BOI 116/4 GENETICS

Semester 1 Academic Session 2018/2019

Lab Practical: Monday
Lab Lecturer: Dr.Manorenjitha
MINI PROJECT 2 : MENDELIAN GENETICS

Group 1 Members:
1.Marilyn H’ng Kai Yuen 142747
2.Michell Lee Yoke Teng 142858
3.Tieu Ling Siew 143813
4.Cheah Mei Kee 141029
5.Illya Maisarah Binti Hesem 144285
6.Lim Ai Wei 141812
7.Intan Nasuha Binti Jamil 144081
8.Choi Yi Lau 140743
9.Chua Tau Er 141331
10.Tan Weng Sim 143657
 Introduction
Objective
1. To identify the gene order and map distance between linked gene by using gene
mapping
2. To investigate the effect on the traits or phenotypes of the Drosophila
melanogaster by studying the sex linked gene
Hypothesis
1) The genetic map of Drosophila melanogaster can be drawn by using the sequence
of gene and the map distance between the genes.

2) Genetic linkage map distance between gene loci can be determined by calculating
recombinant frequency between two genes.

Drosophila Melanogaster
Drosophila melanogaster is a species of flies (the taxonomic order Diptera) in
the family Drosophilidae. The species is known generally as the common fruit fly or
vinegar fly. Drosophila melanogaster continues to be widely used for biological
research in genetics, microbial pathogenesis, and life history evolution. Drosophila
melanogaster is typically used in research because it can be readily reared in the
laboratory, has only four pairs of chromosomes, breeds quickly, and lays many eggs.
Drosophila melanogaster was one of the most widely used and genetically best-known
of all eukaryotic organisms.
This mini project is carried on by using fruit flies named Drosophila
melanogaster that are wild type male Drosophila melanogaster and mutated female
Drosophila melanogaster. The wild type with genotype ‘y’, ‘sn’, ‘w’ has a phenotype
of brown body, normal bristle, and red eyes. While mutant type has phenotype of
yellow body, singed bristle, and white eyes. Another wild type Drosophila
melanogaster with genotype ‘b’, ‘vg’, ‘bw’ has phenotype of brown body, normal
wings, and red eyes. While the mutant type has phenotype of black body, vestigial
wings, and brown eyes.
Gene mapping is the process of locating and identifying genes to create a
genetic map or the determination of the relative locus of genes along a chromosome.
Map distance between the genes can be identified by genetic mapping. The units for
map distance are called map units (mu) or centiMorgans (cM). The larger the distance
of the map distance, the more likely a crossing-over of that gene will happen. Hence,
map distance can be calculated by using the formula below:
(𝑛𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑟𝑒𝑐𝑜𝑚𝑏𝑖𝑛𝑎𝑛𝑡 𝑜𝑓𝑓𝑠𝑝𝑟𝑖𝑛𝑔)
𝑚𝑎𝑝 𝑑𝑖𝑠𝑡𝑎𝑛𝑐𝑎𝑛𝑒 = × 100
𝑡𝑜𝑡𝑎𝑙 𝑛𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑜𝑓𝑓𝑠𝑝𝑟𝑖𝑛𝑔
 Coefficient of coincidence (c.o.c.) is a measure of interference in the formation
of chromosomal crossovers during meiosis. Interference determines how the double
crossover number deviated from the expected value. Positive interference shows a
decrease in observed double cross over number compare to the expected value and vice
versa. Interference c.o.c. can be calculated by using the formula below:
𝑜𝑏𝑠𝑒𝑟𝑣𝑒𝑑 𝑛𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑑𝑜𝑢𝑏𝑙𝑒 𝑐𝑟𝑜𝑠𝑠 𝑜𝑣𝑒𝑟
𝑐𝑜𝑒𝑓𝑓𝑖𝑐𝑖𝑒𝑛𝑡 𝑜𝑓 𝑐𝑜𝑖𝑛𝑐𝑖𝑑𝑒𝑛𝑐𝑒 =
𝑒𝑥𝑝𝑒𝑐𝑡𝑒𝑑 𝑛𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑑𝑜𝑢𝑏𝑙𝑒 𝑐𝑟𝑜𝑠𝑠 𝑜𝑣𝑒𝑟

Interference = 1- coefficient of coincidence (C)

Table 1: The difference between wild type and mutant of Drosophila

melanogaster

Wild Type Mutant

Body colour Yellowish-brown Black or ebony

Eye colour Brick red White or orange eyes

Wing shapes Long and round, smooth edge, Shortened or curled

uniform venation, extend
beyond the abdomen

Bristle Fairly long and smooth Short tick or deform changes in

pattern of distribution

Shape of formed head Has antenna sticking out front Some has no eyes and some
of their red eyes have their antennas on their
forehead.
 Methodology
Apparatus and material:
Drosophila melanogaster which include 6 wild type females,3 ebony body males and
3 vestigial ebony wing males, Drosophila melanogaster food media, Drosophila
melanogaster vial tube, paint brush, ether, ether bottle with cotton plug, cotton wool,
labelling paper, white paper, alcohol.

