns

aaB-

1 aabb Awnless

1. In pumpkin fruit shape is governed by two genes S1 and S2. When these two
gene together in dominant condition produces disc fruit shape. S1 or S2
separately produces sphere shape. When these two genes are in recessive
condition (s1 and s2) produce elongate shape. A cross between disc and sphere
plants produced progenies in the ratio, 3 disc: 4 sphere: 1 elongate. Find out the
genotype of parents and progeny.
2. If a double heterozygous disc is crossed with an elongate, what will be the
genotypic and phenotypic segregation in the progeny?

50
 Summary to epistatic interactions

Genotype A-B- A-bb aaB- aabb Phenotypic ratio

Classical ratio 9 3 3 1 9:3:3:1

Dominant epistasis 12 3 1 12:3:1

Recessive epistasis 9 3 4 9:3:4

Duplicate gene with cumulative 9 6 1 9:6:1

effect

Duplicate Dominant genes 15 1 15:1

Duplicate Recessive genes 9 7 9:7

Dominant and Recessive 12 3 1 13:3

epistasis

Additional questions

1. In summer squash, fruit shape is controlled by two gene A and B. A and B together in
dominant condition produces disc shape. A or B alone in dominant condition produce
spherical shape and double recessive condition produce long fruits. Determine the
genotypes and phenotypes of the progenies from the following crosses
a. AaBb x AaBb
b. AaBB x aaBB
c. AaBB x AAbb
d. AaBB x Aabb

2. In rice, awn production is controlled by two genes (A and B) with duplicate dominant
gene action. Determine the genotypes and phenotypes of the progenies of the following
crosses
a. AaBb x AaBb
b. AaBB x AAbb
c. AaBb x aaBB
d. AaBB x AABb
3. Red kernel colour in wheat is produced by the genotype C- P-, white by double
recessive ccpp. C or P alone in dominant condition (ccP-, C-pp) produce brown kernels.

51
 When a homozygous red variety is crossed to a white, what will be the resulting F1 and
F2?

4. In rice green colour stem is produced by dominant gene I and purple colour by dominant
gene P. gene I is dominant over P. determine the genotypes and phenotypes of the
progenies expected from the following crosses.
a. IIPP x IiPp
b. IiPp x IIpp
c. IiPp x iiPP
d. IiPP x IIpp

5. In an onion variety, there are three different colours of bulb;. red, yellow and white.
when a red bulbed plant was crossed with a white bulbed one, all the progenies have red
bulbs. Upon selfing of F1 produced 119 red, 32 yellow and 9 white bulbed progenies in
F2. What is the mode of inheritance of bulb colour, and how do you account for the
ratio?

52
 Ex. No:16 Determination of linkage – Two point test cross

LINKAGE

The tendency of two or more genes to stay together during inheritance is known as
linkage. This is due to the fact that these genes are located on the same chromosome and the
degree/ strength of linkage depends on the distance between these genes. Recombination % is
used to measure the distance between genes. Smaller the recombination %; more the intensity
of linkage.
Based on the results from dihybrid crosses in sweet pea, Bateson and Punnet put
forward the concept of coupling and repulsion phase linkage.
Coupling phase linkage: In coupling phase linkage, the dominant alleles of both the genes are
in one chromosome and recessive alleles in its homologous chromosome (AB/ab).
Repulsion phase linkage: in repulsion phase linkage, the dominant allele of one gene and
recessive allele of another gene are located on the same chromosome (Ab/aB).

Parental and recombinant gametes will be of different types depending upon how these genes
are linked in the parents.
Coupling phase Parental type AB ab
(AB/ab) Recombinant type Ab aB
AaBb
Repulsion phase Parental type Ab aB
(Ab/aB) Recombinant type AB ab

Estimation of linkage
When genes are located on different chromosomes, during gamete formation they assort
independent of one another (Mendel’s law of independent Assortment). But linked genes on

