Download The Determination of the Genetic Order and Genetic Map

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Kaitie Kudlac
March 24, 2015
Professor Ma
Genetics 356
The Determination of the Genetic Order and Genetic Map for the Eye Color, Wing Size,
and Bristle Morphology in Drosophila melanogaster
Abstract:
In this experiment, Drosophila melanogaster was used as a model organism to
look at the concept of linkage and X linked inheritance through the traits of eye color,
wing size, and bristle morphology. Through the production of an F1 and F2 generation
starting with a P generation of a recessive mutant female and a wild type dominant male,
eight various phenotypes were yielded in order to determine recombination frequencies
and map distances. Ultimately, the gene order was determined, as well as the map
distances, and though the established distances did not exactly correspond to those
determined in the lab, the correct gene order was determined and the genes or traits were
established as linked or located on the X chromosome through a chi square analysis. We
determined the map distances as eye color to bristle morphology being 88.89 map units,
eye color to wing size as 45.6 map units, and wing size to bristle morphology as 43.3 map
units.
Introduction:
The main focus and purpose of this lab is to understand the concept of linkage and
gene mapping in Drosophila or fruit flies. More specifically, to determine the location of
the wing size, eye color, and bristle morphology through the frequency of recombinants
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on a genetic map of a fruit fly. Through these maps, large numbers of genes are related
through the basis of the frequency of crossing over between the various genes. These
goals will be met through working with two fly stocks, wild type and mutant. The first
cross that is made is between a recessive mutant female crossed with a wild type normal
male. This generation is also known as the parent generation or P generation, making the
parents a female fly with all recessive mutant genes and a male fly with all wild type
dominant genes. This then yielded a F1 generation in which the males were all recessive
mutants and the females were wild type. An F2 generation was then bred and created by
crossing a wild type female and recessive male. This cross then yielded eight different
phenotypes whose recombination frequencies were then used to determine the location of
the genes in a genetic map.
In order to create a genetic map and attempt to understand the linkage concept in
a short time, an organism, known, as a model organism is required. A model organism is
an experimental organism helpful to research and whose genetics is studied on the idea
that the findings can then be applied to other organisms such as chimpanzees or humans.
In this lab we use Drosophila melanogaster to experiment with X linked chromosomes,
following Thomas Morgan’s groundbreaking studies on fruit flies. Drosophila
melanogaster is an excellent model organism as it exhibits a variety of traits and features
that make it easy to study. These features include being relatively easy to breed,
comprising a relatively short life cycle, reproducing with large numbers of offspring,
having a fairly straightforward genetic analysis, traits, such as the ones that are studied in
this lab are also easily recognizable, and finally the differences between the sexes of the
flies are easy to distinguish. In this lab there are three different genes or traits being
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studied. The first is the white locus (w), which affects eye color. The dominant or wild
type for this locus is the red eye color while the recessive trait for this locus is white-eye
color. The second gene is the forked locus (f), which affects bristle morphology. Flies
with the recessive forked mutation will have short bristles that will have forked or split
ends while the dominant or wild type flies will have smooth curved bristles. The third and
final gene that this lab is examining is wing size (m). Mutant flies or flies that express the
recessive allele will have miniature wings that do not extend past the tip of the abdomen
while wild type flies or flies that express the dominant trait will have normal size wings.
One of the most important concepts that are being examined in this lab is the idea
of X- linked traits. Not only do the X and Y sex chromosomes carry the genes that
determine sex they also carry other genes for other characteristics, as is seen in
Drosophila melanogaster. Because males have an X and Y chromosome and females
have two X chromosomes, both males and females can inherit X- linked traits since they
each have an X chromosome. Because men only have one X chromosome, if the mother
has the disease or trait, the male has a 100% chance of also getting the trait since they
have to inherit their mother’s X chromosome since they inherit the Y chromosome from
the father. Females are a little bit different. Since they inherit one X chromosome from
their mother and one X chromosome from the father, if the mother is affected they will
also get the disease since both X chromosomes contain the disease but additionally if the
father has the disease they will also get the disease since they get the father’s only X
chromosome (O’Neil, 2012). Another case is if the mother is a carrier for the trait. If the
mother is a carrier than each child has a 50% chance of getting the disease because only
one of the X chromosome’s is affected. However if the mother has the disease and the
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father does not, a female child will not get the disease necessarily but instead will become
a carrier since they will get one X chromosome that contains the mutated gene from the
mother and one normal X chromosome from the father (NIH, 2015). X linked traits
differ from autosomal in that autosomal dominant tend to occur in every generation of an
affected family and occurs when one mutated copy of the gene is enough to be affected
with the disorder (NIH, 2015). This normally occurs when the affected person has one
affected parent. X linked dominant on the other hand is caused by mutations with genes
on the X chromosome (O’Neil, 2012). Females are often more frequently affected than
males. Families that have an X- linked dominant disorder often have affected males and
females in each generation. One of the major characteristics of this X linked inheritance
pattern is that fathers cannot pass these traits to their sons. Autosomal recessive is
different from autosomal dominant in that two mutated copies of the gene are present and
the parents each carry a single copy of the mutated gene or are known as carriers. These
disorders are not seen in every generation (O’Neil, 2012). X- linked recessive as opposed
to autosomal recessive is different in that males are more frequently affected than females
and the chances of passing the disorder on differ from male to female, similar to an Xlinked dominant trait. Families with an X- linked recessive disorder often have affected
males as opposed to affected females in each generation (NIH, 2015).
