Determining Map Distances for Selected Genes in Drosophila melanogaster through a

Three-Point Cross
Jaclyn Wu
Lab partner: Sung (Richard) Park
09/31/14 10/28/14

Abstract
The purpose of this experiment was to gain experience working with the model organism
Drosophila melanogaster and to determine the map distances between three selected genes y,
fb, and cv in the Oregon R fly which produce visibly different mutants. This was achieved by
mating a triple mutant homozygous female with wild-type male flies, collecting the females from
the F1 generation, mating them with triple mutant males, and counting the various offspring
phenotypes. These counts were then used to determine the map distances between the three genes
of interest, which were 44.93 map units (m.u.) between y and fb, 25.28 m.u. from y to cv, and
43.84 m.u. between fb and cv. Inheritance patterns of the three genes also suggested that they are
sex-linked to the X-chromosome. Future experiments could be repeated with a larger number of
offspring to determine more accurate map distances.
Introduction
Drosophila melanogaster has long been utilized as a genetic model organism due to its small
size, rapid generation time, large number of offspring, and easy cultivation. Additionally, due to
its extensive use as a model organism, several of the organisms chromosomes, including the Xchromosome, have been fully sequenced, which facilitates studies of the relationship between
genes. The objective of this experiment was to determine the map distances on the chromosome
of three different genes in D. melanogaster. The first is represented by y, which codes for body
color. Wild-type flies have a dark body color, whereas homozygous recessive mutants are yellow.
The second gene is represented by fb, which determines the bristle pattern on the fly. Wild-type
flies have straight bristles, whereas mutant flies have forked bristles. The third gene we
examined was cv, in which wild-type flies have cross-veins on their wings and mutant flies lack
the cross-vein in their wing. An additional benefit to using D. melanogaster for this experiment

is the wide variety of phenotypes and characteristics with which it presents, which allows for
easy visual scoring and determination of genotype by sight.
To determine the distances between the three genes on the chromosome, a three-point cross was
used. This technique was first developed by Thomas Morgan and Alfred Sturtevant and has been
a simple and fairly accurate method of mapping distances between genes with evident
phenotypes (Grant, 2014). The first mating of the experiment was between a triple mutant female
and a wild-type male. Virgin females were used to ensure that all resulting offspring would be
from the desired mating and not residual from previous matings of the female. The female
offspring from this mating were then mated with triple mutant males. The offspring of this
second mating would present all possible combinations of each of the three traits, resulting in 8
total phenotypes (23). As a control, mutant virgin females were mated mutant males to confirm
that both lines are true-breeding. The number of offspring bearing each of the 8 phenotypes was
then counted and also pooled as class data. Map distances were then calculated between two
genes at a time by dividing the number of recombinant flies for those two genes by the total
number of flies counted. Calculated map distances were determined to be similar to literature
values, although a larger sample size and more accurate scoring could improve results.
Materials and Methods
Materials and methods were followed as provided in the lab protocol (Grant, 2014). An
adjustment was made during data collection in that only female flies were counted due to 1) a
large number of offspring and 2) female fly body color being easier to distinguish.
Results
After the matings were prepared, the phenotypes of the female offspring from the F1
heterozygous female and triple mutant male were counted.

Figure 1: Cross of heterozygous wild-type female with triple mutant male. Total fly counts
during final week of experiment for each phenotype. Individual totals were summed from three
vials.
Phenotype

body color (y)

dark
dark
dark
dark
yellow
yellow
yellow
yellow

bristle (fb)
forked
forked
straight
straight
forked
forked
straight
straight

Individual Total
(n=154)

Class Total from

Both Lab Days
(n=1843)

18
19
13
25
33
6
34
6

106
267
87
308
484
136
318
137

veins (cv)
no cross
cross +
no cross
cross +
no cross
cross +
no cross
cross +

Figure 2: Calculations for map distances between genes for body color (y) and bristle (fb) using
individual totals (top) and class totals (bottom). This was calculated by adding up offspring
which displayed recombination of these two traits from parental phenotypes (dark and forked,
yellow and straight) and dividing by the total number of flies observed. This was repeated for
each combination of the three traits.
18 19 34 6
100cM 50.0m.u.
154
106 267 318 137
100cM 44.93m.u.
1843
Figure 3: Calculations for map distances between genes for bristle (fb) and veins (cv) using
individual totals (top) and class totals (bottom).
13 19 6 34
100cM 46.75m.u.
154
87 267 136 318
100cM 43.84m.u.
1843
Figure 4: Calculations for map distances between genes for veins (cv) and body color (y) using
individual totals (top) and class totals (bottom).
18 13 6 6
100cM 27.92m.u.
154

106 87 136 137

100cM 25.28m.u.
1843
Calculated experimental values were then compared to literature values, with a diagram of the

relevant chromosome shown below.