53
the same chromosometend to stay together during the formation of gametes. As a
consequence, the ratios obtained in F2 and test cross generation are significantly different
from the expected 9:3:3:1 and 1:1:1:1 ratio respectively. Hence F2 and test cross ratios are
used to detect linkage.
Estimation of linkage from test cross data
General procedure
1. Select two parents homozygous for the gene pair under study. P1(AABB) and P2
(aabb). Cross P1 and P2 to produce F1 generation
2. Back cross F1 (AABB) with double recessive parent P2 (aabb)
3. Categorize the test cross progenies in to 4 different phenotypic classes ie.,
AaBb:Aabb:aaBb:aabb, out of these four classes two will be parental type and two
will be recombinant type.
4. Calculate the expected ratios for different phenotypes.
5. Test the deviation of Aa and aa from 1:1 ratio.
6. Test the deviation of Bb and bb from 1:1 ratio.
7. If the alleles of each gene Aa:aa and Bb:bb are segregating in 1:1 ratio, then go for
joint segregation of both the genes A/a and B/b.
8. Test the deviation of AaBb: Aabb: aaBb: aabb from 1:1:1:1 ratio.
9. Draw conclusion: If the gene pair individually show Mendalian segregation but
jointly fail to show independent assortment, then it can be concluded with certainty
that the genes in question are linked.

Example 1
Genes on different chromosome assort independently giving a 1:1:1:1 test cross ratio.
Parents Aa Bb x aa bb
Gametes AB Ab Ab ab ab
F1 ¼ Aa Bb : ¼ Aa bb : ¼ aa Bb: ¼ aa bb
Example 2
Linked genes do not assort independently but tend to stay together in the same
combination as they were in the parents. (Genes to the left of the slash line (/) are on one
chromosome and those to the right are on the homologous chromosome).
Parents AB / ab x ab / bb
Gametes AB ab ab
F1 ½ AB / ab : ½ ab / ab
Large deviations from 1:1:1:1 ratio in the test cross progeny of a Dihybrid indicate
linkage.

54
Recombination
Linked genes do not always stay together because homologous non-sister chromatids
may exchange segments of varying length with one another during meiotic prophase.
Homologous chromosomes pair with one another in the process called ‘synapsis” and the
points of genetic exchange called “chiasmata’ produce recombinant gametes through crossing
over. Crossing over occurs between non-sister chromatids and it involves the breakage and
reunion of only two of the four strands at any given point in the chromosome.

55
When two strand double crossovers occur between two genetic markers, the products is
detected through the progeny phenotypes; are only parental types.

In order to detect these double crossovers, a third gene locus (0) between the outside
markers must be used.

In the diagram, a crossover occurs in the region between the loci A and B. two of the
meiotic products AB and ab have the genes linked in the same way as they were in the
parental chromosomes. These products are produced from chromatids that were not involved
in crossing over and are referred to as non-crossovers or parental types. The other two
meiotic products (Ab,aB) produced by crossing over have recombined the original linkage
relationships of the parent in to two new forms called recombinant or cross over types.
Multiple cross overs
If there is a certain probability that a cross over will form between the A and C loci and
another independent probability of a cross over forming between C and B loci, then the
probability of a double cross over is the product of the two independent probabilities.
Recombination between 2 linked genes does not exceed 50% even when multiple cross overs
occur between them.
Example
If a cross over between the A and C loci occurs in 20% of the tetrads and between C and
B loci in 10% of the tetrads in an individual of genotype ACB/acb, then 2%(0.2*0.1) of the
gametes are excepted to be of double cross over types, AcB and aCb.
Linkage relationships from a Two-point Test cross

56
 Parental combinations will tend to stay together in the majority of the progeny and the
crossover types will always be the least frequent classes. The mode of linkage (coupling or
repulsion) may be determined for the Dihybrid parent.

Example 1
Parents Dihybrid x Test cross parents
Parents
Aa Bb ab ab
(Linkage relationship unknown)
F1 42% Aa Bb
42% aa bb Parental types
8% Aa bb
8% aa Bb Recombinant types
The test cross parent contributes ab to each progeny. The remaining genes come from the
dihybrid parent. Thus “A’ and “B” must have been on one chromosome of the dihybrid
parent and “a” and “b”on the other, ie. The linkage relationship is in the coupling phase
(AB/ab) because these were the combinations that appeared with greatest frequency in the
progeny.

Example 2
Parents Dihybrid x Test cross parents
Parents
Aa Bb ab ab
(Linkage relationship unknown)

F1
42% Aabb
42% aaBb Parental types

8% AaBb
8% aabb Recombinant types

Here ‘A’ and ‘b’ must have been on one chromosome of the dihybrid parent and ‘a’
and ‘B’ on the other, ie. Linkage relationship is in repulsion phase. Ab/aB.