As earlier described, three different genes and traits are examined in this lab,
leading to a three point genetic map or a three point cross. The three point cross is
executed by crossing a wild type male, fruit fly in this case, with a recessive female fruit
fly. This will produce an F1 generation with recessive males and wild type females. The
F1 generation flies are then crossed eventually yielding eight different phenotypes in the
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F2 generation. Then with the F2 generation flies, the parental and recombinants must be
identified. The parental phenotype is most often the phenotype with the largest number of
flies. Conversely, the double crossover phenotype is the phenotype with the lowest
number of flies, and the single crossover is somewhere in between. Once these
phenotypes have been determined, the gene order then needs to be established. This is
normally done through the double crossovers since these are the alleles in the middle that
are flipped. After this the distance between the two genes is then determined through the
recombination frequency, which is calculated by dividing the number of recombinant
progeny by the total progeny. The double crossovers need to be counted twice since they
are part of both sets of gene recombinants. Recombination frequencies need to be
calculated for all three different gene combinations. The recombination frequency is
directly linked to the distance of the gene loci in centimorgans or map units. Once the
recombination frequencies are calculated a genetic map can be drawn, because the higher
the recombination frequency the further apart the genes are or vice versa. In other words
the greater the distance between the two genes the greater chance that the genes will cross
over or have recombination. The official genetic map for Drosophila melanogaster
indicates that correct order of the specific genes that are studied in this lab report are w
(eye color), m (wing size), and f( bristle morphology). The distance between w and m is
34.6 map units, the distance between m and f is 20.6 map units, and the distance between
w and f is 55.2 map units (Chromosome Mapping in Eukaryotes, 2015).
Materials and Methods:
Because the main portion of this procedure and the main work in this lab was
counting and analyzing flies, one of the most important techniques with the manipulation
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of the flies. One of the main skills in this lab is counting and characterizing the various
generations of flies, however, to do this the flies must be anaesthetized. To do this, the
flies are either placed on a fly bed with CO2 gas or they are anaesthetized through a small
CO2 needle that is placed in the vial to put them to sleep before they are counted outside
of the vial. Once the flies are anaesthetized, they are then placed under a dissecting scope
where they are counted according to the different phenotypes that they present. In order
to obtain these different generations, the flies were allowed to mate for up to two weeks.
As the typical life cycle for Drosophila melanogaster is twelve days to reach maturity,
this two-week incubation period allows for slower developing flies to reach maturity so
they are sexually active and ready to produce the subsequent generation. It also allows for
a more accurate fly count. While Dr. Walters completed the parental cross, we removed
the parental flies from the vials so that we would only initially count the F1 generation
flies. Once the F1 generation flies were counted, approximately 5 males and 5 females
were placed into each tube in order to promote reproduction and sexual activity.
Ultimately, only two different generations of flies will be counted and characterized, the
F1 generation and the F2 generation.
In the P generation there are only two phenotypes, females that are mutant
recessive and males that are wild type dominant. In the F1 generation there are also two
phenotypes to be both distinguished and counted; the flies are either males which are
mutant recessive or females that are dominant wild type. The F2 cross is a little bit
different. There are eight different phenotypes that are possible for the flies. These
phenotypes include red eyes, normal wings, and curved bristles, red eyes, normal wings,
and forked bristles, red eyes, miniature wings, and curved bristles, red eyes, miniature
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wings, and forked bristles, white eyes, miniature wings, and forked bristles, white eyes,
miniature wings, and curved bristles, white eyes, normal wings, and forked bristles, and
finally white eyes, normal wings, and curved bristles. At this stage it is no longer
important to determine the sex of the flies since this is the stopping point of the lab.
However, these traits or groups of phenotypes can appear in either males or females.
Collectively, these phenotypes will be determined using a dissecting scope. For the rest
of the lab report, the eye color trait will be referred to as w, the forked bristle morphology
trait will be referred to as f, and the wing size trait will be referred to as m. The letters
will indicate the recessive trait while the + will indicate the wild type trait.
