University of Zimbabwe

Biological Sciences Department

G E N E T I C S

Courses HBZ107 and BSFS115

2016

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CONTENTS OF LABORATORY MANUAL

1. GENERAL INFORMATION ABOUT THE COURSE 3

2. Course description 5

3. Writing Lab Reports 6

4. Laboratory 1: Introduction to D. melanogaster 9

5. Laboratory 2: Variation in humans 17

6. Laboratory 3: Laws of probability 22

7: Laboratory 4: A. Drosophila experiments 30

B. Dihybrid segregation 31

8. Laboratory 5: Mitosis 36

9. Laboratory 6 Drosophila experiments 38

10. Laboratory 7: Chi-Square Test 39

11. Laboratory 8: Population Genetics 50

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 GENERAL INFORMATION ABOUT THE COURSE

LECTURER:

Professor C. Zimudzi Email: czimudzi@science.uz.ac.zw Cell: 0772669975

Office number: 08-3-02

TECHNICAL STAFF:

G.Chiripanhura - Senior Technician Cell: 0775606678

C.Nduna - Technician Cell: 0734544509

The instructors are available to meet with students and discuss matters by appointment
only. PLEASE NOTE: Any student who needs special accommodations in this course is
invited to inform the lecturer as soon as possible.

Recommended texts:
1. Introduction to Genetic Analysis, 9th edition, by Griffiths, Wessler, Lewontin &
Carroll, W.H. Freeman Co., NY, 2008.
2. Hartl D.L. and Jones EW. Essential Genetics: A Genomic Perspective Any Edition.
Jones and Bartlett Publishers, (2006) Sudbury MA.

Skills development:
1. To understand the language and basic concepts of genetics. 2. To understand how
traits are inherited and to use this understanding in analyses (to solve problems and
complete pedigrees) 3. To understand probability concepts and use these concepts to
solve problems (including basic statistical problems) 4. To understand how genetic
problems may lead to disease or lethality 5. To understand current issues regarding
genetics (e.g., cloning, use of transgenic organisms) 6. To understand the workings and
uses of population genetics, variation, and quantitative techniques
Expectations:
HBZ107 is a challenging course and will require strong academic effort. Many students
find genetics to be a particularly difficult subject, in large part because it is a highly
inferential subject. To do well, one must master a basic array of genetics principles, and
then diligently practice application of those principles in solving problems. Students are
responsible for materials presented in lecture and in laboratory. Students must be
prepared to give very strong effort outside of class.
 Regular attendance in lecture is essential. If you miss a lecture, you must rely on
notes from fellow students.

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  I recommend that you allocate time soon after lecture (e.g., that evening) to
review and revise your notes, and to use the text to resolve lecture material that
may not be clear. By quickly resolving any fuzziness or confusion, you consolidate
your understanding and memory of the material.
 Marathon study sessions are poor preparation for exams. You should learn the
material as it is delivered, then review notes and re-read relevant sections of the
text before exams......and get a good night’s sleep.

Course Assesment: You will be examined on material covered in lectures and related
problem-solving skills. Because much of genetics is based on problem solving, expect to
see lots of problem solving in the exams. Important preparation for exams is working
problem sets in lab, in the text, and on the internet. If you regularly work through the
problems and seek help for problems you do not understand, you are likely to do well in
the course. TCA will be assessed by means of two short exams, the first written midway
through the course and the second at the very end. For PCA FOUR labs will be assessed
and it will be the SAME labs for everybody, so please be advised not to miss any.

Laboratory: The HBZ107 laboratory provides hands-on and mind-on experience with
genetics methods, as well as practical reinforcement of material covered in lecture.
Several experiments will be carried out and you will submit lab reports on each of those
experiments. Late submission of reports and homework will cost you marks . Laboratory
attendance and participation is mandatory. If you have to miss a practical due to
circumstances beyond your control, documentary proof will be required; on or before the
date of the missed practical, or as soon as possible thereafter.

Plagiarized answers/homework are entirely unacceptable for all University work.

University policies regarding plagiarism and other academic offenses will be strictly
enforced, with a minimal penalty of zero credit for the work involved.

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 COURSE DESCRIPTION

Mendelian Analysis:
Mendel's experiments, Simple Mendelian Genetics in Humans and Agriculture; Variants and
Genetic Dissection. Chromosome Theory of Heredity: Mitosis and Meiosis; The
chromosome Theory of Heredity; Sex chromosomes and Sex Linkage; The Parallel Behaviour
of Autosomal Genes and Chromosomes; Mendelian Genetics and Life Cycles. Extensions of
Mendelian Analysis: Variations on Dominance; Multiple Alleles; Lethal Alleles; Several Genes
Affecting the Same Trait; Penetrance and Expressivity.Linkage-Basic Eukaryotic
Chromosome Mapping:Discovery of Linkage; Recombination, Sex-Linkage; Linkage Maps;
Three point Test crosses.Introduction to Population Genetics: Phenotypic Diversity and
Genetic Variation; Organisation of Genetic Variation, Allele Frequencies; The Hardy-
Weinberg Principle and its Applications.

Writing Lab Reports

Format:

A scientific report usually consists of the following:

1. Title
2. Abstract
3. Introduction
4. Materials and methods
5. Results
6. Discussion
7. Conclusions
8. Literature cited

Title:

The title should be less than ten words and should reflect the factual content of the
paper. Scientific titles are not designed to catch the reader's fancy. A good title is
straight forward and uses keywords that researchers in a particular field will
recognize.

Abstract:

The purpose of an abstract is to allow the reader to judge whether it would serve his or
her purposes to read the entire report. A good abstract is a concise (100 to 200

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 words) summary of the purpose of the report, the data presented, and the author's
major conclusions.

Introduction:

 Define the subject of the report: "Why was this study performed?"
 Provide background information and relevant studies: "What knowledge already
exists about this subject?"
 Outline scientific purpose(s) and/or objective(s)
 This is usually one to two paragraphs long
 The literature referred to should be cited.

Materials and methods:

 List materials used, how were they used, and where and when was the work done
(especially important in field studies).
 Describe special pieces of equipment and the general theory of the analyses or
assays used.
 Provide enough detail for the reader to understand the experiment without
overwhelming him/her. When procedures from a lab book or another report are
followed exactly, simply cite the work and note that details can be found there.
 For the Drosophila experiments, for example, you would briefly describe the stocks
that you used, the phenotype of the parental flies; and an outline of the procedure(s)
that you used to carry out the experiment. It is important to give the dates of the
various crosses.

Results

 Concentrate on general trends and differences and not on trivial details.

 Summarize the data from the experiments without discussing their implication
 This is not the section for interpreting the data.
 Organize data into tables, figures, graphs, photographs, etc. Data in a table should
not be duplicated in a graph or figure
 Title all figures and tables; include a legend explaining symbols, abbreviations, or
special methods
 Number figures and tables separately and refer to them in the text by their
number, i.e.

1. Figure 1 shows that....

2. The activity decreases after five minutes (fig. 1)

Discussion

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 o Interpret the data; do not restate the results
o Relate results to existing theory and knowledge i.e. Show how your results
relate to theoretical expectations based on background information obtained
from lectures, textbooks or outside reading.
o Discuss how your results relate to the goals of the study, as stated in your
introduction
o Explain the logic that allows you to accept or reject your original hypotheses
 This section is typically the longest part of the report.

Conclusions
o The purpose of this section is to wrap up the lab and summarize what was
reported.
o Include suggestions for improving your techniques or design, or clarify
areas of doubt for further research

References

o Cite only references in your paper and not a general bibliography on the
topic
o Alphabetize by last name of the author.
o Give author(s), date of publication, title of article, journal name, volume and
pages. When citing a book, include its title, name of publisher and place of
publication.
o Cite only material you have actually read.

Example for Journals

Hall, D.J., W.E. Cooper, and E.E. Werner. 1970. An experimental approach to the production
dynamics and structure of freshwater animal communities. Limnology and Oceanography,
15, 839-928.

Example for Books

Gardner, E.J., T.R. Mertens, and R.L. Hammersmith (1985). Genetics Laboratory
Investigations. 8th edition,: MacMillan Publishing Company; New York.

General style

o Strive for logic and precision and avoid ambiguity, especially with pronouns
and sequences
o Keep your writing impersonal; avoid the use of the first person (i.e. I or we)
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 o Use the past tense and be consistent within the report
note: "data" is plural and "datum" is singular; species is singular and plural
o Italicize all scientific names (genus and species)
 Use the metric system of measurement and abbreviate measurements without
periods (i.e. cm kg) Write numbers as numerals when greater than ten (i.e. 156) or
associated with measurements (i.e. 6 mm or 2 g).