57
Problems
1. In maize, coloured seed coat (C) and full endosperm (S) are dominant over
colourless seed coat (c) and shrunken endosperm (s). The F1 of the cross between
a homozygous coloured full and colourless shrunken when crossed to a colourless
shrunken, the progenies produced in the following proportion. 4300 coloured full,
149 coloured shrunken, 4035 colourless shrunken and 152 colourless full. Find
out whether there is linkage? What is the recombination frequency and linkage
phase if there is linkage?
2. Purple coloured flower (P) is dominant over red colour (p). long shaped pollen (L)
is dominant over round (l). F1 of a cross between homozygous purple long and a
homozygous red round when crossed to a double recessive, the following
segregation was observed. 282 purple long, 132 purple round, 106 red long and
260 red round. Calculate the recombination percentage and determine the linkage
phase if there is linkage between these two genes.

58
 Ex. No:17 Determination of linkage – Three point test cross

Three point cross refers to using 3 points (genes) to determine the order and distance
between the genes. In a test cross involving three linked genes, the parental types are
expected to be most frequent and the double cross overs to be the least frequent. The gene
order is determined by manipulating the parental combinations into the proper order for the
production of double crossover types.
Example
Test cross

Parent Trihybrid Recessive

AaBbCc x aabbcc

(Linkage relationship unknown)

F1
36% Aabbcc 9% aabbCc 4% AabbCc 1% AaBbCc
36% aaBbCc 9% AaBbcc 4% aaBbcc 1% aabbcc

72% 18% 8% 2%

The 72% group is composed of parental types because non-cross over gametes are
always produced in the highest frequency. The only contribution the recessive parent makes
to all the progeny is ‘abc’. Thus the trihybrid parent must have had ‘A’, ‘b’ and ‘c’ on one
chromosome and ‘a’, ‘B’ and ‘C’ on the other. But the correct gene order has to be analysed.
Let us study three point test cross step by step.
Case 1. Suppose B locus is in the middle, can we produce the least frequent double cross
over types (2%) ?

x abc/abc = ABc/abC and abC/abc

Double cross overs are ‘ABc’ and ‘abC’ which are not agreeing with the observed double
cross overs ‘ABC’ and ‘abc’.
Case 2. Suppose, ‘C’ locus is in the middle. In this condition, the double cross overs are
‘ACb’ and ‘acB’ which is also not agreeing with the observed double cross overs.

59
 x abc/abc = ACb/acB and acB/acb

Case 3. Suppose, ‘A’ locus is in the middle. In this condition, the double cross overs are
‘bac’ and ‘BAC’, agreeing with the observation. So it can be concluded that the A locus is in
the middle.

x abc/abc = bac/bac and BAC/bac

From this we can note the single cross overs. Let us designate the distance between B
and A as region I and the distance between A and C as region II. Then single cross over in the
region I are baC and BAc. Single cross over in the region II are bAC aand Bac.

Interference and coincidence

One chiasma formation reduces the probability of other chiasma formation in an
immediately adjacent region of the chromosome. The tendency of one cross over to interfere
with the other cross over is called interference. The strength of interference varies in different
segment of the chromosome and is usually expressed in terms of a coefficient of coincidence,
or the ratio between the observed and the expected double cross overs.
% observed double cross overs
coefficient of coincidence =
% expected double cross overs

The coincidence is the complement of interference, so coincidence + interference =

one. When interference is complete (1), no double crossovers will be observed and
coincidence becomes zero. Coincidence value ordinarily varies between 0 to 1.

Problem
1. In a three pointtest cross of a triple heterozygote CDE/cde;
a. What are the parental type progenies?
b. What are the single crossover progenies between C &D?
c. What are the single crossover progenies between D& E?
d. What are the double crossover progenies?

2. In Drosophila, yellowbody (y), cut wing (ct) and vermilion eye (v) are three mutant
genes located in the x chromosome and these 3 genes are recessive to grey body (+),
non-cut wing (+) and red eye (+) respectively. A yellow cut vermilion female is

60
 crossed to normal male and F1 female is crossed to a yellow cut vermilion male. The
offsprings of the test are as follows;

 Grey non-cut red = 3562 (P) (+ + +)

 Yellow cut vermilion = 3425 (P) (y ct v)
 Grey cut vermilion = 941 (+ ct y)
 Yellow non-cut vermilion = 884 (y + +)
 Grey non-cut vermilion = 592 (+ + v)
 Yellow cut red = 592 (y ct +)
 Grey cut red = 107 (+ ct +)
 Yellow non-cut vermilion =96 (y + v)
a. Which of the classes contain crossover between y and ct?
b. Which of the classes contain crossover between ct and v?
c. Give the total number of crossovers in each region and figure the
percentage?
d. Give the distance between y and ct, ct and v?
e. Construct a chromosome map giving the location of genes
3. Assume that there is 10% crossover between the loci A and B and 15% between A
and C. the order of loci being A, B and C. what would be the expected % of double
cross overs. Assume that there is no interference.
4. A cross was made between a stalks with genotype CCshshWxWx and ccShShwxwx.
The F1 was test crossed and the following phenotypes were obtained.