Once the flies are counted and characterized, the final portion of the experiment is
to determine the gene order and the map distance calculations using the number of flies
from the F2 generation. The first step in determining the gene order is to determine
which phenotypes are the parentals and which phenotypes are the double crossovers.
Most often, the phenotype with the largest number of flies is the parental while the
phenotype with the smallest number of flies is the double crossover; the single crossovers
are in the in between zone. After these different groups are distinguished, recombination
frequencies for each different gene combination are calculated to determine the percent
recombination. The percent recombination is able to determine the gene order because
the percentage is equal to the gene distance in map units or centimorgans. Then by using
the fact that the double crossovers are the alleles in the middle that are flipped, the gene
order can be determined based on the distances between the two genes and the double
crossover gene being in the middle of the two alleles.
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Results:
The first cross that was conducted in this experiment was the P generation. This
cross was between a recessive female who had white eyes, miniature wings, and forked
bristles and she was crossed with a wildtpye male who had red eyes, normal wings, and
curved bristles. These two flies are expected to produce a F1 generation of flies that will
have males that are recessive mutants and females that are dominant wildtypes.
Table 1: Phenotypes and Number of Offspring in the F1 Generation
Phenotype
Number of Offspring
+++- red eyes, normal wings, curved
36
bristles
Wmf- white eyes, miniature wings, and
26
forked bristles
Though the sex of the flies is not shown in this table, all flies that have the recessive
mutant alleles are males and all flies that have the wildtype dominant allele are female.
This table indicates the phenotype of the F1 generation and the number of offspring that
the P generation produced. These flies will then be crossed, in a test cross, to create the
F2 generation of flies.
Once the F1 flies are categorized and counted they are then used to mate the F2
generation. In order to mate the F2 generation a male recessive mutant with white eyes,
miniature wings, and forked bristles is crossed with a dominant wildtype female with red
eyes, normal wings, and curved bristles. This then produces the following fly counts and
phenotypes.
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Table 2: Numbers and Phenotypes of All F2 Offspring Resulting from the F1 Female
+++ x wmf male
Phenotype
Number of Flies
Red, normal wings, curvy bristles(+++)
23
Red, normal wings, forked bristles(++f)
12
Red, miniature wings, curvy bristles (+m+) 11
Red, miniature wings, forked bristles (+mf) 3
White, miniature wings, forked
7
bristles(wmf)
White, miniature wings, curvy
2
bristles(wm+)
White, normal wings, forked bristles(w+f) 10
White ,normal wings, curvy bristles (w++) 13
This table indicates the eight different phenotypes that are produced as a result of
the cross between a wildtype dominant female and a recessive mutant male. These
numbers and these flies will be used to determine the genetic order and the genetic map
for these three traits. The phenotype with the most flies is the parental and the phenotype
with the smallest number of flies is the double crossover, the single crossovers are
everything else that is not the parentals or double crossovers. However, instead of using
the smallest number of flies for the double cross over, because we lost two vials of flies
in this experiment, the double crossovers will be the phenotypes in which there are two
traits that vary from the parentals. So in this case those will be +m+ and w+f. The
parentals will be +++ and wmf. Normally the data would follow the number pattern as
previously outlined, however, due to a loss of flies, the data does not adhere to the correct
phenotypes. All recombinant calculations will be made based on this information.
With these numbers, a chi square analysis will be used to determine if
independent assortment exists within these genes or if these traits or genes are linked and
inherited through sex chromosomes as opposed to autosomal chromosomes.
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Table 3: Calculation of Chi Square using Expected Ratio of Dominant to Recessive for
Each Trait in Monohybrid Test Cross
Trait 1: Wild- Type (Red Eyes) vs. White Eyes
Phenotype
Observed
Expected
(O-E)^2
(O-E)^2/E
Red Eyes
49
60.75
138.0625
2.273
White Eyes
32
20.25
138.0625
6.8179
This table shows the calculation of a chi-square that for the eye color gene. The chisquare that is calculated is equal to 9.091, which allows for the hypothesis to be rejected
since the chi square value is more than 3.841 (when p is 0.05).
Trait 2: Wild Type (Curved Bristles) vs. Forked Bristles
Phenotype
Forked Bristles
Curved Bristles
Observed
32
49
Expected
20.25
60.75
(O-E)^2
138.0625
138.0625
(O-E)^2/E
2.273
6.8179
This table shows the chi-square calculation for the bristle morphology gene. The
chi-square is equal to 9.091, which rejects the null hypothesis according to the degrees of
freedom, which says the chi square value should be less than 3.841.
Trait 3: Wild Type (Normal Size Wings) vs. Miniature Wings
Phenotype
Observed
Expected
(O-E)^2
(O-E)^2/E
Miniature
23
20.25
7.5625
0.3734
Wings
Normal Wings 58
60.75
7.5625
0.1244
This table shows the chi- square calculation for the wing size trait. The chi-square is
equal to 0.4979, which is less than 3.841, so the hypothesis is accepted for this trait.
