Determination of gene location, order, and linkage in Drosphila melanogaster for body color,
eye color, bristle shape, and wing shape
Shannon Ceperich, Lindsey Levine, and Marybeth Norton
Introduction
Heredity is the passing of traits, controlled by genes, from parents and ancestors to
offspring. It can lead to species evolution and adaptation. Thomas Hunt Morgan showed
that genes are linked in a series on chromosomes and are responsible for identifiable, hereditable
traits (Morgan 1916). The purpose of this experiment was to examine the inheritance of
Drosophila melanogaster and the genes responsible for ebony body, white eyes, miniature
wings, and singed bristles.
The wild-type eye color of D. melanogaster is red (w+) and a mutant phenotype is white
(w). White eyes can result from a sex-linked mutation on the X chromosome. This was first
observed by Morgan in 1910 when he saw a male fly with white eyes. He crossed that whiteeyed male with a red-eyed female and both sexes of the F1 progeny had red eyes. He crossed the
F1 progeny and found that half of the F2 males had red eyes and the other half had white eyes.
The F2 females had red eyes. The eye color mutation occurred in males, leading him to believe
that eye color was sex-limited. Further breeding proved that the white-eyed mutation occurred in
a sex-linked gene (Morgan 1910). Because the F1 females were heterozygous and expressed the
red eye phenotype, the wild type red eyes trait is dominant and the mutant white eye trait is
recessive (Reed and Reed, 1948). The "w" gene that leads to white eye color is located at 1.5
MU from the end of the X chromosome (Morgan 1910.) The heterozygous F1 female progeny
resulting from reciprocal crosses between female wild-type, pure breeding, red-eyed (w+),
normal-bristled (sn+), long-winged (m+), tan-bodied (e+) flies and male mutant, pure breeding,
white-eyed (w), singed-bristled (sn), miniature-winged (m), ebony-bodied (e) flies are expected
to show the dominant red eye trait and the hemizygous F1 male progeny are expected to have the
same eye color phenotype as the mother because it is an X-linked trait.
The mutant ebony body is due to an autosomal recessive mutation in the ebony gene (e)
located on the third chromosome in Drosophila melanogaster at 70.7 mu (Bridges 1923). This
mutation was first discovered by E. M. Wallace in 1990 (Wallace 1990). The wild-type (e+)
allele of the gene results in a tan-colored body. The ebony mutation affects the quantity of
pigments formed as well as the time of their appearance making the adult fly body black
(Waddington, 2009). The wild- type tan body allele is dominant and the mutant ebony body
allele is recessive. The F1 progeny resulting from the reciprocal crosses of this experiment are
expected to show the dominant tan body trait because all flies, both male and female, are
heterozygous for this autosomal gene.
Wing size is another phenotype being observed. Morgan was the first to discover the
miniature wing (m) mutant in 1910. Miniature wings are a recessive, X-linked trait that is
controlled by a gene located at 36.1 mu on the X chromosome (Bridges and Morgan 1923). The
mutant miniature wings are smaller in size than the wild-type wings, but can be similar in shape
(Waddington, 2009). The flies in this experiment that have miniature wings are expected to have
rounder wings. The heterozygous F1 female progeny of this experiment are expected to express
the dominant wild-type wing and the hemizygous F1 male progeny are expected to have the
same wing phenotype as the mother because it is an X-linked trait.
Mohr was the first to discover the singed bristle mutant in 1922 and found the responsible
gene (sn) to be X-linked and mutant allele recessive. The location of this gene is 20.9 mu from
the chromosome end (Mohr 1922). The singed bristle phenotype appears gnarled and kinky and
is due to the lack of actin filament bundles in both large and small bristles. The DNA of flies
with singed bristles contains a TATA-box deficient (TATA-less) promoter (Roiha 1988).
Reciprocal crosses between pure-breeding wild-type females and hemizygous mutant
males and pure-breeding mutant females and hemizygous wild-type males were used to
determine what were dominant traits and if a gene was sex-linked or autosomal (Figure 1). The
F1 females would express the dominant traits because they were heterozygous. For example, if
the F1 females all have normal wings, then normal wings are dominant and miniature wings are
recessive. Females have two X chromosomes and males have one X chromosome and a Y
chromosome. This means that the male inherits his X chromosome from his mother. In this
experiment, F1 male flies resulting from pure breeding mutant females and pure breeding wildtype males were observed to determine sex linkage. The F1 male progeny resulting from this
experiment’s reciprocal crosses between pure breeding flies (Figure 1) should have the same
phenotype as his mother if the gene causing the phenotype is X-linked as his only X
chromosome is from her. If the gene causing the phenotype was autosomal, then the F1 male
progeny had a 75% chance of expressing the dominant phenotype and a 25% chance of
expressing the recessive phenotype.