Laboratory 1

Drosophila melanogaster experiments

Introduction: Drosophila melanogaster is a fruit fly, a little insect about 3mm long, of
the kind that accumulates around spoiled fruit. It is also one of the most valuable of
organisms in biological research, particularly in genetics and developmental biology.
Drosophila has been used as a model organism for research for almost a century, and
today, several thousand scientists are working on many different aspects of the fruit fly.

OBJECTIVES: At the completion of the exercise you should be able to

 Identify Drosophila melanogaster.

 Conduct a genetic experiment which spans a number of generations
 Discuss the life cycle of Drosophila melanogaster
 Differentiate between male and female Drosophila melanogaster
 Adeptly handle fruit flies and be able to recognize wild-type flies and mutants.

Background Information on Drosophila melanogaster

Figure 1:Drawing of the fruit fly Drosophila melanogaster

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Why work with Drosophila?

So much is already known about it that it is easy to handle and well-understood - and part
of it is practical: it's a small animal, with a short life cycle of just two weeks, and ischeap
and easy to keep large numbers. Fruit flies are hardy with simple food requirements and
occupy little space. Fruit flies produce large numbers of offspring to allow sufficient
data to be collected. Examination and data collection is easy because the flies can be
quickly and easily immobilized for examination. Many types of hereditary variations can
be recognized with low-power magnification and stock cultures can be readily obtained.
Drosophila has a small number of chromosomes (four pairs). Much research about the
genetics of Drosophila over the last 50 years has resulted in a wealth of reference
literature and knowledge about hundreds of its genes.

Life cycle of Drosophila

It is important to realize that a number of factors determine the length of time of each
stage in the life cycle. Of these factors, temperature is the most important. At room
temperature (about 25oC), the complete cycle takes 10 to 12 days.

The eggs. The eggs are small and ellipsoid and have two filaments at one end. They are
usually laid on the surface-of the culture medium and, with practice, can be seen with the
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naked eye. The eggs hatch into larvae after about a day.

The larval stage. The worm like larva eats almost continuously, and its black mouthparts
can easily be seen moving back and forth even when the larva itself is less distinct Larvae
channel through the culture medium while eating; thus channels are a good indication of
the successful growth of a culture. The larva sheds its skin twice as it increases in size.
In the last of the three larval stages, the cells of the salivary glands contain giant
chromosomes, which may be seen readily under low-power magnification after proper
staining. The larval stage lasts about 6 days.

The pupal stage. When a mature larva in a laboratory culture is about to become a pupa, it
usually climbs up the side of the culture bottle or onto the paper strip provided in the
culture. The last larval covering then becomes harder and darker, forming the pupa case.
Through this case the later stages of metamorphosis to an adult fly can be observed. In
particular, the eyes, the wings, and the legs become readily visible. The pupal stage lasts
approximately 4 days.

The adult stage. When metamorphosis is complete, the adult flies emerge from the pupa
case. They are fragile and light in color and their wings are not fully expanded.
These flies darken in a few hours and take on the normal appearance of the adult
fly. They live a month or more and then die. A female does not mate for about 10 to
12 hours after emerging from the pupa. Once she has mated, she stores a
considerable quantity of sperm in receptacles and fertilizes her eggs as she lays
them. Hence, to ensure a controlled mating, it is necessary to use females that have
not mated before. These flies are referred to as virgin females.

The Drosophila genome

Drosophila has four pairs of chromosomes: the X/Y sex chromosomes and the autosomes
2,3, and 4.

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Polytene Chromosomes

These are the magic markers that first put Drosophila in the spotlight. As the fly larva
grows, it keeps the same number of cells, but needs to make much more gene product.
The result is that the cells get much bigger and each chromosome divides hundreds of
times, but all the strands stay attached to each other . The result is a massively thick
polytene chromosome, which can easily be seen under the microscope. Even better, these
chromosomes have a pattern of dark and light bands, like a bar code, which is unique for
each section of the chromosome. As a result, by reading the polytene bands, you can see
what part of the chromosome you are looking at. Any large deletions, or other
rearrangements of part of a chromosome can be identified, and using modern nucleic acid
probes, individual cloned genes can be placed on the polytene map.

Handling the Flies

Anaesthetizing
Flies need to be sexed and sorted both for making the matings and for counting their
subsequent offspring. To immobilize flies we will use ether¨, a safe anaesthetizing agent
that will knock the flies out. There will be a demonstration on immobilising the flies in the
first lab. Observe the flies in the jar. When they stop moving leave them for another 5 -
15 seconds, and drop them on your counting plate. Flies can be sorted under a dissecting
microscope. To sort or transfer flies, use a camel-hair brush or fine forceps. Lift them by
the wings, not by the legs or body. Take special care with vestigial-winged flies.

Do not try to handle too many flies on your plate at one time. Put some in empty vials
provided, and re-etherise them when you are ready for them.

If you have a mixture of flies with different phenotypes, it is convenient to line the flies in
the centre of the counting plate, separate them into the various categories, and then count
the numbers in each category.

If the flies begin to reawaken on the counting plate, they may be re-etherised by placing
the re-etheriser over them.

N.B. If you re-etherise them more than once they can die or get sterilised from over-
exposure to ether.

You can distinguish over-etherised flies by examining the angle of the wings to the body -
the wings of over-etherised (dead) flies will be raised at an angle of about 90C above the
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abdomen. Males tend to be more susceptible to the effects of over-etherisation than
females.

After the flies are no longer needed, they should be dumped in the fly morgue.

Sexing

Careful sex determination is essential in preparing matings for genetics studies. Sexes
can be distinguished soon after emergence and, with practice, even in the pupal case.
Males are usually smaller than females. Males have dark, blunt posteriors, whereas the
females have lighter, pointed posteriors. The males have sex combs, which are groups of
black bristles on the uppermost joint of the forelegs, whereas the females do not. The
most distinguishing characteristic is the presence of the genital arch on the male flies.
The genital arch can be seen when the flies are placed on their backs and the posterior
area is examined for the tan colored arch. Newly emerged flies lack pigmentation and the
genital arch will be the only clear difference between the sexes.

Virgin Females
Genetics experiments require that the parents of the offspring are known. Because
females can store sperm for weeks after a single mating, it is important to isolate
females before they have had the opportunity to mate. Virgin females are usually obtained
by clearing stock cultures of all flies and then isolating the females which emerge within
the following 8 hours.You only need this method to obtain virgin females; males used in
matings do not need to be virgins.

Mating
After you have selected about six or eight virgin females of one genotype and the same
number of males of the second genotype, you are ready to gently transfer the flies to a
vial that has been previously prepared with media. Do not place the flies directly on the
media. Rather with the vial lying on its side, place the flies along one dry plastic side of
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the vial and insert the foam plug. Let the vial remain on its side until the flies are awake
and active. Be sure to label the vial with the date, parental genotypes, your name, and lab
section.

Procedure

1. Sexual Variation and Mutants in Drosophila

(a) Distinguishing males and females Make sure that you are able to distinguish between
the males and females. Refer to the section on `Background information on Drosophila
melanogaster'.

(b) Identifying Mutant Strains

Before you observe their mutants you need to be familiar with the appearance of the wild-
type Drosophila - the commonest naturally occurring form. Mutant strains are those which
show differences from the wild-type.
Mutant Wild Type

white eye red eye

vestigial wing long wing

(c) Use information from (b) above to identify flies in all the vial provided.

2. Obtain the vials containing experimental flies. This vial contains male and female
flies, which are the parental generation for your study. They have already mated
and eggs and larvae are on the surface of the culture medium. The eggs (or maybe
larvae now) represent the first filial, F1, generation and will be emerging from
their pupa cases in about a week.
a) Record the date the cross was made in your lab notebook. Use this date to
estimate when the adults will emerge.
b) Immobilize the adult flies and observe them carefully under magnification.
Separate the males from the females, making a pile of each sex.
c) Study each sex and identify the mutant. Observe and describe the mutation and
assign a name and genetic symbol in your lab notebook. Note whether the mutation
is associated with the males or females. Please confirm these observations with
your instructor.
d) Place the parents in the morgue (jar containing alcohol).
e) Label the vial with your name.
f) Record the date the parental generation was put in the morgue.

Second Week: (See instructions under laboratory 4)

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Third Week:Remove the F1 flies from the vials and place them into the morgue. The F2
generation is in the form of eggs, larvae, and pupae in the vial. Place the vial back in the
incubation area. Record the date the F1 generation was put in the morgue.