While the charts above use the expected ratio of dominant to recessive individuals
for each trait in a monohybrid cross, this lab looks more specifically at the trihybrid ratio
and how the three genes are mapped in relation to each other. So rather than looking at
the individual monohybrid cross, it would be more productive to look at the trihybrid
cross.
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Table 4: Calculation of Chi Square Assuming Three Traits are Assorting Independently
(Ratios used from Trihybrid Test Cross)
Phenotype
Observed
Expected
(O-E)^2
(O-E)^2/E
+++
23
34.17
124.77
3.65
++f
12
11.39
0.3721
0.0326
+m+
11
11.39
0.1521
0.0133
+mf
3
3.79
0.6241
0.1647
Wmf
7
1.265
32.89
26
Wm+
2
3.796
3.2256
0.8497
W+f
10
3.79
38.56
10.174
W++
13
11.39
2.59
0.227
This table shows the chi square calculation based on the hypothesis that these three traits
are assorting independently through the assumption of the expected rations used for the
trihybrid test cross. The chi square value that was calculated based on this information
was 41.111 leading to the rejection of the null hypothesis and indicating that these traits
are linked and not independently assorted.
Figure 1: Genetic Map:
W
45.6
M
Distance from W to F is 88.9
Legend Key:
w- eye color, m- wing size, and f- bristle morphology
43.3
F
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This genetic map displays the distance in map units between the three different traits or
gene loci. Because the genes are generally far apart this allows for a greater chance of
crossover and a greater propensity of recombination.
The final piece of data that was collected for this experiment was the interference
value. The interference value is calculated by subtracting 1 from the coefficient of
coincidence. The coefficient of coincidence is calculated by dividing the observed double
crossovers by the expected double crossovers. The coefficient of coincidence in this case
is 1.383 so the interference value is negative .383.
Discussion
Generally the theoretical background of the predicted outcome of the experiment
is that for any genetic cross, including this one, the further apart the genes are or the
greater the distance between the two genes the larger chance of recombination and the
higher recombination frequency between the two loci. In other words, because they genes
or linked or rather if the genes are linked the greater the distance the more recombination
is likely. The three traits were determined to be X-linked through the P generation and the
F1 generation. Because the P generation started with a cross between a recessive mutant
female and wildtype dominant male and yielded a F1 generation of all recessive mutant
males and all dominant wildtype females, and because a male inherits his X chromosome
from his mother, these traits are X linked because of the swap over the two generations.
In other words because of the fact in the first generation that the males were dominant
and recessive in the second generation, we can say that this trait is X linked or sex linked.
The established genetic order for these three traits is w( eye color), m(wing size)
and finally f( bristle morphology). The distance between w and m is 34.6 map units, the
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distance between m and f is 20.6 map units and the distance between w and f is 55.2 map
units. While these distances do not ultimately correspond to the distances that were
determined in this lab they are relatively close. In the lab we determined the distance
from w to f as 88.89 map units, the distance between w and m as 45.6 map units, and the
distance between m and f as 43.3 map units. Though the distance were a little off, the
genetic order that was determined was correct. Because our distances were not exactly
the same as the established genetic map, there are a few reasons that might account for
these discrepancies. One of the main reasons that our data was skewed was because
between the F1 and the F2 generation two vials of our flies died. Because of this we did
not have as many flies for our statistical analysis and these flies could have improved our
numbers and increased or decreased the recombination values and thus changed the
calculated distances for the genetic map. Another possible source of error was that
ultimately we did not have large numbers of flies. In order for statistical analysis to be
useful, a large total needs to be used so that small changes or errors are not detected in
the overall result. A final possible source of error was that in some cases there were more
single crossovers than double crossovers, attributing tot the difference in recombination
frequency and genetic distances.
In conclusion, the main findings of this experiment were that the traits of eye
color, bristle morphology, and wing size are linked and are found on the X chromosome.
They are in the order of wmf and are a total of 55.2 map units apart, though we found
them to be 88.9 map units apart (Chromosome Mapping in Eukaryotes).
References:
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"Chapter 5 Chromosome Mapping in Eukaryotes." Chromosome Mapping. 1 Jan. 2015.
Web. 26 Mar. 2015.
NIH. "What Are the Different Ways in Which a Genetic Condition Can Be Inherited?"
Genetics Home Reference. 1 Jan. 2015. Web. 26 Mar. 2015.
O'Neil, Dennis. "Biological Basis of Heredity: Sex Linked Genes." Biological Basis of
Heredity: Sex Linked Genes. 1 Jan. 2012. Web. 26 Mar. 2015.