C - colour c – colourless
Sh - plump sh – shrunken
Wx – starchy endosperm wx- waxy endosperm

Coloured shrunken starchy - 2538

Colourless plum waxy - 2708
Coloured plum waxy - 116
Coloured shrunken starchy - 113
Coloured shrunken waxy - 601
Colourless plum starchy - 626
Coloured plum starchy - 4
Colourless shrunken waxy - 2

Calculate the cross overpercentage between C and sh and between sh and Wx.
Calculate the % of double cross over and coefficient of coincidence. Draw a linkage map
showing the genes in the chromosome.

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 Ex. No:18 Study on models for DNA and RNA structure

Structure of DNA
DNA is composed of sequences which encode for all the genes which determine how
an organism will look like and react to its environment. The milestone work in determining
the nature of hereditary information was done by Friedrich Miescher (1871). He studied the
pus cells isolated from the bandages. Most of those cells were white blood cells and primarily
composed of nuclei. He called this nuclear material nuclein. He further characterized this
material chemically and revealed that it contained acidic (nucleic acid) and basic (histone
proteins) components. Later Altmann renamed it as “nucleic acid’. Further he discovered the
existence of two types of nucleic acids- Deoxyribo Nucleic Acid (DNA) and Ribo Nucleic
Acid (RNA).
Fred Griffith (1928), Avery, MacLeod and Mc Carty (1944) and Hershey and Chase
(1952) conducted experiments and put forward the final proof that DNA is the genetic
material. It should also be noted that in some organisms like RNA viruses, RNA act as
genetic material.
The famous discover, DNA double helical structure was discovered by two scientists,
James Watson and Francis Crick (1953). They analysed chemical and physical aspects of
DNA and determined that, DNA is double stranded, these strands are in anti-parallel
orientation in which purines always pair with pyrimidines in the opposite strand.
However rather than carrying out new experiments in lab, Watson and Crick mostly
collected and analysed existing pieces of data, putting them together in new ways. Some of
their most crucial clues to DNA’s structure came from Erwin Chargaff and Rosalin Franklin.
Chargaff Rules: E. E. Chargaff (1950) formulated important generalization about DNA
structure: These generalizations are called Chargaff’s Rules. They are summarised below.
 The purines and pyrimidines are always in equal amount, ie. A+G = T+C
 The amount of adinine is always equals to that of thymine, and the amount of guanine
is always equal to that of cytosine, ie. A = T and G = C
 The base ratio A+T/G+C may vary from one species to another, but is constant for a
given species.
 The deoxy ribo sugar and phosphate component occurs in equal proportion.

Physical structure
W. T. Atsbury (1938) performed X- ray diffraction studies to suggest 3 – D configuration.
By 1947, he had detected a periodicity of 3.4 A0 repetitions within the structure of molecule.
H.F.Wilkins and Rosalind Franklin in 1950 further confirmed this by obtaining improved X-
ray data from more purified samples of DNA. It suggested that the DNA resembled a tightly
coiled helix and was composed of two or three chains of nucleotides.

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Structure of DNA

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Watson and crick published their analysis of DNA structure in 1953. By building models
based on the above mentioned parameters, they arrived at double helical form of DNA.
 Two polynucleotide chains are coiled around a central axis, forming a right handed
helix.
 The two chains are antparallel to each other.
 The bases of both chains are flat, lying perpendicular to the axis; they are stacker on
one another, 3.4 A0 apart on the onside of the double helix.
 The nitrogenous bases of opposite chains are paired as the result of hydrogen bonding
(A-T, G – C).
 Each complete turn of the helix is 34 A0 long, each turn consist of ten bases.
 A large major groove alternating with a smaller minor groove winds along the length
of the molecule.
 The double helix has a diameter of 20 A0.