Fourth Week: (See instructions under laboratory 8).

Laboratory 2

Variation in humans
This lab is designed to reinforce some of the concepts of variation that have been
introduced in the lectures.

(a) Weight, height and waist size.

Using the equipment provided record your weight, height and waist size. Enter these data in
appropriate tables drawn on the board. Copy the completed tables and prepare histograms
to show variation in: Weight, Height and Waist size for the class.

(b) Ability to Taste PTC (Phenyl thiocarbamide)

You will be supplied with a small piece of filter paper that has been impregnated with a
PTC solution. Place this paper on the tip of your tongue; if you do not obtain a
characteristic and distinctive taste, chew the paper. Generally, if you are a taster, you
will immediately be able to taste a very bitter taste. Even though it has been recently
demonstrated complex inheritance of this trait, the ability to taste
PTC is apparently controlled by a dominant gene. A taster could be either homozygous or
heterozygous, while a non-taster would be homozygous recessive
Taster: TT or Tt Non-Taster: tt

(c) Interlocking Fingers

(I = left on top, i = right on top) Interlock your fingers and observe which thumb you
“naturally” place on top. If your left thumb is on top you have the dominant allele. If
right is on top you are homozygous recessive for the trait

(d) Tongue Rolling

Extend the tongue and determine whether or not you are able to roll the edges up into a U-

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shaped figure. Tongue Roller: RR or Rr Non-Tongue Roller: rr

(e) Dimpled Cheeks

The presence of dimples in the cheeks is inherited as a phenotypically dominant trait.

The traits shows considerable variability, with dimples sometimes in both cheeks,
sometimes in only one cheek, and occasionally even two dimples in the same cheek.
Dimples: DD or Dd No Dimples: dd

(f) Ear Lobes

Ear lobes may be attached, which means they form nearly a straight line where they
intersect the side of the head, or they may be free, which means they are pendulous, or
hanging down. Have your lab partner examining you, and pass judgment on your ear lobes.
Free Ear Lobes: EE or Ee Attached Ear Lobes:

(g) Tongue Folding

The ability to fold the tip of the tongue upward and backward, without touching the
upper teeth or the roof of the mouth is quite rare, and is inherited as a phenotypically
dominant trait.
Tongue Folder: FF or Ff Non-Folder: ff

(h) Sex

The sex of an individual is a inherited trait, and is determined by one of the twenty-
three pairs of the chromosomes that humans have: the X chromosome and the Y
chromosome.
Female XX Male XY
Double-jointed Thumbs (hypermobility)

(i) The condition of having loose ligaments, which permits unusual movement of the
thumbs, even permitting them to be bent almost completely back to the wrist and be
“thrown out of joint”, is due to the presence of a dominant gene. Its recessive allele
results in tighter ligaments and more limited thumb movement.
Double-Jointed Thumbs: DD or Dd Tight Thumbs: dd

Write up

Present your write-up in the form of a scientific paper with all the necessary subheadings.
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 In the introduction the examiner would want to test your understanding of the
concept of variation, what it is, how its partitioned, why its important to study
variation and how variation is related to genetics. In this section you are required to
cite important references (Books and journals). Make sure you cite the references
correctly.

In the discussion you need to explain what accounts for the variation observed among
individuals? You also need to explain why we do not get any meaningful ratios of the
individual traits in the class.

Table 1. Variation in some human genetic traits in the 2016 genetics class
Phenotype Insert your Give your genotype Number in class
phenotype
- PTC taster - - -
-Non PTC taster - - -
Left thumb on top - - -
Right thump on top
Thumbs d jointed - - -
Not d.jointed
-Tongue roller - - -
-Non roller - - -
-Cheeks dimpled - - -
-Not dimpled - - -
-Lobes attached - - -
-Not attached - - -
-Tongue folder
-On-folder
-Female - - -
-Male - - -

Laboratory 3

Laws of Probability

Few concepts have had greater effect on the science of genetics than the laws of
probability. Probability refers to the chance of something happening. Under normal
conditions, probability calculations can give us good ideas of what to expect from
different genetic combinations. A thorough understanding of probability was
instrumental in leading Gregor Mendel to his basic conclusions about genetics, and these

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same laws of probability play an essential role in genetics today.

Objectives:
 Explain the role of sample size in estimating probability
 Calculate the probability of occurrence of a single event.
 Calculate the probability of simultaneous occurrence of two independent events.
 Use probability principles in solving problems concerning
- independent events occurring simultaneously;
- binomial expansion;
- mutually exclusive events;
 Apply the fundamental principles of probability to genetic problems

Fundamental probability principles underlying Mendel`s basic law of heredity will be considered
in today`s laboratory work. Much of this work will be problem solving. You will investigate the
probability of independent events occurring simultaneously and the probability of either one or
the other of two mutually events occurring. You will learn to expand the binomial (a + b) n to
calculate probability for certain combinations of events. Finally, you will use probability
principles as a genetics counselor might use them to advise parents of the chance that they will
have defective offspring from certain matings within the pedigree.

THE IDEA OF CHANCE

First of all, consider a simple demonstration of the operation of chance (i.e., probability) in the
tossing of coins. It is usually impossible to measure or control the many factors involved when a
coin is tossed and allowed to return to the resting position. The end result is said to occur by
chance. The coin returns to a resting position with one or the other of its faces upturned, that
is, heads or tails. When a single coin is tossed many times, it is expected that about half of the
tosses will result in heads facing up and other half in tails.

PART I: PROBABILITY OF THE OCCURRENCE OF A SINGLE EVENT

1. Toss a coin 20 times. Have your partner count how many times it lands head up and
how many times it lands tails up.
2. Write the totals under the observed column for 20 toss in Table 1.
The law of probability states that when a procedure can result in two equally
likely outcomes (in this case, heads or tails), the probability of either outcome
occurring is 1/2 or 50 percent.
3. Using the law of probability, decide how many times out of 20 tosses you would
expect heads to appear and how many times you would expect tails to appear.
4. Write your answer in the expected column for 20 tosses in Table 1.
5. Calculate deviation by subtracting the expected number from the observed
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number.Record these in the deviation column for 20 tosses in Table 1. Make all numbers
positive (absolute value).
6. Have your partner toss the coin 30 times. You count how many times heads and tails
will appear.
7. Record the observed numbers in the observed column for 30 tosses in Table 1.
8. Calculate the expected number of heads and tails and record them in the proper
column in Table 1. Then calculate the deviates, and enter these in the proper column.
9. Now you repeat step 1, but toss the coin 50 times. Your partner should keep track of
the appearance of heads and tails.
10. Record the observed numbers, expected numbers and deviations in the columns for 50
tosses in Table 1.
11. Add the observed numbers of heads and tails and the expected numbers of heads and
tails from the three trials and record the totals in the Totals column in Table 1. Then
calculate the deviations and record these numbers.
12. What was the expected ratio of heads to tails for tosses of a single coin?
Did your results always agree with the expected ratio? If not, what would be a
reason for the deviation?

TABLE I. Probability of the Occurrence of a Single Event

HEADS TAILS
20 Tosses Observed
Expected
Deviation
30 Tosses Observed
Expected
Deviation
50 Tosses Observed
Expected
Deviation
Total Observed
Expected
Deviation

13. Compare the deviations from the expected for 20, 30 and 50 tosses. What seems to
be the relationship between the sample size and deviation?
14. What biological and genetic situations are analogous to, and result in, the 50:50
expectations one observes when flipping a coin? _____________

PART II: Probability of Independent events occurring simultaneously

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1. Toss two coins 40 times simultaneously. Have your partner keep track of how many
times heads-heads, heads-tails, tails-heads, and tails-tails occur. Count tails-heads and
heads-tails together.
2. Record the numbers for each combination in the observed column in Table 2.
3. Calculate the percent of the total that each combination (heads-heads, heads-tails, or
tails-tails) occurred and record it in the proper column. To find the percent, divide each
observed number by 40 and multiply by 100.
4. According to the law of probability, when there are four equally likely outcomes from a
procedure, the probability that one of the outcomes will occur is 1/4 or 25%. We can see
how this is calculated. For example, we know that in tossing two coins, the probability off
heads occurring on one coin is 1/2. The possibility of heads occurring on the other coin is
also 1.2. The probability of heads occurring on
both coins in one toss is 1/2 x 1/2 = 1/4.
5. Using the law of probability, predict the expected outcomes of tossing two coins.
6. Record the expected numbers in the proper column in Table 2.
7. Calculate the percent of the total that each combinations expected to occur. Enter
these numbers in the proper column.
8. Calculate the deviation by subtracting the expected from the observed and enter your
results in Table 2.

