In a cross between the cinnabar and brown eye color genes of Drosophila
melanogaster, crossing over does not occur, producing an absence of
double mutants.
University of Wisconsin- Whitewater
Department of Biology
Amy Henderson  Alexis Hinkle  Amanda King  Hesther Perez
April 26, 2012
Abstract
The assortment of eye pigment in the D. melanogaster collection was established in the deposition through
ommochrome and pteridine pathways. Previous studies of eye pigmentation in Drosophila melanogaster
delivered convincing evidence that in a double hybrid cross between cinnabar and brown genes, it would
not support a Mendelian prediction in regards to eye color in the F2 offspring for a 9 (wild type): 3 (brown):
3 (cinnabar): 1 (double mutant) ratio. Here we report the crosses conducted and their phenotypic ratios to
define what phenotype a double mutant would possess, if any. It was determined that both cn and bw genes
exist on the 2R chromosome but at separate locations. This would typically increase the frequency in
crossing over recombination; however, crossing over does not occur between bw and cn males. The
interaction between cinnabar and brown causes a lack of pigmentation which supports our value of the
pathway deposition based on the number of studies conducted for eye color where no double mutant results
from crossing the brown gene with the cinnabar gene.
Introduction
Based on previous predictions of Mendel, we hypothesized that through a dihybrid cross
with cinnabar and brown eye color mutants. The F1 generation is predicted to be all wild
type. The F2 generation would show a 9:3:3:1 ratio with 9/16 wild type, 3/16 brown, 3/16
cinnabar and 1/16 very dark red (the predicted double mutant), where the cinnabar (cn)
gene will trump the brown (bw) gene. We then broke this ratio up by sex, with a
predicted 9 female wild type (brick red): 9 male wildtype:3 female brown:3 male
brown:3 female cinnabar: 3 male cinnabar:1 female very dark red (double mutant):1 male
very dark red (double mutant).
The mutations that cause the differences in genes became the focus of Thomas Hunt
Morgan (Dressen, Johnson, and Henikoff, 1988). Scientists use model organisms in
order to translate the basic concepts that can be used to understand other complex
organisms. Drosophila melanogaster, or more commonly known as fruit flies, are used
as model organisms for a number reasons including; easy maintenance in the lab, a short
generation time, and only 4 pair of chromosomes. They also contain large polytene
chromosomes which are found in the salivary glands, holding 1,000's of copies of each
chromosome (Dressen, Johnson, and Henikoff, 1988).
Drosophila melanogaster has 4 homologous pairs of chromosomes. They consist of two
large pairs of autosomes; one pair being smaller than the other, one pair of really small
autosomes and sex chromosomes. Female D. melanogaster have two X chromosomes
and males have an X and a small Y chromosome. The sex of the fruit fly is determined
by the X to autosome ratio, and not the existence or lack of the Y chromosome
(Granadino et al, 1991). When there are two X chromosomes to two sets of autosomes, it
equals a 1:1 ratio, and will develop into a female fly. When there is one X chromosome
and two sets of autosomes, a 1:2 ratio, the fly will be male. When a fly has one X and no
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Y (XO), the fly will be male and sterile due to the lack of a Y chromosome but will
appear normal externally. If a female is XXY, it is considered normal (Preston and
Engeles, 1996).
Upon further research on our cross between cn and bw fruit flies, it has been revealed that
the temperature the flies are incubated at may cause certain mutations to develop or
become suppressed. One paper indicated that fl(2)d^1 and fl(2)d^2 were temperature
sensitive mutations and could involve the cross between cn and bw. Fl(2)d^1 is lethal in
female and semi-lethal in males and fl(2)d^2 is lethal in both female and male fruit flies.
In females, this mutation causes the temperature sensitive period to continue throughout
the entirety of development. In the same study, it was shown that at 18 degrees Celsius
females were still able to produce larva and pupae. Lethality in germline cells was
observed when the flies were incubated at 29 degrees Celsius. We incubated our fruit
flies at 25 degrees Celsius (Granadino et al, 1991).
Cn gene and bw gene are found on the 2R chromosome arm. According to another study,
the majority of crossovers on chromosome arm 2R occurred within the 4-kb region
containing the P element (Preston and Engeles, 1996). Cinnabar gene and brown gene
are on opposite ends of the 2R chromosome arm. In 23,188 flies, the male recombination
frequency was found to be 1.05%. This was the same between reciprocal crosses, cn
bw+ and cn+ bw. The only circumstance when the recombination frequency decreased
was when the temperature of the gen-0 males is decreased to 19°C (Preston and Engels,
1996).