A test cross between F1 heterozygous virgin females and hemizygous recessive males
who were heterozygous for the autosomal (e/e+) gene, was used to determine map distances
between genes as well as whether genes were linked to each other or followed The Law of
Independent Assortment (Figure 2). The Law of Independent Assortment, first described by
Gregor Mendel, the father of genetics, states that allele pairs separate independently of other
allele pairs during gamete formation. On the other hand, genes may be linked and inherited
together during meiosis because they are located near each other on the same chromosome.
These linked genes are less likely to be separated by a crossover event. In heterozygotes, if two
phenotypes are correlated, or always appear together, they can be assumed to be resulting from
linked genes (Sutton 1903); therefore, the F2 progeny were screened for gene linkage.
As shown in Figure 2 and based on the hypothesis that for both male and females all Xlinked traits are linked and will not separate in a crossover event, 3/8 of the F2 progeny are
expected to be completely wild-type, 1/8 are expected to be wild-type for the X-linked traits and
to express the mutant ebony body, 3/8 are expected to be mutant for the X-linked traits and to
express the wild-type body, 1/8 are expected to be completely mutant, 1/2 of are expected to
express the wild-type of the X-linked genes, and 1/2 are expected to express the wild-type of the
autosomal body color gene.
A)
w
sn
m
P1
e+
X
w
sn
m
e+
e
w+ sn+ m+
e+
w
e
F1
B)
w+ sn+ m+
e
w
sn
m
e+
&
sn
m
w+ sn+ m+
e+
w+ sn+ m+
e+
w+ sn+ Mv
e+
P2
e
w
sn
m
e
X
F1
e
w+ sn+ m+
e+
&
w
sn
m
e
e
Figure 1. Results expected from reciprocal crosses between wild-type, pure breeding, red-eyed
(w+), normal-bristled (sn+), long-winged (m+), tan-bodied (e+) flies and mutant, pure breeding,
white-eyed (w), singed-bristled (sn), miniature-winged (m), ebony-bodied (e) flies. The w,sn,
and m genes are X-linked and the e gene is autosomal. A) In the P1 cross, pure breeding mutant
females (ww snsn mm ee) were crossed with the pure breeding wild-type males (w+ sn+ m+ //\
e+e+). The resulting F1 females were expected to be heterozygous for all traits and to express the
dominant allele. The resulting F1 males were expected to be hemizygous mutant for the X-linked
traits as they inherited their X chromosome from their homozygous wild-type mothers. They
were expected to be heterozygous for the autosomal body color gene and thus express the
dominant wild-type of the gene. B) In the P2 cross, pure breeding wild-type females (w+w+
sn+sn+ m+m+ e+e+) were crossed with pure breeding mutant males (w sn m //\ ee). The resulting
F1 females were expected to be heterozygous for all traits and to express the dominant allele.
The resulting F1 males were expected to be hemizygous wild-type for the X-linked traits. They
were expected to be heterozygous for body color and thus express the dominant wild-type.
e+
w
sn
m
+
+
+
m
e
m+
e+
F1
F2
w
sn
m
e+
X
w
sn
w+
sn+
e
w+
sn+
m+
e+
&
3/8
w
sn
m
e or
e+
w+
sn+
m+
e
e or
e+
w+
sn+
m+
e
&
1/8
w
sn
m
e
w
sn
m
e+
e
w
sn
m
e+
&
3/8
w
sn
m
e or
e+
w
sn
m
e
e or
e+
w
sn
m
e
&
1/8
w
sn
m
e
e
Figure 2. An F1x F1 cross was conducted between the heterozygous F1 females (w+w sn+sn
m+m e+e) and the F1 males (w sn m //\ e+e) resulting from the P1 reciprocal cross (Figure 1).
This cross will help determine map distances between the genes if genes are linked to each other.
Based on the hypothesis that for both male and females all X-linked traits are completely linked
and will not separate in a crossover event, 3/8 of the F2 progeny are expected to be completely
wild-type, 1/8 are expected to be wild-type for the X-linked traits and to express the mutant
ebony body, 3/8 are expected to be mutant for the X-linked traits and to express the wild-type
body, 1/8 are expected to be completely mutant, 1/2 of are expected to express the wild-type of
the X-linked genes, and 1/2 are expected to express the wild-type of the autosomal body color
gene.
Results
Table 1. Progeny resulting from reciprocal crosses between wild-type, pure breeding, red-eyed
(w+), normal-bristled (sn+), long-winged (m+), tan-bodied (e+) flies and mutant, pure breeding,
white-eyed (w), singed-bristled (sn), miniature-winged (m), ebony-bodied (e) flies. In the P1
cross, pure-breeding mutant females (ww snsn mm ee) were crossed with the pure breeding, wildtype males (w+ sn+ m+ // e+e+). In the P2 cross, pure breeding wild-type females
(w+w+ sn+sn+ m+m+ e+e+) were crossed with pure breeding mutant males (w sn m // ee).