Structure of RNA
Some plant viruses (TMV, Turnip yellow mosaic viruses), animal viruses ( Influenza
viruses, rous sarcoma viruses, reoviruses) and bacteriophages (MS2) contains RNA as
genetic material. Just like DNA, RNA is polymeric nucleic acid of four monomeric
ribonucleiotides. Each ribonucleiotide contains a pentose sugar, a molecule of phosphate
group and a nitrogenous base. RNA consists of two purines (adenine and guanine) and
two pyramidines ( cytosine and uracil).
The organisms which have only RNA as genetic material is called genetic RNA.
While the organism which have DNA along with RNA, they use the RNA in carrying the
orders of DNA and called as non-genetic RNA since they has no genetic role. The non-
genetic RNA is heterogeneous and includes the following three genera: ribosomal RNA (r
RNA), transfer RNA (t RNA) and messenger RNA (m RNA). Each of these non-genetic
RNA has DNA- dependent replication of itself, that is, it is not self-replicating like DNA
and is transcribed by DNA.
In eukaryotic cell RNAs are formed in the nucleus and pass into the cytoplasm
through nuclear pores. In prokaryotic cella RNAs are released by the nucleoid directly
into the cytoplasm after synthesis.
Ribosomal RNA (r RNA)
It constitute large (up to 80%) part of the cellular RNA. It is a stable or insoluble
component of RNA. It appears to be single polynucleotide strands which are unbranched
and flexible.
There are three kinds of rRNAs in each prokaryotic cell namely 23S, 16S and 5S
rRNAs. The 23S and 5S rRNAs occur in the 50S subunit and 16S in the 30S subunit of
70S ribosome in prokaryotes. The eukaryotic cells have four kinds of rRNAs, namely
28S rRNA, 18S rRNA, 5.8S and 5S rRNAs. The 28S rRNA, 5.8S and 5S rRNAs occur in

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60S ribosomal subunit, while 18S r RNA occurs in 40S ribosomal subunit of 80S
ribosome of eukaryotes.

Schematic representation of RNAs

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 Schematic representation of transfer of genetic information

Messenger RNA (mRNA)

M RNA gets its name from the fact that it carries the message from DNA in the
nucleus to the cytoplasm where it gets transferred to proteins. The message is in the form
of triplets called as codon. The mRNA molecule is linear and does not show hydrogen
bonding. The coding sequence lies in the middle of mRNA molecule, on either side it has
non-coding sequence which include a cap and a leader sequence at 5’ end and a trailer
sequence at 3’ end. Thus eukaryotic mRNA consists of:

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Cap leader (not translated) coding sequence (translated) trailer (not
translated) poly A tail
Prokaryotic mRNA molecules are similar to those of eukaryotic cells. However, a
single mRNA molecule may have many coding sequences, each separated from the next
by a short non coding spacer sequence. Each coding sequence is translated into a different
polypeptide.
Monocistronic mRNA: mostly the mRNA carries the codons of single cistron (ie. Code
for one complete protein molecule) of the DNA. Such mRNA molecule is called
Monocistronic mRNA.
Polycistronic mRNA: Polycistronic mRNA is a mRNA that encodes several proteins and
is characteristic of many bacterial and chloroplast mRNAs. Polycistronic mRNAs consist
of a leader sequence which precedes the first gene. The gene is followed by an
intercistronic region and then another gene.

Transfer RNA (tRNA)

Robert Holley and co- workers (1965) reported the complete sequence of tRNA
isolated from yeast. It led them to propose the two dimensional clover leaf model of
tRNA.
 All tRNAmolecules have guanine residue G at the 5’ terminal end and unpaired
(single stranded) CC-A sequence at the 3’ end. This is called amino acid

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 attachment site, because the amino acids becomes covalently attached to adenylic
acid or A of CCA sequence during polypeptide synthesis.
 The amino acid stem or helix consists of seven paired bases.
 The T-stem is composed of five paired bases – the last (nearest the T-loop) is C –
G. T- loop contains seven unpaired bases and is involved in the binding of tRNA
molecule to the ribosomes.
 The anticodon stem includes five paired bases. The anticodon loop consists of
seven unpaired bases, the 3rd ,4th and 5th of which (from the 3’ end of the
molecule ) constitute the anticodon. It permits temporary complementary pairing
with three bases (triplet codon) on mRNA.
 The base on the 3’ side of anticodon is a purine.
 Immediately adjacent to the 5’ side of the anticodon, uracil and another
pyrimidine occurs.
 A purine, often dimethylguanylic acid, is located in the ‘corner’ between the
anticodon stem and the D stem.
 The D- stem is composed of three or four base paires. The DHU loop or D loop is
also variable in size containing 8-12 unpaired bases. This helps in binding of
amino-acyl synthetase.
 The extra arm is variable in nucleiotide composition and is lacking entirely in
some tRNA.

Clover leaf structure of rRNA

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