800 ommatidia, which are a radial element that help compose a compound eye, are what
make up the compound eye of the Drosophila melanogaster, and each of these contains
eight photoreceptor cells of 3 morphologically and physiologically definitive classes.
They are also blue and UV sensitive (Harris and Stark, 2012). The double mutation cn
and bw as well as the single mutation w (white) are both able to eliminate screening
pigments from the eye without involving the function of the photoreceptor cells. While
most visual mutants are found on the first chromosome, cn and bw are found on the
second, making them easier to use to construct a white-eyed multiple mutant.
Photoreceptor cells in retinal degeneration mutations are responsible for their own
degradation. The rdgB mutants’ retinal degeneration is restrictive due to its light
sensitivity (Harris and Stark, 2012).
The brown gene is necessary for the deposition of pteridine pigments in the compound
eye of D. melanogaster and encodes for the protein of 675 amino acids. Pteridine is a
chemical compound composed of pyrimidine and pyrazine. Evidence has shown that the
brown and white gene products are necessary for the uptake of pteridine precursor and
with that proof; we can conclude that the brown and white proteins interact (Dressen,
Johnson, and Henikoff, 1988).
In D. Melanogaster there are two types of major classes of eye screening pigments;
pteridines and ommochromes. Pteridines control the expression of the red and yellow
pigments while ommochromes control the expression of the brown pigments (Dressen,
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Johnson, and Henikoff, 1988). The pigments work on pathways to make chromosome
eye colors. If the mutant is brown, the mutation has occurred in the red/yellow pathway
due to a lack of pteridines. Brick red eye color is produced from both the brown and
red/yellow pathways, whereas a cinnabar mutation occurs from a lack of ommochromes
on the brown pathway. Eye pigmentation is deposited during late pupal and young adult
stages of life (Dressen, Johnson, and Henikoff, 1988).
The cinnabar gene is a protein coding gene (as in Flybase from Lindsley and Zimm,
1992) which encodes for the kynurenine 3- hydroxylase protein or enzyme (Flybase,
2012). The defect in the function of the gene blocks the synthesis of the kynurenine to 3hydroxykynurenine in the eye discs and the Malpighian tubules (Ghosh and Forrest,
1967). The cinnabar phenotype is a bright red eye color with clear ocelli (Flybase, 2012).
The cinnabar gene causes a defect in the ommochromes which blocks the pathway so that
the brown pigment cannot be expressed, and that is why the phenotype is bright red eyes
(Butenandt et al, 1949).
The other gene that we focused on in our D. melanogaster cross was the brown eye color
gene. The biological process that is involved within this gene is the ommochrome
biosynthetic process. It is said that the brown gene has a molecular function of ATPase
activity, which aids in the movement of substances. We found out that if flies are
homozygous for the brown gene that this usually causes lethality (Flybase, 2012).
However, if it is homozygous and survives, the fly will have brown eyes. The specific
allele that causes this lethality is bw5 (Flybase, 2012). However, if the brown eye color
gene interacts with the cinnabar eye color gene, the fly will have white eyes.
Ommochromes and drosopterins are two types of pigments that deposit in the pigment
granules; found to cause the typical red eye color in D. melanogaster. Other eye colors
of D. melanogaster encode elements of either one of these two pathways. There is a third
group of mutations that affects both kinds of pigments and is comprised of a specific
collection of eye color genes (Sevrioukov et al, 1999). These genes are classified into a
“granule group” and are supported by a network of genetic interactions. An example of
this would be carnation (car) that genetically interacts with deep orange (dor), light, and
garnet (Sevrioukov et al, 1999).
For this study, the prediction for the F1 phenotypes all containing wild type eyes was also
supported with the knowledge that both brown gene and cinnabar gene are located on
chromosome 2R which is not sex linked. We will test this hypothesis by performing 2
crosses, one with parent generation true breeding flies, using virgin females, and the
second cross with the offspring produced from the first cross. By observing the F2
generation we could determine the double mutant (cn;cn/bw;bw) and its phenotype. One
dihybrid cross that we performed had a P generation consisting of a brown mutant male
(bw;bw/cn+cn+) and a cinnabar mutant female(bw+;bw+/cn;cn). The reciprocal
dihybrid cross that was performed had a P generation comprised of a brown mutant
female (bw;bw/cn+cn+) and a cinnabar mutant male (bw+;bw+/cn;cn).