Methods:
Methods to anesthetise the fruit flies:

1. Ether is dropped on the cotton which placed under the ether bottle cap and the bottle
is closed for a few seconds until the ether gas is entirely filled the bottle.
2. Then, the base of the vial tube containing the flies is knocked lightly on the surface
of the table so that the flies will drop to the bottom of the tube.
3. The flies are then quickly transferred into the ether bottle to immobilized them for
a while.
4. As soon as the flies are incapacitated, the flies are transferred onto a piece of white
paper. The flies cannot be exposed to ether for too long as the flies will die from
over-etherized.
5. The flies are placed into the newly fresh cultured vial tube by using soft paint brush.
6. The vial tube is placed horizontally as to prevent the flies from drowning in the
food media.
Methods to differentiate the sex of Drosophila melanogaster

The sex of Drosophila melanogaster is distinguished by observing the characteristics

under dissecting microscope.

1. Size of the fly

Male are generally smaller than females.(Based on diagram: The bottom one is female
and the top is male)

2. Abdomen
Females have a pointed posterior while males bears a round terminus posterior.
Females also have 7 distinct segment of the abdomen, whereas males have 5
abdominal segments and several terminal that are fused together.
3.Genital apparatus
The genitalia is the easiest characteristic used to determine the sex of fruit flies. The
round and circle of darkly pigmented part is male. Meanwhile, the tip of the female’s
abdomen is lightly coloured and pigmented and pointed shaped.

Methods to differentiate virgin female

Females have the ability to store sperm after a single mating, so if the female for a
cross is not a virgin, you will not know the genotype of the male used for your cross.
So, that is why differentiating the virgin females is crucial in this experiment.

1. To ensure that F1 generation gene are in a heterozygous form and are a virgin. It is
crucial to be able to recognize the virgin female.
2. To find a virgin female Drosophila, we are required to always observe the vial to
remove the virgin female Drosophila within 8-10 hours after it has matured.
3. Virgin female Drosophila are much larger than older female.
4. Virgin female Drosophila does not have dark coloration.
5. Virgin female Drosophila will also have a greenish dark spot on the right top side
of the abdomen, meconium.
 The diagram shows the female flies that has the meconium (arrow)

Methods of making crosses:

1. In one of the vial tube, 3 female Drosophila melanogaster with yellow body, single
bristles and white eyes (y sn w / y sn w) are crossed with 3 wild type male
Drosophila melanogaster (+ + + / 0).
2. In another vial tube, 3 female Drosophila melanogaster with black body, vestigial
wing and brown eyes (b vg bw / b vg bw) are crossed with another 3 wild type
Drosophila melanogaster (+ + + / + + +).
3. All vial tubes are then labelled.
4. The vials are incubated at room temperature and away from the sunlight.
5. After one week, the parent flies are removed and the F1 generation are left to
mature.
6. After few days, all the flies from the cultured vial are cleared from the vial tube and
the virgin females are collected a few hours later. Note that females remain virgins
for only 8-10 hours after enclosure and must be collected within this time frame.
7. On 15th day, two cross breed are carried out. For the first cross breed, we crossed
the virgin heterozygous with male flies (b vg bw/b vg bw) together.
8. Meanwhile, in the second cross breed, the female flies ( + + + / y sn w) are crossed
with male flies ( y sn w / 0)
9. After one week, F1 parents are removed and F2 generation are left to mature.
Methods of counting the F2 generation

1. All of the F2 generation are killed by over-etherized.

2. The dead flies are transferred onto a piece of paper and the phenotypes of the F2
generation are observed.
3. The flies are separated according to their phenotypes.
4. The number of flies are recorded in a table and the Chi-square test is carried out to
determine if the result follows the Mendel’s Law.