Figure 5: Map of D. melanogaster X-chromosome from Bridges and Anderson, 1925. In the
diagram, fb is denoted as f.

Figure 6: Comparison of map distances determined from individual, class, and literature results.
Genes
y and fb
fb and cv
cv and y

Individual (m.u.)
50.0
46.75
27.92

Class (m.u.)
44.93
43.84
25.28

Literature (m.u.)
56.8
42.8
14.0

Discussion
The total fly counts were pooled from both lab days in order to achieve a larger sample size for
the map distance calculations. These results were then compared to our individual totals. In both
cases, the largest number of offspring were found to have the parental genotypes (all wild-type or
triple mutants), which was expected as the genes were determined to be located fairly close to
each other on the same chromosome. This thus diminishes the likelihood of crossover events
occurring between these three genes and resulting recombination. Along those lines, double
crossovers (ex. dark, forked, cross-veinless or yellow, straight, cross-vein) were also found to be
in the smallest frequency, as the chances of this recombination are even less likely.
Each of the three genes produces a different protein product that leads to the phenotype observed
in the experiment. We hypothesized that the yellow gene y encodes a protein that blocks

synthesis of the normal dark pigment in wild-type flies, thus resulting in flies that are yellow in
color. We hypothesized that the gene for bristles, fb, encodes a protein that normally bundles the
bristles together. In the forked mutants, if this bundle protein is dysfunctional, the bristles will
split and assume a forked appearance. Finally, we hypothesized that cv encodes a protein that is
important for vein branching to form cross-veins. Without the ability to branch, only straight
veins would be observed, which is seen in the cross-veinless flies.
The map distances determined using our class data were closer to literature values for the
distances between fb and cv (43.84 m.u. class vs. 42.8 m.u. literature) as well as cv and y (25.28
m.u. class vs. 14.0 m.u. literature), whereas the map distance for y and fb was closer using
individual data (50.0 m.u. individual vs. 56.8 literature) (Bridges and Anderson, 1925). The
larger sample size from the pooled class data provides a more accurate representation, which is
likely why the majority of class data distances were closer to literature values. Ideally the
distance from y to cv and cv to fb should equal the distance from y to fb; however, our map
distances were slightly larger than the literature values. This error could arise from our
interpretation of each of the phenotypes. Within our group, it was the first time either of us had
worked with D. melanogaster and there was some difficulty in discriminating between body
color and forked or straight bristles. Additionally, since each lab partner counted half of the flies,
it is possible that different thresholds were used for body color assignments (dark or yellow),
which would affect the accuracy of the data.
This experiment could also be repeated with a larger sample size to obtain more accurate results.
Additionally, we only counted females and adding the data from male offspring would provide a
larger sample size. Further experience with the model organism would also increase accuracy of
the reported results.

Questions
1. Yes, the three genes, y, fb, and cv are sex-linked to the X-chromosome. This was observed by
examining the offspring of the mating of true-breeding triple mutant females to wild-type males.
The resulting offspring were all either wild-type females or triple mutant males. This suggests
that the mutation is recessive and having one copy of the non-mutant genes from the father
allows for heterozygous female offspring to present the wild-type phenotype. However, since
triple mutant female thus has two copies of mutant genes, she is only capable of passing on the
mutated X-chromosome to the male offspring. Since the male offspring must receive a Ychromosome from the father, their phenotype will be determined by the X-chromosome from the
mother. Therefore, with a triple mutant mother that has the mutations present on the Xchromosome, all male offspring will be mutants, which was observed in our mating results.
Thus, sex-linkage to the X-chromosome explains why only 2 different phenotypes were observed
in the cross between triple mutant females and wild type males.
2. The order of the genes on the chromosome is y, cv, and fb. That is, the cv gene for cross-veins
lies between the y and fb genes. The distances determined based on class data is 44.93 m.u.
between y and fb, 25.28 from y to cv, and 43.84 m.u. between fb and cv. See Results section for
corresponding calculations and Discussion for error discussion.
References
Bridges, CB and Anderson, EG. Crossing over in the X Chromosomes of Triploid Females of
DROSOPHILA MELANOGASTER. Genetics. 1925 Sep;10(5):418-41. PMID:
17246283.
Grant, S. (2014). Drosophila Lab Three Point Cross. Lab Protocol.

Grant, S. (2014). Mapping Genes by Recombination Frequency. Powerpoint.