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Materials & Methods
Treatments and Controls:
The crosses were done in a controlled manner in glass vials, filled with cornmeal agar
and yeast that make up a media for the larvae and adult flies to live off of, which makes
them easy to maintain. The flies are anesthetized in an anesthetizing unit for a limited
amount of time, approximately 10 minutes, to allow for sorting with a paint brush or
pipet tip and identification of phenotypes by epi-illumination of a stereomicroscope. The
anesthesia used is known as FlyNap. If the flies are over anesthetized they could die or
become sterile. Once placed in the vials, they are stored in an incubator at a constant
25°C.
Parental Cross:
We performed a dihybrid cross with two true breeding mutant strains of Drosophila
melanogaster, focusing on the character of eye color. For the Parent generation, the
initial cross was between 5 female virgin brown flies and 7 male cinnabar flies, as well as
a cross with the reciprocal, with 5 female virgin cinnabar flies and 6 male brown flies.
The cross between brown female flies and cinnabar male flies was successful in
producing progeny on the first attempt, whereas the cross between cinnabar female flies
and brown male flies failed to produce progeny. We theorized that this may have been
due to sterility from over anesthetization and performed the cross again with success.
F1 Cross:
The Parental cross offspring of both dihybrid crosses produced all wild type flies. From
the many offspring produced, we performed a second series of crosses. The first cross
performed was between 25 female wild type flies and 27 male wild type flies. The
reciprocal cross used 15 male wild type flies and 25 female wild type flies. We expected
to find the 9:3:3:1 ratio from the F2 offspring this generation will produce as stated in our
initial (null) hypothesis. The same procedure was followed with the F1 crosses as was
done in the parental crosses in regards to the methods and materials used.
Chi-Squared Test:
We used our tabulated data to perform a chi-squared test that will determine if our null
hypothesis should or should not be rejected. This test compares the number of expected
phenotypes to the actual number of observed phenotypes in each category to see if our
results distributed are a good fit to our predictions. We separated them by sex when
counting phenotypes but grouped them while calculating our chi squared data, since
neither cinnabar nor brown are sex-linked (Shown in Figures 3 and 4). Also, we grouped
amber eye colored flies in with the brown eye colored flies since we were determining if
the very dark red was in fact our double mutant or a form of variance. In both crosses we
then compared our p values to determine if the chance is higher or lower than 5% of
getting the value we predicted in our null hypothesis.
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Results
Our initial hypothesis failed to be rejected after the F1 phenotypes proved to show what
was expected, all wild type mutants (shown below in Figure 1). Therefore, the
hypothesis did not need to be revised. The parent generation in both crosses resulted with
all wild type offspring, also known as brick red. We initially thought the P cross between
brown female D. melanogaster and cinnabar male D. melanogaster was spoiled, showing
many phenotypes in the F1 offspring, ranging from brown, wild type, and cinnabar (see
Figure 2).
F1 Generation: Phenotype
Key for Vertical Columns
Figure 1:
1- Female offspring
of cinnabar females
X Brown males
2- Male offspring of
cinnabar females X
Brown males
3- Female offspring of
cinnabar males X
Brown females
4- Male of offspring
of cinnabar males
X Brown females
Number of Flies
25
20
15
10
5
0
1
2
3
4
Phenotype; Brick Red (wild-type)
Figure 1: Illustrates the number of D. melanogaster observed in the F1 generation for both reciprocal crosses. The only
phenotypes: wild-type (which is a brick red eye color).
Drosophila melanogaster : F2 Phenotypes
Number of Fruit Flies
250
200
150
Brick Red
100
Brown (With Amber)
50
0
Cinnabar
Dark Red (Double Mutant)
Figure 2: Number of D. melanogaster with varying phenotypes for eye color in the F2 generation of a dihybrid cross. The
study on the left crossed brown virgin female flies with cinnabar male flies. The results show both male and female values as
well as combined. The set of graphs on the right represent the reciprocal study crossing cinnabar virgin female flies with
brown male flies. The combined values are also shown.
