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Three Point Mapping in Drosophila melanogaster
Andra Postu
February 18, 2013
TA: Dr. Walters
Lab Partner: Eli McComb
Abstract:
Over the span of five weeks, a P1 generation of true breeding mutant females and
true breeding wild type males were crossed to give rise to the F1 generation that consisted
of all mutant males and heterozygous wild type females. The F1 generation was crossed
to produce the F2 generation that consists of eight different phenotypes. In the F2
generation it was possible to observe single and double crossover events, determine the
gene order, and calculate map distance. It was determined that traits in the wmf gene
order are x-linked. The map distance was determined to be 57 m.u. The null hypothesis
was rejected because there was great deviation from the expected ratio of offspring in the
F2 generation. There was also a large discrepancy between the experimental map and
actual map at the second loci that codes for wing length. The experimental distance was
found to be 29 m.u. from the locus for eye coloration, where as in the actual map it was
found to be 36.1 map units away.
Introduction:
The phenomenon of linkage was discovered after the time of Mendel. Linkage
demonstrates that there are exceptions to Mendel’s law of independent assortment. This is
clear because when large numbers of mutant genes are examined, genes that are located
on the same chromosome show evidence of linkage. Genetic linkage occurs due to
recombination by the process of crossing-over. Crossing over occurs when homologous
chromosomes exchange genetic information. The amount of recombination is dependent
on the distance between gene loci. Therefore, frequency of crossing over is a function of
distance. The greater the distance is, the higher the frequency of recombination that is
occurring. Genes that have loci that are close to each other are not likely to be separated
on to different chromatids during cross over events that begin during the pachytene sub
stage of Prophase 1 in Meiosis. These crossover events are a form of genetic
recombination that led to a great deal of variability, far beyond that produced by random
assortment of chromosomes. By observing and then combining information about linkage
as it effects large numbers of genes, one can create linkage maps (Klug, 2011).
A genetic linkage map is a map of a species or population that demonstrates how
genes are related to each other by determining the frequency of crossover events
occurring between them. It is important to note that linage maps show the position of
known genes relative to each other. The positions are identified by recombination
frequency, not physical distances on chromosomes. Thus, linkage maps are not physical
maps; they are calculated based off of frequencies of recombination. Map distances are
relative measures of distance and 1 map unit (m.u.) is 1% recombination frequency and is
equal to 1 centiMorgan (cM). Recombination frequencies between linked genes are
additive. The probability of a single cross over event between two loci is a linear function
of the distance between them. Therefore, the smaller the physical distance between genes
on chromosomes, the lower the frequency of recombination between the two loci.
However, double crossover events also take place. During a double cross over event two
independent separate exchanges occur at the same time. Therefore, the chance that both
are occurring is the product of the individual probabilities. The frequency of double
crossovers is very low, especially in cases where the genes are located close to each other.
However, large numbers of offspring are still required to observe a double cross over
event taking place (Carlini, 2013)(Klug, 2011). The fruit fly is an ideal organism when it
comes to genetics.
Drosophila melanogaster, commonly known as the fruit fly, has been an essential
organism in the study of genes and gene interactions. Drosophila melanogaster is ideal
and flawlessly engineered for genetic experimentation because it breeds easily in the
laboratory, is small which makes it easy to raise large populations in a laboratory, has a
short life cycle of about 10 days at 25°C, and are also large enough to make the detection
of mutations rather uncomplicated (Carlini, 2013).
X-linked traits are often observed in Drosophila. X-linked recessive inheritance
occurs when a gene or trait is located on the X chromosome. Females have two X
chromosomes, where as males have one X chromosomes and a Y chromosome.
Therefore, if an affected female has a trait there is 100 % chance that she will pass the
trait to her male offspring and that he will be affected as well and show the phenotype of
the mother (Carlini, 2013) .