TABLE II. Probability of Independent Events Occurring Simultaneously

Combinations Observed % Expected % Deviation

Heads-Heads

Heads-Tails or
Tails-Heads

Tails-Tails

Total

8. Note that the situation is similar to what one sees in a simple monohybrid cross when Aa
produces gametes, the probability is that ½ of the gametes will contain the gene A and ½
will contain a. When an Aa female is crossed with an Aa male and the progeny are produced,
the probability is 1/4 that an A egg and an A sperm will come together to produce and AA
offspring. Similarly, the probability is ½ for Aa and 1/4 for aa progeny. In studying this
monohybrid cross you are considering a situation in which independent events (the union of
different kinds of gametes) occur simultaneously. Thus, a basic probability principle
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underlines Mendel`s first law.
9. The same law of probability applied to the experiment with two coins may be used
to calculate the expected ratios when 3, 4 or more coins are tossed together.
a. For example, calculate the expected results from tossing three coins together. Let
H represent the individual coins that fall heads and let T indicate the tails for the
same coins. Complete Table 3, showing the various possible combinations of three
chances of their occurring. The first combinations of three heads, is worked out as
an example. Finally toss three coins 80 times and record the frequency of
occurrence each of the classes. Calculate the expected numbers and deviations.

Table 3. Expected Results From Tossing Three Coins Together

Classes Combinations Probability Observed Expected Deviation

Of each class Number Number
occurring
3 heads HHH ½X½X½=⅛
2 heads HHT,HTH,THH
1 tail
1 head
2 tails
3 tails
totals 80

Write in the letters (H and T) to represent the coins in the "Combinations" column, and calculate
the probability for each class resulting when three coins are tossed. Toss three coins 80 times
to obtain the observed numbers.

b. A parallel situation would be observed is you were to study families consisting of three
children. If you were to select randomly 160 families in each of which there were three children,
in how many would the three children be expected to be boys? _________Two boys and girl?
_____________ One boy and two girls? ____________ Three girls? __________________.

III. BINOMIAL EXPANSION

Expectations for various combinations in groups of given size (n) can be obtained mathematically.
Mendel and others recognized that combinations can be calculated by expanding the binomial
(a+b)n, in which "n" is the size of the group, "a" is the probability of the first event, "b" is the
probability of the alternative event, and a + b = 1. In the example involving babies, a = probability
of girls = ½ and b = probability of boys = ½. Now if you wish to consider the case in which four
babies are born in the hospital in 1 day, expand (a+b)4 = a4 + 4a3b + 6a2b2 + 4ab3 + b4.
Expectation of 4 girls = a4 = (½)4 = 1/16
Expectation of 3 girls: 1 boy = 4a3b = 4(½)3(½) = 4/16 = ¼.
20
 Expectation of 2 girls: 2 boys = 6a2b2 = 6(½)2(½)2 = 6/16 = 3/8.
Expectation of 1 girl : 3 boys = 4ab3 = 4(½)(½)3 = 4/16 = ¼.
Expectation of 4 boys = (½)4 = 1/16
Problem 1:
In dogs, black fur color is dominant to white. Two heterozygous black dogs are mated.
What would be the probability of the following combinations of offspring?
A. A litter of six pups, four with black fur and two with white fur.
B. A litter of six pups, the firstborn with white fur, and among the remaining
five pups, two with white fur and three with black fur.
C. A first litter of six pups, four with black fur and two with white fur, and
then a second litter of seven pups, five with black fur and two with white fur.
D. A first litter of five pups, four with black fur and one with white fur, and
then a second litter of seven pups in which the firstborn is homozygous, the second
born is black, and the remaining five pups are three black and two white.

IV: MULTINOMIAL EXPANSION EQUATIONS

In the problem above, the binomial expansion equation was used in situations where there
are only two possible phenotypic outcomes. When there are more than two possible
outcomes, it is necessary to use a multinomial expansion equation to solve a problem
involving an unordered number of events. A general expression for this equation is:
n!
P= p a qb r c . . .
a !b !c! . . .
where P = the probability that the unordered number of events will occur.

n = total number of events

a+b+c+...=n
p+q+r+...=1

(p is the likelihood of a, q is the likelihood of b, r is the likelihood of c, and

so on)
The multinomial expansion equation can be useful in many genetic problems
where there are more than two possible combinations of offspring. For example, this
formula can be used to solve problems associated with an unordered sequence of events
in a dihybrid experiment. This approach is illustrated next.
Problem 1:
A cross is made between two heterozygous tall plants with axial flowers ( TtAa), where
tall is dominant to dwarf and axial is dominant to terminal flowers. What is the
probability that a group of five offspring will be composed of two tall plants with axial
flowers, one tall plant with terminal flowers, one dwarf plant with axial flowers, and one
dwarf plant with terminal flowers?

PART V: EITHER-OR SITUATIONS (MUTUALLY EXCLUSIVE EVENTS)

An additional probability principle should be called to your attention. The probability of either

one or the other of two mutually exclusive events occurring is the sum of their individual

probabilities.

21
Three examples will illustrate how this principle has implications for genetic studies.

1. What is the probability that an individual with the genotype Cc will produce either C

or c gametes? ____________________________
1
Note that the theoretical expectation from the cross Cc X Cc is a 3:1 ratio. Yet when only four

progeny are produced, the probability is actually less than one half (27/64) that the expected

3:1 ratio will be obtained! Thus, geneticists generally favour experimental organisms that

produce many progeny so that theoretically expected ratios can be more readily approximated.

Obviously the answer must be 1 (i.e 100%); that is, Cc can produce only two kinds of gametes -

those containing C and those containing c. On the basis of the principle just present, ½ =

probability that the gamete will contain C and ½ = probability that the gamete will contain c.

Therefore, the probability that the gamete will contain either C or c is ½ + ½ = 1.

2. If Aa is mated to Aa, what is the probability that the offspring will have either the

genotype AA or the genotype Aa? __________ ____________________

The probability for AA = ¼; that for Aa = ½. Therefore, ¼ + ½ = 3/4 = probability of either
AA or Aa.
3. A heterozygous pea plant that is tall with yellow seeds, TtYy, is allowed to self-
fertilize. What is the probability that an offspring will be either tall with yellow seeds,
tall with green seeds, or dwarf with yellow seeds?
V. PROBABILITY AND GENETIC COUNSELLING
In many genetic counseling situations the counsellor will prepare a pedigree for the family or
families seeking advice. As far as possible, the counsellor will determine the phenotype and the
genotype for each person in the pedigree. The counsellor can then apply probability principles to
determine the probability that a defective child will be produced among the offspring of a
certain marriage.

To illustrate the application to these concepts, consider the pedigrees shown in the
following figures.

1. The following pedigree shows the pattern of inheritance for a rare human disease.
Filled symbols indicate individuals with the disease: open symbols indicate normal
individuals.

22
(a) What is the most probable mode of inheritance (dominant or recessive) for this trait?
On what do you base your answer?

(b) What are the most probable genotypes of individuals I-1 and I-2?

(c) What are the most probable genotypes of individuals II-9 and II-10?

(d) If individuals II-1 and II-2 have another child, what is the probability that it will
have the disease?
2. The following human pedigree shows a family affected by a specific disease. Assume
that the individuals marked with an asterisk (*) do not carry any allele associated with
the affected phenotype and that no other mutation spontaneously occurs. Also assume
complete penetrance

a) State the most likely mode of inheritance for this disease.

b) Write all possible genotypes of the following individuals in the pedigree. Use the
uppercase “A” for the allele associated with the dominant phenotype and lowercase “a”
for the allele associated with the recessive phenotype.

All possible
Individuals Genotypes
#1
23
 #2
#4

c) What is the probability that Individual 5 will be a carrier?

d) The following human pedigree shows a family affected by a different disease. Assume
that the individuals marked with an asterisk (*) do not carry any allele associated with
the affected phenotype and that no other mutation spontaneously occurs. Also assume
complete penetrance. State the most likely mode of inheritance for this disease.

24
25
 *

*
6
2nd Pedigree

26
e) Individual 3 from the 1st pedigree has a second marriage with Individual 6 from
the 2nd pedigree. They have a son and a daughter
i) What would be the genotype of their son for the two disease genes?

i) What would be the genotype of their daughter for the two disease genes?