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After reviewing the adult flies the following week, the conclusion was made that perhaps
the pigments had not been completely deposited or were a variation of expressivity or codominance. The parent flies in the original cross between brown female and cinnabar
male flies were in fact wild type. However, in case we were incorrect at identifying
phenotypes and taking into account eye pigment deposition timing, we continued the
cross and placed 15 male and 15 female F1 adult flies into the large glass jar. After one
week, there were only 5 male and 13 female adult flies remaining. This lead us to think
that either there is a lethality gene when crossing brown and cinnabar or that there is
some form of cannibalism where the females are devouring the males.
BW:F x CN:M
Phenotypes
Brick Red
Brown (With Amber)
Cinnabar
Double Mutant (Dk. Red)
Chi Square Value
Degrees of Freedom
P Value
Observed
Expected
(O-E)²/E
Sex
Male
Female
Total
101
98
199
299
33.44
Male
Female
Total
91
120
211
100
123.21
Male
Female
Total
40
44
84
100
2.56
Male
Female
Total
16
22
38
33
0.76
159.97
3
P<0.005
REJECT
Figure 3: Chart of the observed and expected number of D. melanogaster for the F2 Generation, based on the null hypothesis
of a 9:3:3:1 phenotypic ratio for eye color. The values above are the results of a dihybrid cross of female virgin brown flies and
male cinnabar flies. The far right column represents the (observed-expected) ²/expected. These values are then summed and
compared to other values on a chart, proportional to the degrees of freedom (# of phenotypic categories minus 1). If P>0.05
Fail to Reject the null hypothesis and if P<0.05 Reject the null hypothesis. In the above cross the P value was <0.05, so the null
hypothesis was rejected.
532 of the F2 offspring (bw F x cn M) were counted and their sex and phenotype
recorded. The reciprocal counted 561 total flies and the same process of recording
occurred. The mutant Drosophila melanogaster ranged from brick red, brown, amber
(light orange-brown), cinnabar, and very dark red. These results are shown in Figure 2
where brick red (wild type) is the majority with a total of 199 (101 male, 98 female) and
264 (113 male, 151 female) respectively; with brown (including a lighter orange-brown
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color we refer to as amber) close behind with 211 (91 male, 120 female) and 106 (47
male, 59 female). Cinnabar follows with a total of 84 (40 male, 44 female) and 151 (57
male, 94 female).
Lastly, the least number of flies is represented by our initial hypothesized very dark red at
38 (16 male, 22 female) and 40 (14 male, 26 female). We recorded the sex of the flies as
well, even though the two genes in our cross are not sex linked. Figure 2 shows
phenotypes recorded based on the initial hypothesis.
CN:F x BW:M
Phenotypes
Brick Red
Brown (With Amber)
Cinnabar
Double Mutant (Dk. Red)
Chi Square Value
Degrees of Freedom
P Value
Observed
Expected
(O-E)²/E
Sex
Male
Female
Total
113
151
264
316
8.56
Male
Female
Total
47
59
106
105
0.01
Male
Female
Total
57
94
151
105
20.15
Male
Female
Total
14
26
40
35
0.71
29.43
3
P<0.005
REJECT
Figure 4: Chart of the observed and expected number of D. melanogaster for the F2 Generation, based on the null hypothesis
of a 9:3:3:1 phenotypic ratio for eye color. The values above are the results of a dihybrid cross of female virgin cinnabar flies
and brown male flies. The far right column represents the (observed-expected) ²/expected. These values are then summed and
compared to other values on a chart, proportional to the degrees of freedom (# of phenotypic categories minus 1). If P>0.05
Fail to Reject the null hypothesis and if P<0.05 Reject the null hypothesis. In the above cross the P value was <0.05, so the null
hypothesis was rejected.
Based on the chi square and p values shown in Figure 3 and the reciprocal values in
Figure 4, we were able to reject the initial hypothesis that the dihybrid cross would result
in a 9 female wild type (brick red): 9 male wild type: 3 female brown: 3 male brown: 3
female cinnabar: 3 male cinnabar: 1 female very dark red (double mutant):1 male very
dark red (double mutant) offspring phenotypic ratio for eye color. The hypothesis
predicted the double mutant would show a phenotypic eye color of very dark red so all of
the values reflected the prediction to reject or fail to reject if that was the case. After
further research, we found the double mutant to actually have the phenotypic eye color
resembling amber, a light orange-brown. This was due to a lack of crossing over
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amongst cn and bw genes in males. The reason for this is unknown (Personal
communication with Dr. Brown, 2012).