The lab takes place over the course of five weeks. The parental generation (P1) is
wild type recessive homozygous males x mutant homozygous recessive females at w f
and m loci. The mutant forms are white eyes (w), forked bristles (f), and miniature wings
(m). Both parents in the P1 generation are true breeding. During week 1, the adults from
the P1 cross are removed from the vials in order to prevent a backcross and thus isolates
the F1 generation that is preparing to hatch. The P1 generation is disposed of in a 95%
ethanol solution after being counted. During the second week, the heterozygous F1
generation from the P1 cross is removed and transferred to four fresh vials that contains
fly food and they are then allowed to mate in order to make the F2 generation. The goal is
to produce a large F2 population so that experimental map distance match established
map distances. During the third week, the adults from the F1 crossed in order to prevent
back cross and the F2 generation is left to develop. During the fourth week, the F2 is
allowed more time to develop before the three point crosses are counted and calculated
during the fifth week(Carlini, 2013 .
Materials & Methods:
It is essential to handle Drosophila with care because otherwise it will not be able to
successfully reproduce. In order to handle or transfer the flies, they must be handled very
gently. When moving the flies on the fly pad, they must be guided with a small camel’s
hair brush. This applies for sorting (by sex, traits, etc), and when observing them under a
microscope. In order to observe them they are anesthetized, otherwise they would escape
into the surrounding environment. If they are being counted or observed without killing,
CO2 gas is used as an anesthetic. In order to administer it properly, the gas valves are
turned to the “ON” position slightly. The flow of CO2 from the blunt needle is checked by
holding the needle 3 inches away from the cheek. The airflow should be felt only slightly.
If the flow is still felt at 6 inches from the face, the flow rate must be adjusted by turning
the valves. The vial of Drosophila should be held horizontally in one hand, with the blunt
needle in the other hand. The needle must be pushed into the side of the vial between the
cotton and side of the vial itself. After a few moments pass, the flies should be
anaesthetized and not moving. After they have fallen, the cotton ball is removed and the
flies are placed on the fly pad to be observed. The valve for the needles should be turned
“OFF”, while the valve for the fly pad should be left “ON” to prevent escape. Unwanted
flies that are being disposed of in a 95% ethanol solution should still be counted and
handled with care (Carlini, 2013).
The parental generation (P1) is mated for a week, there were three males and three
females used. The F1 generation is then mated for another week. There are two males and
three males in each of the 4 vials for the F1s. The P1 generation consisted of mutant
homozygous recessive females (genotype wfm)(white eyes, forked bristles, miniature
wings phenotype) and recessive homozygous dominant males that were wild type
(genotype +++)(red eyes, smooth curve bristles, long bristles phenotype). The F1
generation was all heterozygous because the parents were homozygous. The females are
heterozygous dominant at each loci and have a wild-type phenotype. The males are also
heterozygous, however they express mutant phenotype because they had to receive an
affected X chromosome from the mother and they got a normal Y chromosome from the
father. The F1s (2 males, 3 females in each of the four vials) were mated for a week. The
F2 generation had eight different phenotypes: +++, wfm, ++m, wf+,+fm, w++,w+m, and
+f+. It is important to note the difference between autosomal and X-linked genes.
Autosomal genes are not located on sex chromosomes; therefore they do not carry the
same impact as X-linked genes when it comes to mother-son inheritance. With X-linked
chromosomes, it must be taken into account that if the male will only receive one Xchromosome, and if the mother is affected so will the male offspring (Carlini, 2013).
Males and females are separated by distinguishing certain traits. The simplest way
to determine between a male and female is by the pattern on the abdomen of the male and
the genitalia. The male genitalia form a distinct dark ring. Males also have a sex comb
used in copulation. Females have an abdomen that is elongated at the time, where as the
males have a rounded tip. The male abdomen is solid black at the tip, where as the female
abdomen s stripped. The females also have seven segments that are clearly visible under a
microscope, where as men only have five. There are two possible phenotypes at each of
the three loci. The phenotype can either be mutant or wild type. For eye color, the mutant
eye color is white where as the wild type is red. The locus for bristle morphology is
forked in the mutant. The bristles are short in this case and has forked ends. The mutant
bristles also form more of an S curve, where as the normal bristles show a smooth curve.
The phenotype for wing size is expressed in either the mutant or wild type form. The
mutant forms have wings that are miniature and do not extent past the tip of the abdomen,
where as the wild type has normal long wings (Carlini, 2013).