Solve the following problems on probability

1. You and your spouse have no children. You stand to inherit a sizeable fortune from
your Uncle if you can produce three daughters in your family of three children.
a) What is the probability of doing just that?
b). If you could convince Uncle that simply having three children all of the same sex
would do, then what would be the probability of your receiving the inheritance?
c). In quest of the family stipulated in #b above, you produce a boy as your first child.
Now what is the probability of inheriting the fortune?
d). Why are the answers to #b and #c the same?
e). Finally, you have convinced Uncle that you will agree to try for at least three girls
out of four children. How likely are you to become wealthy given those conditions?
2. The ability to taste the chemical PTC is determined by a single gene in humans
with the ability to taste given by the dominant allele T and inability to taste by
the recessive allele t. Suppose two heterozygous tasters (Tt) have a large
family.
a. Predict the proportion of their children who will be tasters and nontasters.
Use a Punnett square to illustrate how you make these predictions.
b. What is the likelihood that their first child will be a taster? What is the
likelihood that their fourth child will be a taster?

c. What is the likelihood that the first three children of this couple will be
nontasters?
3. A man with hemophilia (a recessive , sex-linked condition has a daughter of
normal phenotype. She marries a man who is normal for the trait. What is the
probability that a daughter of this mating will be a hemophiliac? A son? If the
couple has four sons, what is the probability that all four will be born with
hemophilia?
4. Red-green color blindness is caused by a sex-linked recessive allele. A color-
25
blind man marries a woman with normal vision whose father was color-blind. (a)
What is the probability that they will have a color-blind daughter? (b)What is the
probability that their first son will be color-blind? (Note: the two questions are
worded a bit differently.)
5. For the purpose of this problem assume that in humans the gene for brown eyes
is dominant to that for blue eyes.
a. A brown-eyed man marries a blue-eyed woman, and they have eight brown-eyed
children. What are the genotypes of all the individuals in the family?
b. What is the probability that the first child produced in parents who are both
heterozygous for brown eyes will be blue eyed?
c. If the first child is a brown-eyed girl (same parents as in b), what is the
probability that the second child will be a blue eyed boy?
d. Again referring to the marriage in b, what is the probability that the first three
children will be blue-eyed girls and the fourth a brown-eyed boy?

Laboratory 4
Drosophila experiments
Procedure:
Begin collecting the F1 flies. Immobilize and examine all flies. Divide the flies into
piles of males and females. Record the following data for the F1 generation:
the date of the count
the number of wild-type male flies
the number of mutant male flies
the number of wild-type female flies
the number of mutant female flies
Record the data in your notebook. Place seven pairs of flies in a fresh culture
bottle. For this mating the females need not be virgins. Label the new vial F1.

Lab Report:
Describe your mutation. Was the mutation found in the male or female flies? What
is the name of the observed mutation?
Record the following dates: the date of the original cross and the date that the
parental generation was sent to the morgue. What is the significance of the date
the parental generation was sent to the morgue?
What is your phenotypic ratio?
26
Construct Punnett squares using the symbols ++ for wild-type and the symbols you
assigned your mutation. Assume the following modes of inheritance for your
mutation:
Autosomal dominant
Autosomal recessive
Sex-linked dominant
Sex-linked recessive
Calculate the expected genotypic ratios for the crosses.
Compare the observed phenotypic ratio to calculated genotypic ratios. Is your
mutation(s) dominant or recessive? Is it sex-linked or autosomal? State evidence
for your answer.
Did you notice a difference in the male/female distribution? What does this
suggest?
Did the phenotypic ratio deviate from what was expected? Give possible reasons
this might happen.
Using your genetic symbol design a Punnett square for your F1 cross. Use proper
notations for dominant/recessive and sex-linked/autosomal mutations. What is the
predicted genotypic ratio?

B. Dihybrid Segregation

A dihybrid cross involves a study of inheritance patterns for organisms differing in

two traits. Mendel invented the dihybrid cross to determine if different traits of
pea plants, such as flower color and seed shape, were inherited independently. Our
objective is to understand the principles that govern inheritance of different
traits in a dihybrid cross that led Mendel to propose that alleles of different
genes are assorted independently of one another during the formation of gametes.

In today’s lab you will study dihybrid inheritance by analyzing phenotypes of an F2

generation of maize grains.

Procedure:

Working in pairs, obtain one ear of maize. Do not remove any grains from the ears.
Notice that some grains are purple and others are yellow. The purple colour is

27
produced by a pigmented layer within the grains. If the layer is not pigmented (is
colourless), the yellow colour of an inner tissue shows through. Sweet maize grains
can be recognized because they wrinkle upon drying while starchy grains remain
smooth. An individual grain may be purple and starchy, purple and sweet, yellow and
starchy, or yellow and sweet.

RECORDING DATA

Working in pairs, count and record in Table 1 the number of grains of each
phenotype. One person should call out the phenotypes while the other records
them in the table.

Put a coloured marker pin in the end of one row of grains and count and record the
phenotypes. Put an uncoloured row marker pin in the next row and continue
counting. After each row is completed, move the row marker pin to the next row
until you return to the row marked by the coloured pin.

When finished, combine the totals for each phenotype counted by your team and
record them under "Phenotype Class" in Table 2. Then record the total number of
grains counted.

DATA INTERPRETATION

Examine the totals obtained by your team. Which are the dominant phenotypes?

………………… and ………………... How do you know? …………………………………….

The maize grains are an F2 generation resulting from a cross between a

homozygous purple and starchy maize plant (R/R Su/Su) and a maize plant that is
homozygous yellow and sweet (r/r su/su). To better understand how these ears
were produced, fill in the following by placing the symbol for an allele in each blank.

R = gene for colour (purple); r = gene for colourless (yellow)

Su = gene for starchy; su = gene for sweet

/ / X / / Parents

28
 / / F1

/ / X / / F1 Cross

/ / / / / / / /

/ / / / / / / / F2

/ / / / / / / /

/ / / / / / / /

Thus, we see that the F1 gametes can combine in ……… ways to give rise to the
F2, and that the expected phenotypic ratio of the F2 is 9 …….
: 3 ….. :3 ….. :1…….

compare your "Numbers of Individuals" (actual counts) with the numbers that
would be expected for your sample size. To do this, divide the "Total Maize Grains
Counted" by the number of possible gamete combinations in the F2 generation.
Multiply this number (the dividend) by, respectively, 9,3,3 and 1 to fill in the
"Expected Numbers" of Table 2. Do the numbers of phenotypes you observed
seem to be close approximations of the "Expected Numbers"? …..
What factors could cause variation from the “Expected Numbers"? ………

Table 1.Number of grains of each phenotype

Purple Starchy Purple Sweet Yellow Starchy Yellow Sweet
Total Ear 1
Total Ear 2
Totals Ears 1&2

29
 Table 2. Actual and expected grain types

1 2 3 4 Classes
Phenotype Purple& & Purple& Yellow& Yellow& Total(1-4)
class Starchy Sweet Starchy Sweet
Number of
Individuals
(actual count)
Expected
Number

Analyse your results using an appropriate statistical test.

Dihybrid cross problems

Steps for solving dihybrid cross problems using Punnet squares:

1. Figure out the genotypes of the parents.

2. Figure out what kinds of gametes the parents can produce.
3. Set up a Punnett Square for your cross. Remember that what you put at the
head of each column and row represents what can go in the gametes, so you
have to account for both chromosomes in each row or column.
4. Fill in the offspring inside the table.
5. Figure out the genotypic ratio for your predicted offspring.
6. Figure out the phenotypic ratio for your predicted offspring.
7. Answer the question you've been asked.

Steps for solving dihybrid cross problems using probability (easier once you get
the math):

1: Figure out the genotypes of the parents.

2: Treat each trait separately as a monohybrid cross problem.
3: Determine the decimal percentage of each phenotype for trait A, and for trait
B. (For example, 75% BB or Bb and 25% bb translates to 0.75 BB or Bb and 0.25
bb)
4: Multiply the decimal percentage for the desired phenotypes to get the

30
probability of inheriting both phenotypes. (example: If the odds of inheriting the
dominant trait A is 0.75 and the odds of inheriting the dominant trait B is 0.75,
then the odds of inheriting both traits is 0.75 x 0.75 = .56, or 56% which can also
be expressed as 9/16)

A. About 70% of Americans perceive a bitter taste from the chemical

phenylthiocarbamide (PTC). The ability to taste this chemical results from
a dominant allele (T) and not being able to taste PTC is the result of having
two recessive alleles (t). Albinism is also a single locus trait with normal pigment
being dominant (A) and the lack of pigment being recessive (a). A normally
pigmented woman who cannot taste PTC has a father who is an albino taster. She
marries a homozygous, normally pigmented man who is a taster but who has a
mother that does not taste PTC.
a) What are the genotypes of the possible children ?
b) What percentage of the children will be albinos?
c) What percentage of the children will be non-tasters of PTC?
B. Consider the following crosses in Drosophila. The two traits being investigated
involve eye color and the presence or absence of wing crossveins. The outcomes of
four crosses are shown below.