The observed offspring did not fit the predicted 9/16 wild type, 3/16 brown, 3/16
cinnabar, 1/16 very dark red ratio (x²=159.97, df=3, p<0.005). For the reciprocal
dihybrid cross, the observed offspring did not fit the predicted 9/16 wild type, 3/16
brown, 3/16 cinnabar, 1/16 very dark red ratio (x²=29.43, df=3, p<0.005).
After rejecting our null hypothesis, we decided to calculate a number of scenarios using
the chi squared test to find if there was any eye colored combinations that would in fact
fit the chi squared test from our actual results. We first ran the chi squared test with
amber as our double mutant with a chi square value of 89.55 where p<0.005, so we
rejected amber as our double mutant.
Next, we recalculated using 5 phenotypes and a degree of freedom of 4, with amber and
dark red counted separately. This too was rejected with a chi squared value of 43.07
where p<0.005. Lastly, we calculated a chi squared test with only 3 phenotypes, amber
and dark red both included with the values for brown, assuming different shades of
brown are being expressed, known as expressivity. We were then able to fail to reject the
p value of 0.22 in one cross but rejected the other cross with a p value of 0.004. This is
the closest we have come to finding an actual explanation for our results.
Discussion
The D. melanogaster F1 generation for the cross with a P generation of male (brown
eyes) cn+;cn+/bw;bw mated with female (cinnabar eyes) predictions were supported by
the results because all of the phenotypes had brick red (wild-type) eyes like suggested in
the predictions. The D. melanogaster F1 generation for the cross with a P generation of
female (brown eyes) cn+;cn+/bw;bw mated with male (cinnabar eyes) cn;cn/bw+;bw+
predictions were supported by the data because all of the F1 generation had a phenotype
of brick red (wild-type)eyes like suggested in the predictions. The F1 generation’s results
supported our prediction that the cinnabar gene and brown gene were not sex linked.
Based on the results from the chi square test, the null hypothesis is rejected. The
experiment did not prove that there would be a 9:3:3:1 phenotypic ratio from a dihybrid
cross of brown and cinnabar eye color.
It is important to note that the data itself could contain some error due to few different
factors. For example, we must take into account that the variance of the expression in
eye color could have played a role when observing and categorizing the F2 generation’s
phenotypes. Also, we think it is important to note that since there were so many flies in
the F2 generation we chose to only count some of them, which adds to the chance that we
could have collected some slightly skewed data.
The p values are so small that the percentage of certainty in the correlation of the two
genes studied is far below the allotted 5%. In this particular cross, we then assumed that
the amber eye color would in fact be the double mutant and the very dark red would then
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be included in the values with the brown eye color. Further chi square tests were done to
test this and in every possible scenario, the null hypothesis was rejected, or not proved,
nearly every time. This lead us to conclude that there is no double mutant when crossing
bw and cn eye colored flies.
We are questioning not only the lethality but also if the particular sex of the D.
melanogaster plays a role in the outcome of the fly’s eye color. We believe that we could
have over looked the fact that there may be some lethality of the D. melanogaster when
crossing the brown gene and the cinnabar gene. The cinnabar gene can cause defects in
the ommochrome biosynthetic process (Flybase, 2012). This then does not let kynurenine
convert to 3-hydroxykynurenine (Flybase, 2012). We noticed that the F2 generations of
both crosses were more female dominated, possibly concluding that there is some relation
to lethality in males and the double mutant. Research showed that the males were less
viable at temperatures around 29°C and since our flies were kept in a 25°C incubator, we
assume that was the reason many of our male flies died after the F1 cross. This would
disprove our theory of cannibalism since females are more viable at temperatures above
18°C.
We concluded that crossing over did not occur in cn and bw males (Personal
communication with Dr. Brown, 2012). We are unsure of why this has happened, but
have a few different theories. We ruled out sex linkage because the phenotypes were
carried on through both sexes. They were involved in the recessive gene in our wild type
F1 generation; indicating that there was no dominant or recessive lethality. Research has
indicated that penetrance was not the cause of our rejected hypothesis; it does not fit the
situation presented when the F2 generation was counted. This is what led us to research
this topic in particular. Our brown flies demonstrated expressivity in that the color brown
was several different shades, causing us to question which one was actually the double
mutant and that the brown gene was in fact epistatic to the cinnabar gene. The results of
this experiment definitely told us there is more to consider when looking at how precisely
the cinnabar genes and the brown genes interact with each other such as crossing over,
lethality factors, and inheritance patterns.
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