Cross
P1
P2
Parents
Mutant Females and
Wild-type Males
Wild-type Females and
Mutant Males
Phenotypes of F1 Progeny
Observed
Number
Observed
Females
Males
Red eyes, normal bristles, long wings,
and tan body
57
0
White eyes, singed bristles, miniature
wings, and tan body
0
43
Red eyes, normal bristles, long wings,
and tan body
55
45
Table 2. Progeny resulting from a cross between heterozygous red-eyed (w+), normal-bristled
(sn+), long-winged (m+), tan-bodied (e+) F1 females (w+w sn+sn m+m e+e) and the F1 males
(w sn m // e+e) resulting from the P1 reciprocal cross and expressing white-eyes (w), normalbristles (sn), long-wings (m), and tan-bodies (e+) carrying the autosomal recessive allele for
mutant ebony bodies (e).
F2 Progeny Phenotypes
Origin
Body
color
Eye
color
P
P
P
P
DCO
DCO
DCO
DCO
SCOI
SCOI
SCOI
SCOI
SCOII
SCOII
SCOII
SCOII
Tan
Ebony
Tan
Ebony
Tan
Ebony
Tan
Ebony
Tan
Ebony
Tan
Ebony
Tan
Ebony
Tan
Ebony
Red
Red
White
White
Red
Red
White
White
Red
Red
White
White
Red
Red
White
White
Bristle
Shape
Normal
Normal
Singed
Singed
Singed
Singed
Normal
Normal
Singed
Singed
Normal
Normal
Normal
Normal
Singed
Singed
Numbers
Expected
If all 4 genes
Total
independently
assorting **
107
38
39
13
4
38
52
13
6
38
11
13
57
38
7
13
30
38
7
13
23
38
25
13
20
38
9
13
8
38
0
13
Numbers observed
Wing
Shape
Long
Long
Miniature
Miniature
Long
Long
Miniature
Miniature
Miniature
Miniature
Long
Long
Miniature
Miniature
Long
Long
Males
Females
49
18
0
15
1
6
24
1
14
5
11
8
3
5
4
0
58
21
4
37
5
5
33
6
16
2
12
17
17
4
4
0
** If all four genes were independently assorting, then there was a ½ chance that the F2 progeny would
express red eyes, a ½ chance they would express normal bristles, and a ½ chance they would express
long wings. There was a ¾ chance they would express a tan body and a ¼ chance they would express a
mutant white body color.
Statistical Analysis and Conclusion
Reciprocal crosses between pure-breeding wild-type females and pure-breeding males
who were mutant for white-eyes, singed-bristles, miniature-wings and ebony body color or purebreeding white-eyed, singed-bristled, miniature-winged, ebony bodied females and purebreeding wild-type males were used to determine if a gene was sex-linked or autosomal and
which allele was dominant. All of the F1 females resulting from both reciprocal crosses (Table 1)
were heterozygous for all traits and were tan-bodied, red-eyed, normal-bristled, and longwinged; therefore, the wild-type phenotype for all traits was dominant. The progeny resulting
from the reciprocal crosses also showed that the gene controlling body color was autosomal and
the genes for eye color, bristle shape, and wing shape were X-linked. The genes for eye color,
bristle shape, and wing shape were determined to be X-linked because the hemizygous F1 males
had the same phenotypes for these traits as their mother. Mothers with red eyes, normal bristles,
and long-wings had sons with red eyes, normal bristles, and long-wings. Mothers with white
eyes, singed-bristles, and miniature-wings had sons with the same phenotypes for those traits.
The gene controlling body color was determined to be autosomal because a cross between a
pure-breeding ebony female and a pure-breeding tan male resulted in F1 male progeny with a tan
body color. Because the gene controlling body color is autosomal and the genes controlling eye
color, bristle shape, and wing type are on the X chromosome, the body color gene is not linked to
the other genes.
A cross between F1 heterozygous females and F1males hemizygous for white eyes,
singed-bristles and miniature-wings and heterozygous for body color (Figure 2) was used to
determine whether all four genes were linked to each other or followed the Law of Independent
Assortment. The number of progeny in each phenotypic category was predicted based on the
hypothesis that all four genes assorted independently (Table 2). The observed number of progeny
differed significantly from expectations. A chi square test with 15 degrees of freedom resulted in
a value of 306.69, which indicates a probability less than 0.01; therefore, all four genes were not
independently assorting and some may be linked.