(a) Which eye color is dominant and which is recessive?

(b) Which wing vein type is dominant and which is recessive?
(c) What is the most likely genotype of each parent for each of the four crosses?
C. Two of the traits Mendel studied in peas were flower position and pod color.
From monohybrid crosses, he determined that axial flowers (A) is dominant to
terminal (a), and green pods (P) is dominant to yellow (p). Suppose that you pollinate
the flowers of a plant with axial flowers and green pods with pollen taken from a
31
plant with terminal flowers and yellow pods. Both of these parent plants are known
to be true-breeding. F1 plants are then allowed to self pollinate to produce the F2
generation.
(a) What are the genotypes and phenotypes of the parents (P generation)?

(b) What genotypes and phenotypes are possible in the F1 plants?

(c) Using a Punnett square, determine which genotypes and phenotypes are possible
in the F2 generation and calculate the expected ratio of each.

D) In tomatoes, yellow fruit and dwarfed vine are due to recessive alleles of genes
which produce the more common red fruit and tall vine. If pollen from the pure–line
dwarf plant bearing red fruit is placed on the pistil of a pure–line tall plant bearing
yellow fruit, what type of plant and fruit would be expected in the F1 ? If these
are crossed among themselves, what results would be expected in the F2 ?
E) In Drosophila, vestigial wings and ebony colour are due to two separate
recessive genes. The dominant alleles are normal (long) wings and normal (gray)
body colour. What type of offspring would you expect from a cross between a
homozygous vestigial ebony female and a normal double homozygous (long–winged,
gray–bodied) male? If the F1 are allowed to breed among themselves what types of
offspring would you expect in the F2? Show complete genotype and phenotype of
both generations.

Laboratory 5

Mitosis

In a growing plant root, the cells at the tip of the root are constantly dividing to
allow the root to grow. Because each cell divides independently of the others, a
root tip contains cells at different stages of the cell cycle. This makes a root tip
an excellent tissue to study the stages of cell division.

Objectives:
a. to better understand the process and stages of mitosis.
b. to prepare your own specimens of onion root in which you can visualize all of the
stages of mitosis.
32
Procedure:

1. Preparation of Mitotic Chromosome from Bean Root Tip

Method

i) Place the apical 3mm of the root tip into HCl/alcohol in watch glass. Leave
for 3 min on top of your microscope lamp. The material must not dry out.
ii) Transfer to acetic alcohol fixative for 5 min.
iii) Place a small drop of stain (the size of a finger nail) on a clean slide and add
root tip. Using the ground end of the glass rod provided, tap the tip - (use only
vertical movements) - until fragmented. Remove any large pieces.
iv) Leave for 20 mins on top of your microscope lamp. Add stain to prevent drying
out, if necessary.
v) Carefully add two tiny drops of ferric acetate and stir.
vi) Add cover glass. Fold in half a piece of filter paper and place slide in fold.
Holding filter paper firmly with one hand press firmly on cover glass (note: too
much pressing will ruin the preparation).
vii) Ring cover slip with nail varnish.
iii) Observe slide under X40 object.
ix) Make several more slides if necessary to see all the stages of mitosis.

Observing the slides:

1. Place the slide on the microscope stage.

2. Use the low-power objective to find the preparation, and focus it with the
coarse adjustment until it is clearly visible.
3. Look for the region that has large nuclei relative to the size of the cell; among
these cells will be found cells displaying stages of mitosis.
4. Center the image, and then progressively switch to higher power.
5. Observe the box-like cells that are arranged in rows. The chromosomes of the
cells have been stained to make them easily visible.
6. Find and draw a cell showing each stage of mitosis.
7. What is a distinguishing visible feature of each stage of mitosis?

1. Mitosis prepared slides

33
In this section we will be observing the steps of mitosis under the microscope in
the root tips of plants and the blastula of animals. The process is similar in both
plants and animals.

In both organisms, locate cells in each phase of mitosis. Magnify the cells up to
400x and make a drawing of each of the stages. If you can identify them, label the
nucleus, spindle fibers and chromosomes with their centromeres.

WRITE-UP

The assessment of this practical will be based on the quality of your slides and
accuracy of your drawings. Please be warned against reproducing drawings from
your books.

Laboratory 6

A.Drosophila experiments

Begin scoring the F2 flies. As the F2 flies emerge from their pupa cases, score
them as to sex and the presence or absence of the mutation observed in the
parental flies. The greater the number of flies collected, the more reliable the
data will be so you will need to use the class data for all your calculations and
conclusions.
Lab Report:
Record the date you set up the F1 cross. What is the latest date the F1 generation
can be sent to the morgue?
Why was it necessary for the females of the parental generation to be virgins?
Why was it not important for the females of the F1 generation to be virgins when
they were mated?
Record the following data for the F2 generation in the appropriate column on the
board for the class data:
The number of wild-type male flies
The number of mutant male flies
The number of wild-type female flies
The number of mutant female flies
34
Tally the data from the F2 generation. What is your phenotypic ratio?
Does the observed phenotypic ratio agree with the genotypic ratio you predicted
from the Punnett square you designed for the F1 generation (lab 4 report, question
#9).
Is your mutation(s) dominant or recessive? Is it sex-linked or autosomal? State
evidence for your answer.
What Mendelian ratio is exemplified by the phenotypic ratios in this cross?
Postulate an hypothesis which is compatible with your observations.
Carry out an appropriate statistical test to assess the validity of the
hypothesis.
Briefly comment on your results.
If the analysis does not support your hypothesis, revaluate your data and
formulate a new explanation. Construct a Punnett square to represent your
conclusions diagramatically.
Does your conclusion match the conclusion you made after analyzing the F1 data?
If not, what changed your conclusion?
13.When you submit your write-ups, be sure to include all Data Sheets, Punnett
squares, and statistical analyses.

Laboratory 7

The Chi-Square Test

The purpose of the chi-square (X2)* test is to determine whether experimentally

obtained data constitute a good fit to or a satisfactory approximation of a
theoretical expected ratio: that is the X2 test enables one to determine whether
it is reasonable to attribute deviation from a perfect fit to chance. Obviously, if
deviations are small, they can be more reasonably attributed to chance than if they
are large. The question we try to answer with the X2 test is "How small must the
deviations be to be attributed to chance alone?" The formular for X2 is as follows:

35
 where 0 = the observed number of individuals in a
particular phenotype. E = the expected number in that phenotype and Ʃ = the summation
of all possible values of (0 - E) 2/E for the various phenotypic categories.
Consider the following example for practice: In a cross of tall tomato plants to dwarf
ones, the F1 consisted entirely of tall plants and the F2 consisted of 94 tall and 36
dwarf plants. Do these F2 data fit a ratio of 3:1? To answer this question, X2 was
calculated: the calculations are summarized in Table 1.