Summary of methodology:

Day

Day 1
12 Nov 2018 b vgbw/b vgbw X +++/+++ ysn w/y sn w X +++/O
(Parental cross)

Day 8
19 Nov 2018 Remove parent flies Remove parent flies

Day 10 - 14
21 -25 Nov Collect virgin female Collect virgin female
2018

Day 15
26 Nov 2018 virgin heterozygous X b vgbw/b vgbw +++/y sn w X ysn w/O
(F1 cross)

Day 21
3 Dec 2018 Remove F1 flies Remove F1 flies

Day 28
10 Dec 2018 Count F2 Count F2
 RESULT AND DISCUSSION
Cross 1 Inheritance pattern

P1 genotype: b vg bw b+ vg+ bw+

b vg bw X b+ vg+ bw+

Gametes: b vg bw b+ vg+ bw+

F1 genotype: b vg bw
b+ vg+ bw+

F1 cross: b vg bw b vg bw

b+ vg+ bw+ X b vg bw

Gametes: b vg bw

b+ vg+ bw+

b vg+ bw+

b+ vg bw b vg bw

b vg bw+

b+ vg+ bw

b vg+ bw

b+ vg bw+
F2 genotype and phenotype:

b vg bw b+ vg+ bw+ b vg+ bw+

b vg bw b vg bw b vg bw
black body, vestigial brown body, normal black body, normal
wings, brown eyes wings, red eyes wings, red eyes

b+ vg bw b vg bw+

b vg bw b vg bw

brown body, vestigial black body, vestigial

wings, brown eyes wings, red eyes

b+ vg+ bw b vg+ bw b+ vg bw+

b vg bw b vg bw b vg bw

brown body, normal black body, normal brown body, vestigial

wings, brown eyes wings, brown eyes wings, red eyes
Map distance between genes

Genotype Number of F2 Frequency of F2 Remarks

generation generation

Parental b vg bw 3 0.007 No Cross

Over
b+ vg+ bw+ 122 0.298 (NCO)

Recombination of b with b vg+ bw+ 33 0.080 Single

vg and bw Cross
Over
b+ vg bw 8 0.020 (SCO)

Recombination of bw b vg bw+ 63 0.154 Single

with b and vg Cross
Over
b+ vg+ bw 7 0.017 (SCO)

Recombination of vg b vg+ bw 31 0.076 Double

with b and bw Cross
Over
b+ vg bw+ 143 0.349 (DCO)

Total 410 1

Note:
b+ :brown coloured body/ wild type
b :black coloured body/ mutant type
vg+ :normal or full wing/ wild type
vg :vestigial wing/ mutant type
bw+ :red eye/ wild type
bw :brown eye/ mutant type

Map distance between b and vg:

33 + 8 + 31 + 143
× 100% = 52.44𝑚𝑢
410
Map distance between vg and bw: Genetic map:
63 + 7 + 31 + 143 52.44mu 59.51mu
× 100% = 59.51𝑚𝑢
410
b vg bw

Map distance between b and bw:

111.95cmu
52.44 + 59.51 = 111.95𝑚𝑢

Observed Double-crossover: 0.076 + 0.349 = 0.425

Expected Double-crossover: 0.5244 × 0.5951 = 0.312
0.425
Coefficient of Coincidence(C): 0.312 = 1.3621

Interference: 1 − 1.3621 = −0.3621

Cross between mutant and wild Drosophila

Parents: y sn w y+ sn+ w+
y sn w
Gamete: y sn w y+ sn+ w+

F1 genotype: y sn w
y+ sn+ w+

F1 cross: y sn w y sn w
y+ sn+ w+

Gametes: y sn w
y+ sn+ w+
y sn w+
y+ sn+ w
y sn+ w+
y+ sn w
y sn+ w
y+ sn w+
 F2 genotype and phenotype

y sn w y+ sn+ w+ y sn+ w+ y+ sn w

y sn w y sn w y sn w y sn w

yellow body, single brown body, yellow body, brown body, single
bristle, white eyes normal bristle, red normal wings, red bristle, white eyes
eyes eyes

y+ sn+ w
y sn w
brown body,
y sn w+
normal bristle,
white eyes
yellow body, +
y sn w+ y sn+ w y+ singlesnbristle,wred
y
y
sn
sn
w+
w y sn w+
y
y
sn
sn
w+
w y eyes sn w+
y sn w
yellow body, single yellow body, brown body, single
yellow body,
bristle, red eyes normal bristle, red bristle, white eyes
single bristle, red
yellow body, eyes
yellow body, yellow body,
eyes
single bristle, red single bristle, red single bristle, red
eyes eyes eyes

y sn w+ y sn w+ y sn w+

yellow body, yellow body, yellow body,

single bristle, red single bristle, red single bristle, red
eyes eyes eyes
Map distance between genes

Genotype Number of F2 Frequency of F2 Remarks

generation generation

Parental y sn w 56 0.286 No Cross

Over
y+ sn+ w+ 41 0.209 (NCO)