The progeny resulting from the same F1 x F1 cross were used to determine gene order,
distances between the genes, and where crossovers occurred if the genes controlling eye color,
bristle shape, and wing shape were X-linked. The genes controlling eye color, bristle shape, and
wing shape were determined to be in the same order as previously reported (Figure 3); however,
the map distances were not at the same distances as reported previously in the literature (Morgan
1910; Bridges and Morgan 1923; Mohr 1992). Using a three point test cross and previously
recorded map distances of genes, the expected number of progeny in each phenotypic category
was predicted and compared to the observed progeny (Table3). There was a significant
difference that was reflected in a large chi square value of 168.34, of less than 0.01; therefore,
the hypotheses that the genes would be separated by the same map distances as previously
reported in the literature was not supported. The difference between predicted progeny and
resulting progeny was likely due to a screening error for bristle shape, which caused a higher
count of flies in the double cross category. The screening error was distinguishing flies with
singed-bristles versus flies with normal bristles. The least frequent pair of categories should have
been double crossovers (w+w snsn m+m and ww sn+sn mm), but we observed 64 progeny in one
category (ww sn+sn mm) of double crossovers when the expected value was 12. If the number of
progeny with white eyes, normal bristles, and miniature-wings was reduced significantly to
match the expected value, the chi-square value would be lower and support the hypothesis better.
The F2 progeny were reevaluated by looking at the number as a two point test cross
involving the genes controlling eye color and wing shape. Expected frequencies were again
predicted using previously reported map distances and compared to the observed progeny
(Table4). This two gene comparison eliminated the distortion of the double cross over category.
The chi square value was 11.4 with 3 degrees of freedom, which gives a probability less than
0.01. This probability is too low meaning there was a significant difference between observed
and expected map units. This may have been due to the fact that there was a larger number of
red-eyed progeny than white-eyed progeny (229:176) when there should have been a 1:1 ratio of
progeny with red eyes and white eyes. This means that red-eyed flies were surviving more than
white-eyed flies. If the number of progeny with red eyes and white eyes was the same, then the
map units would have been more similar to the map distances reported previously in the
literature (Morgan 1910; Bridges and Morgan 1923; Mohr 1992). The map distances may have
been different due to interference. There was a high incidence of double crossovers observed as
the value of interference was -2.375.
Overall, the gene controlling body color was determined to be autosomal and the genes
controlling eye color, bristle shape, and wing shape were X-linked. The wild-type allele was
dominant for all traits. All four genes were not independently assorting. The distances between
genes controlling eye color, bristle shape, and wing shape were calculated and chi square
analysis showed that they were different than the expected distances recorded in previous
literature, but the order of the genes was correct (Figure 3).
Table 3. Three point test cross evaluation of progeny resulting from a cross between
heterozygous red-eyed (w+), normal-bristled (sn+), long-winged (m+). F1 females (w+w sn+sn
m+m) and the F1 males(w sn m) resulting from the P1 reciprocal cross and expressing white-eyes
(w), normal-bristles (sn), and long-wings (m).
Phenotypes
Category
Eye color
Bristle shape
Numbers
Wing shape
Observed Expected *
P
Red
Normal
Long
146
140
P
Ebony
Singed
miniature
56
140
DCO
Red
Normal
Long
17
12
DCO
Ebony
Singed
miniature
64
12
SCO I
Red
Normal
Long
37
32
SCO I
Ebony
Singed
miniature
48
32
SCO II
Red
Normal
Long
29
26
SCO II
Ebony
Singed
miniature
8
26
*Numbers expected if genes controlling eye color, bristle shape, and wing shape were linked and
did not assort independently. Values were obtained using a distance of 19.4 mu between w and
sn and a distance of 15.2 mu between sn and m.
Table 4. Two point cross analysis for genes controlling eye color and wing shape using progeny
resulting from a cross between heterozygous red-eyed (w+) and long-winged (m+) F1 females
(w+w m+m) and the F1 males (w m) resulting from the P1 reciprocal cross and expressing whiteeyes (w) and long-wings (m).
Phenotypes
Category
Eye
color
Wing
shape
Number
Observed Expected *
Parent
Red
Long
163
132
Parent
White
Miniature
120
132
Recombinant
Red
Miniature
66
70
Recombinant
White
Long
56
70
*Numbers expected if genes controlling eye color and wing shape were linked and did not assort
independently. Values were obtained using a distance of 34.6 mu between w and m.
A) ___w___19.4___sn___15.2___m___
__e__
B) ___w____41____sn___29.1___m___
__e__
Figure 3. Map distances between genes investigated for inheritance of white eyes (w), singedbristles (sn), and miniature-wings (m). A) The expected map distances and order based on
previous literature (Morgan 1910; Bridges and Morgan 1923; Mohr 1992). B) The observed and
calculated map distances and order based on progeny resulting from a testcross between F1
heterozygous virgin females and hemizygous recessive males who were heterozygous for the
autosomal (e)/(e+) gene.
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