What does this X2 value of 2.0548 mean? If the observed numbers (0) were exactly
equal to the corresponding theoretically expected number (E) the fit would be perfect
and X2 would be zero. Thus a small value of X2 indicates that the observed and
expected ratios are in close agreement whereas a large value indicates marked deviation
from the expected ratio. Because chance deviations from the theoretical values are
expected to occur, the question to be answered is "Are the observed deviations within
the limits expected by chance?" Generally, statisticians have agreed on the arbitrary
limits of odds of 1 chance in 20 (probability = 0.05) for drawing the line between
acceptance and rejection of the data as a satisfactory fit to the expected ratio.
An X2 value of 3.841 for a two term ratio corresponds to a probability of 1 in 20 or 0.05.
One would obtain an X2 value of 3.841 due to chance alone in only about 5% of similar
trials if the hypothesis is true. When X2 exceeds 3.841, for a two term ratio the
probability that the deviations can be attributed to chance alone is less than 1 in 20.
The hypothesis of compatibility between the observed and expected ratios is thus
rejected. In the practice example X2 = 2.0548 which is considerably less than 3.841 the
maximum allowable value for a two-term ratio associated with the probability of 1 in 20.
Therefore, you can attribute the deviations to chance alone and accept the data as a
suitable fit to a 3:1 ratio.
 Table 1. Summary of the calculation of X2 in a Hypothetical Illustration
Phenotype Genotype 0 E (0-E) (0-E)2 (0-E)2/E

Tall T- 102 109.5 -7.5 56.25 0.5137

Dwarf tt 44 36.5 7.5 56.25 1.5411

Totals 146 146 X²=2.0548

Where did the value of 3.841 come from? Statisticians have published extensive tables
listing X2 values (see Table 2).
Table 2.  Table X2 Values*
p 0.995 0.99 0.975 0.95 0.90 0.10 0.05 0.025 0.01 0.005
df
1 --- --- 0.001 0.004 0.016 2.706 3.841 5.024 6.635 7.879
2 0.010 0.020 0.051 0.103 0.211 4.605 5.991 7.378 9.210 10.597
3 0.072 0.115 0.216 0.352 0.584 6.251 7.815 9.348 11.345 12.838
4 0.207 0.297 0.484 0.711 1.064 7.779 9.488 11.143 13.277 14.860
5 0.412 0.554 0.831 1.145 1.610 9.236 11.070 12.833 15.086 16.750
6 0.676 0.872 1.237 1.635 2.204 10.645 12.592 14.449 16.812 18.548
7 0.989 1.239 1.690 2.167 2.833 12.017 14.067 16.013 18.475 20.278
8 1.344 1.646 2.180 2.733 3.490 13.362 15.507 17.535 20.090 21.955
9 1.735 2.088 2.700 3.325 4.168 14.684 16.919 19.023 21.666 23.589
10 2.156 2.558 3.247 3.940 4.865 15.987 18.307 20.483 23.209 25.188

Notice that across the top of Table 2 are probability (p) values: down the left-hand side
are "degrees of freedom" values (n = 1,2, ...10). The number of degrees of freedom (d.f)
is generally one less than the number of terms in the ratio. In the practice example
with two terms in the ratio (3:1) you have one degree of freedom in interpreting X2.
Thus, on the one-degree-of-freedom row and under the 0.05 column you find the X2
value of 3.841 the maximum value of X2 that you are willing to accept and still attribute
the deviations observed to chance alone.

In the hypothetical example, X2 was calculated to be 2.0548. Looking on the one-

degree-of-freedom row you see that this value of X2 falls between the .10 (X2 = 2.706)
and the .20(X2 = 1.642) probability columns. This means that the probability that the
deviations observed may be attributed to chance alone is between 10% and 20% :, that
is, if you were to do the same experiment 100 times, you would expect to observe
deviations as large as you obtained or larger in between 10 and 20 of the 100 trials due
to chance alone.

With this background in the meaning, calculation, and interpretation of X2 you can apply
the test to some data you have collected yourself.

Objectives

Upon completion of this practical, the student should be able to:

1. calculate X2 to determine whether a given set of data approximates a

theoretically expected ratio;

2. interpret a calculated X2 value given the appropriate number of degrees of

freedom and a table of X2 values.

1 APPLICATION OF THE CHI-SQUARE TEST

A. Application of Chi-Square to New Data

Coloured and white beans were carefully selected for equality of size and uniformity of
shape. Equal quantities of each colour were thoroughly mixed and placed in a single
container.
Remove a random sample (one petri dish cover full) of this mixture. Segregate and
count the beans of the different colours. Record your data in Table 3 and then
calculate the expected numbers based on the size of the sample and the known ratio of
coloured to white beans in the entire population. Complete Table 3 and calculate X2.

1. How many degrees of freedom do you have in the interpretation of this X2 value?

...........................................

2. Using Table 2, determine what X2 values lie on either side of the X2 calculated in
Table 3. Record these values here.
..............................................................................................................

Table 3: Calculation of X2 for a sample Removed From a Large Population Consisting of

Equal Numbers of Coloured and White Beans

Classes Observed Expected Deviations (0-E)2 (0-E)2/E

(phenotypes) 0 E (0-E)
Coloured
White
Totals X²=

3. What probability values are associated with these table values of X²?

...............................................................................................................

4. In the space provided, write a brief interpretation of the X2 value you have just
calculated?

......................................................................................................................................................................

Chi-Square: Practice Problems

Calculate X2 in the problems that follow. The X2 values you should obtain are given in
the first two problems. If the value you obtain does not agree with the given, repeat
the calculation until agreement is reached. Notice how the X2 calculated is interpreted.

1. A zookeeper hypothesizes that changing the intensity of the light in the primate
exhibits will reduce the amount of aggression between the baboons. In exhibit A, with a
lower light intensity, he observes 36 incidences of aggression over a one month period.
In exhibit B, with normal lights, he observes 42 incidences of aggression. Should he
support or reject his hypothesis?

2.At a high school, students can choose to enter one of three doors. Custodians noticed
that door #3 was always getting broken and suggested that more students use that door
because it has a hands-free opener. Science minded students counted the number of
students entering each door to see if the custodians were right. Door #1 had 60
students enter | Door #2 had 66 students enter | Door #3 had 80 students enter.
Were the custodians right?

3. A scientist predicts that the kittens born with a congenital birth defect will be 25%
based on the hypothesis that it is caused be a recessive gene in that breed of cat. After
surveying several litters, he found that 44 out of 125 kittens had the defect. Is his
hypothesis correct?
4. A student makes a monohybrid cross with Drosophila (fruit flies). She crosses two
heterozygotes for the white eye. Ww x Ww. She expects to see a 3:1 phenotypic ratio
of Red eyes (WW and Ww) to white eyes (ww) – this is her null hypothesis. She rears
the next generation through to adult flies and counts the following numbers:
White eyes 210
Wild type (red eyes) 680
Perform a chi square analysis on these results and find out if it is close enough to 3:1 to
fail to reject her null hypothesis. Make sure to show all work and explain your
conclusions.
5. In the garden pea, yellow cotyledon color is dominant to green, and inflated pod shape
is dominant to the constricted form. Considering both of these traits jointly in self-
fertilized dihybrids, the progeny appeared in the following numbers:
193 green, inflated
184 yellow constricted
556 yellow, inflated
61 green, constricted
Do these genes assort independently? Support your answer using Chi-square analysis.

Laboratory 8

Population Genetics-the Hardy-Weinberg Law

The Hardy–Weinberg principle is normally stated as a mathematical equation:

p2 + 2pq + q2 = 1.The frequencies of the dominant and recessive alleles are represented
by p and q, respectively. For example, if a diploid individual has two alleles, “A” and “a”,
at a particular locus, only three possible genotypes can be the result: AA, Aa, and aa.
The probability of receiving the “A” alleles from both parents is p x p, or p2; for the “a”
alleles, q x q, or q2. Those who received the Aa combination are described by 2pq, since
it is possible for the “A” or the “a” to come from either parent, thereby doubling the
chance.

To apply the principle, at least one of the allele frequencies must be known. If the
relationship between p and q are constant through randomly mating generations, the
population is said to be in Hardy–Weinberg equilibrium; no evolution occurs. However,
five evolutionary forces act on a population to affect it: mutation, migration, non-random
mating, genetic drift, and natural selection. If any of these conditions are present, the
proportions of heterozygotes and homozygotes can differ. Therefore, the Hardy–
Weinberg principle is a useful tool for measuring the degree of genetic change or
evolution occurring in a population. Mutation is a fairly uncommon occurrence, which will
not significantly change allele frequencies itself, but is the main source of change and
therefore evolution. Migration of individuals in or out of a population is the cause of
gene flow, which introduces new alleles to a group and removes others. Non-random
mating can be the result of either inbreeding or an organism’s ability to choose a mate
based on certain characteristics such as size, coloration, or lifestyle. In either case, the
proportion of homozygotes can increase, upsetting the equilibrium. Genetic drift refers
to the possibility that by chance, certain alleles could be eliminated from a population.
For example, two heterozygous parents, Aa, could possibly have two children, both AA,
thus eliminating the aa allele from the next generation. Normally this is balanced by
similar events that might eliminate the A allele from another family, but in small
populations it is possible to lose the same allele at a large number of loci, causing
evolution to take place. Natural selection is the predominant force in creating
evolutionary change. An allele can increase or decrease in frequency based on its effect
on reproduction and survival. This can change from one generation to the next, and from
one area to another, since a trait that is harmful to its carrier in one circumstance could
be beneficial in another.
OBJECTIVES
In this experiment, you will
• Investigate a genetically inherited trait and apply the Hardy-Weinberg Principle to a
population.
• Calculate allele frequencies and genotypes for a population using the Hardy-Weinberg
formula.
• Demonstrate the stability of allele frequencies over five generations in an ideal Hardy-
Weinberg population.
Examine the effects of natural selection, heterozygous advantage, and genetic drift on
allele frequencies in a simulated mating exercise.
PROCEDURE
Part I Calculating Allele Frequencies Using the Hardy–Weinberg Principle
Testing different individuals’ ability to taste PTC (phenylthiocarbamide) is a good way to
demonstrate the Hardy–Weinberg principle. Homozygous-dominant (AA) and
heterozygous (Aa) individuals can taste this bitter chemical, although homozygous-
recessive (aa) individuals cannot. Use your class as a representative population to
calculate the frequencies of the two alleles with the Hardy–Weinberg equation
1. Obtain a piece of PTC test paper. Note: Use each strip of PTC and control test paper
only once. Do not share test papers with other students in your class.
2. Place it on your tongue and note whether you can detect a bitter taste. Note: Used
test papers may be disposed of with general waste.
3. Obtain a piece of control paper and place it on your tongue. Comparing the control
paper with the PTC paper will help determine whether you detected a taste on the PTC
paper.
4. Record your results in Table 1.
Table 1:
Taster Non-taster
PTC