Recombination of y with y sn w+ 14 0.071 Single

w and sn Cross
y+ sn+ w 5 0.026 Over
(SCO)

Recombination of sn with y sn+ w+ 27 0.138 Single

y and w Cross
y+ sn w 42 0.214 Over
(SCO)

Recombination of w with y sn+ w 6 0.030 Double

y and sn Cross
y+ sn w+ 5 0.026 Over
(DCO)

Total 196 1.000

Notes:

y+ :brown coloured body/ wild type

y : yellow coloured body/ mutant

sn+ :normal bristle/ wild type

sn :singed bristles/ mutant

w+ :red eye/ wild type

w :white eye/ mutant

Map distance between y and w:

27 + 42 + 6 + 5
× 100% = 40.81𝑚𝑢
196

Map distance between w and sn: Genetic map:

14 + 5 + 6 + 5 40.81mu 15.31mu
× 100% = 15.31𝑚𝑢
196 y w sn

56.12mu
Map distance between y and sn:

40.81 + 15.31 = 56.12𝑚𝑢

Observed Double-crossover: 0.030 + 0.026 = 0.056

Expected Double-crossover: 0.4081 × 0.1531 = 0.062

0.056
Coefficient of Coincidence(C): 0.062 = 0.9032

Interference: 1 − 0.9032 = 0.0968

 Discussion
For the cross of Type 1, the interference calculated is -0.3621, a negative value which
shows a negative interference. This indicates that a crossover enhances the chances of a second
crossover event occurring in the region. However, negative interference only occurs in some
rare cases such as in more complex organism since crossing over require extra energy.
Therefore, the negative value obtained for the value of interference for Cross Type 1 might due
to 2 factors, which are environmental factor and mankind error. For example, the temperature
for breeding and hatching may not always consistent as the experiment is ongoing. The
suddenly cold and hot condition in the lab may cause the death of large number of the flies
which will then affects the currently result. The bacteria growth in the food medium may also
affect the immune system of Drosophila melanogaster and lead to dead. Besides that, mankind
error occur during counting of F2 generation may also affects the results.

For Cross Type 2, the interference calculated is 0.0968, which is a positive value which
showing a positive interference. Thus, this indicates that a decrease in observed Double-
Crossover (DCO) compared to the expected as a crossover decrease the chances of a second
crossover event occurring in the region.
 Conclusion
Through this experiment, we are able to identify the the gene order and the map distance
between genes of Drosophila melanogaster and hence construct a genetic map. The distance
between two genes can use to indicate the percentage of recombinant. We are also able to
identify each mutated and wild type traits and their differences when handling the flies. Besides
that, by calculating the coefficient of coincidence and the interference, we are able to determine
how greatly a crossover in the gene of Drosophila melanogaster may interfere with the
formation of another crossover.

From the result obtained in b vg bw test (cross type1), we are able to conclude that the
cross gives a negative interference which indicates that one crossover event increase the
likelihood of a second crossover event. The observed double cross over is more than the
expected double cross over. This situation is rare in nature and may cause by mankind error.
For example, some of our vial tube’s cover had been pushed inside and this caused the death
of some flies. This has greatly affected the data that we collected and lead to deviation from
usual result. Besides, some of our group members didn’t know the exact procedure when
handling the flies and yet didn’t ask for help from the other but chose to continue to carry out
the experiment therefore caused some practical error in this experiment.

From the result obtained in y w sn test (cross type 2), we are able to conclude that the
cross gives a positive interference which indicates that in fact interference occurring that is
preventing a crossover event. The observed double cross over is less than the expected double
cross over which is more prevalent in genetic study. The reason we assigned the gene sequence
as y w sn because it is a more correct sequence compare to y sn w, although we were using y
sn w in our result for crossing. According to our research on internet, the correct sequence shall
be y w sn but we have no further evidence to prove that this is the exact correct sequence.
Therefore in our opinion, since w stands for the eye colour of Drosophila melanogaster while
sn stands for the body bristles. Eye colour is more visible and easier to observe compare to
body bristle. Therefore the sequence shall be y w sn.
 References
1. “Introduction of “Drosophila melanogaster” retrieved from
https://www.sciencedirect.com/topics/neuroscience/drosophila-melanogaster

2. “Methods to differentiate the sex of Drosophila melanogaster” retrieved from

http://depts.washington.edu/cberglab/wordpress/outreach/an-introduction-to-fruit-flies/

3. “Interference and Coefficient of Coincidence” retrieved from

https://www.chegg.com/homework-help/definitions/interference-and-coincidence-14