Control

5. Fill in the results for each individual in the class and enter the results in Table 2.
Table 2:
Phenotypes Allele frequency based
on the H-W equation
Class Tasters Non-tasters p q
population (p2+2pq) (q2)
No. % No. %

6. Tally the results for the entire class and calculate the frequencies for each allele,
using the Hardy-Weinberg equation. Include details on your calculations.
Part II Testing the Hardy–Weinberg Principle

Case A: Testing an Ideal Population

1. Obtain four index cards. Label two “A” and two “a”. These will be your haploid
chromosomes.
2. Randomly pair off with another student for “breeding”. Choose any other student;
your gender, and your partner’s, doesn’t matter in the simulation.
3. Turn your cards upside down and shuffle them. Turn over the top card in your pile.
Pair this card with your partner’s card; this will be the genotype of your first offspring.
Record this on your data sheet.
4. Turn over the next card in your pile. Pair this card with your partner’s card; this will
be the genotype of your second offspring. Record this on your data sheet.
5. Now begin the second generation, taking the genotype of your offspring. For example,
if you produced offspring with the genotype AA, begin the next generation with four A
cards. Record the results in the Analysis section of the lab.
6. Repeat the above procedure for the second generation. Record the genotypes in the
Analysis section.
7. Repeat the procedure for three more generations, for a total of five generations.
Record the genotypes for each.
8. Combine the genotype of your fifth generation results with the rest of the students’
fifth generation results and enter the totals in the Analysis section.
9. Using the table in the Analysis section, calculate the allele frequencies after five
generations of random mating.
Case B: Selection
The previous exercise was conducted with ideal parameters. For a more realistic
situation, selection must be used. There is 100% selection against homozygous-recessive
offspring. If offspring are recessive (if they receive two mutated alleles), they will
never live long enough to reach a reproductive age; offspring that are either
heterozygous or homozygous dominant will survive long enough to reproduce.
10. Follow the same procedure as the previous exercise, with one difference: If
offspring is produced with the genotype aa, this offspring will not survive; eliminate the
alleles from the population. To maintain population size, you must produce two surviving
offspring. If two alleles are eliminated, draw two new alleles from the extra cards.
11. Repeat the procedure for a total of five generations, selecting against homozygous-
recessive offspring in each generation. Record the genotypes after every generation in
the Analysis section.
12. Combine your fifth generation results with the rest of the students’ fifth generation
results and record in the Analysis section.
13. Using the table in the Analysis section, calculate the allele frequencies after five
generations of random mating.
Case C: Heterozygote Advantage
The previous exercise showed how selection against homozygous-recessive individuals
clearly alters the allelic frequencies in a population. Another form of selection that
operates within a gene pool is diseases, such as a deadly form of malaria, that affect
homozygous-dominant individuals more severely than heterozygous individuals. The
heterozygote is therefore favored in
a population.
14. Follow the same procedure, eliminating homozygous-recessive individuals as before.
In addition, if a homozygous-dominant individual is produced, flip a coin. If the result is
heads, the offspring dies; if it is tails, the offspring survives.
15. Repeat the procedure for a total of five generations. Record the genotypes after
every generation in the Analysis section.
16. Combine your fifth generation results with the rest of the students’ fifth generation
results and record in the Analysis section.
17. Continue the procedure for five more generations, for a total of ten generations,
this time starting with the genotypes from the end of the fifth generation. Record the
genotypes in the Analysis section.
18. Using the table in the Analysis section, calculate the allele frequencies after ten
generations of random mating.
Case D: Genetic Drift
Genetic drift is a phenomenon where an allele is lost solely from chance instead of
through selection. The most important factor in genetic drift is population size; smaller
populations have a much greater potential for genetic drift.
19. Your instructor will divide the class into several smaller populations. Within your
smaller population, follow the mating procedure, as in the first exercise, for a total of
five generations. Record the genotypes after every generation in the Analysis section.
20. Combine your group’s fifth generation results with those of the other small
populations and calculate the new allele frequencies.
ANALYSIS

Part II Testing the Hardy–Weinberg Principle

Case A
Testing an Ideal Population
Initial Class Frequencies: AA:__________ Aa:__________ aa:__________
My Initial Genotype: ________
F1 Genotype: ________
F2 Genotype: ________
F3 Genotype: ________
F4 Genotype: ________
F5 Genotype: ________
Final Class Frequencies: AA:__________ Aa:__________ aa:__________
p:________ q:________

Number of A alleles present at the fifth generation:

Number of offspring with genotype AA ____________ x 2 = __________ A alleles

Number of offspring with genotype Aa _____________x 1 = __________ A alleles
Total = __________ A alleles
p = TOTAL number of A alleles = _____________

TOTAL number of alleles in the population:

Number of a alleles present at the fifth generation

Number of offspring with genotype aa ____________ x 2 = __________ a alleles
Number of offspring with genotype Aa ____________ x 1 = __________ a alleles
Total = __________ a alleles
q = TOTAL number of a alleles = _____________

TOTAL number of alleles in the population

Case B
Selection
Initial Class Frequencies: AA:__________ Aa:__________ aa:__________
My Initial Genotype: ________
F1 Genotype: ________
F2 Genotype: ________
F3 Genotype: ________
F4 Genotype: ________
F5 Genotype: ________
Final Class Frequencies: AA: ________ Aa: ________ aa:__________
p:________ q:________
Case C
Heterozygote Advantage
Initial Class Frequencies: AA:__________ Aa:__________ aa:__________
My Initial Genotype: ________
F1 Genotype: ________
F2 Genotype: ________
F3 Genotype: ________
F4 Genotype: ________
F5 Genotype: ________
Final Class Frequencies:
(After five generations) AA:__________ Aa:__________ aa:__________
p:________ q:________
F6 Genotype: ________
F7 Genotype: ________
F8 Genotype: ________
F9 Genotype: ________
F10 Genotype: ________
Final Class Frequencies:
(After ten generations) AA:__________ Aa:__________ aa:__________
p:________ q:________
Case D
Genetic Drift
Initial Class Frequencies: AA:__________ Aa:__________ aa:__________
p:________ q:________
My Initial Genotype: ________
F1 Genotype: ________
F2 Genotype: ________
F3 Genotype: ________
F4 Genotype: ________
F5 Genotype: ________
Final Class Frequencies: AA:__________ Aa:__________ aa:__________

QUESTIONS
1. In the first exercise in Part B, ‘Testing an Ideal Hardy-Weinberg Population’, what
would be the expected values of P and Q after the five generations?
2. How close were your class’s results to an ideal population.
3 What do you think would happen if you carried this simulation out for 10 more
generations?
4 How do the frequencies of p and q compare after the factor of selection was added to
the simulation?
5. What do you think would happen if you carried this simulation out for another
10 generations?
6. Do you think that the a allele would ever be totally eliminated from the population?
Why or why not?
7. Suppose there was a medical advance that allowed most individuals with the double
recessive condition to survive and reach reproductive age. How would this affect the
allele frequencies?
8. Explain the difference in the results of the simulation showing selection to the
simulation favoring heterozygotes.
9. Why is the heterozygous condition important in maintaining genetic variation within a
population?
10. In the simulation demonstrating genetic drift, what do the resulting allele
frequencies suggest about population size as an evolutionary force?