Three point crosses are used to determine the loci of three genes. There are three general
rules when it comes to three point mapping in Drosophila. The genotype of the organism
producing the recombinant gametes must be heterozygous at all loci. Each phenotypic
class must also reflect the gentotypes of the parents producing it. There must also be
enough offspring generated so that the sample size is large enough to obtain all the
recombinant classes. When considering three point linkage data, the coefficient of
interference (C) and interference (I) but be taken into consideration. The predicted and
observed frequency of double crossover events often differs. The observed frequency can
be less than the expected frequency. This occurs due to interference, or a crossover in one
region of the chromosome that inhibits a second event in nearby regions. Genes that are
not located near to each experience less interference. This means that expected and
observed crossovers are equal or nearly equal. The coefficient of interference is the
observed frequency of double crossovers divided by the expected frequency of double
crossovers. The corresponding interference is then 1-C. In Drosophila, interference is
somewhere between 0 and 1(Carlini, 2013)(Klug, 2011).
Results:
The results show that there is X-linkage occurring with the traits. The original
phenotypes of the P1 generation was females that were mutant at each loci (white eyes,
forked bristles, miniature wings) and males that were true breeding wild type at all loci
(red eyes, normal bristles, normal wings). X-linkage is clear because the all of the males
in the F1 generation show the mutant phenotypes. The males received a Y chromosome
from their father, and the female parent could only offer a mutant X chromosome to the
male offspring in the F1 generation. The females in the F1 generation are heterozygous
because they received an X chromosome from each parent in the P1 generation, and are
thus showing the wild type phenotype. Table1 clearly demonstrates inheritance of the
mutant and wild type phenotype in the F1 generation.
Table 1: Phenotypic Frequency and Numbers of the F1 generation
Sex
Phenotype
Number and Frequency
Male
Mutant (white eyes, forked bristles, miniature
wings)
2x4=8 (40%)
Female Wild Type
3x4=12 (60%)
The frequency of the mutant phenotype in males is 40%, where as the wild type
phenotype in females is 60%. There are a total of 8 males and 12 females present. This
clearly demonstrates that if linkage is occurring, it X-linked. If it were not X-linked, the
males in the F1 generation would have had the wild-type phenotype according to the
regular rules of dominance. The F1s were crossed and gave rise to the F2 generation. The
F2 generation shows the phenotypes of the P1 generation, single cross over phenotypes,
and double cross over phenotypes. The largest numbers of phenotypes are the non-
crossover phenotype (mutant or wild type at every allele). The smallest number of
phenotypes is the double crossover phenotypes because they have smaller recombination
frequency. Table 2 depicts a clear image of how the F2 generation appears phenotypically,
Table 2: Phenotypic Frequencies in Males and Females of F2 Generation
Males
Group
Phenotypes
1
2
3
4
5
+++
5
8
4
8
14
wfm
2
6
2
6
27
++m
0
3
4
5
10
wf+
2
1
1
3
0
+fm
1
8
2
1
8
w++
1
0
1
4
3
w+m
2
0
2
2
8
+f+
1
3
3
2
1
Phenotypes
1
2
3
4
5
+++
3
8
12
19
10
wfm
4
2
5
0
6
++m
3
2
10
2
7
wf+
6
0
1
0
3
+fm
1
1
4
0
7
w++
2
0
3
2
8
w+m
6
5
0
11
5
+f+
3
3
5
2
3
Females
Group
The non-crossover phenotypes (+++ and wfm) are the most frequenct and
occurred 151 times in this sample size (352). There is a .43 frequency for non-crossover
phenotypes. There is a .26 frequency for the( +++) phenotype. There is a .17 frequency
for the (wfm) phenotype. There is a .13 frequency for the (++m) single crossover
phenotype. There is a .05 frequency for the (wf+) phenotype. There is a .09 frequency for
the (+fm) phenotype. There is a .11 frequency for the (w++) phenotype. There is a .12
frequency for the (w+m) frequency. There was a .07 frequency for the( +f+) phenotype.
The double cross over phenotypes are the least common and have the smallest frequency.
Therefore, the (wf+) and (+f+) are the double cross over phenotypes. The (+++) and
(wfm) phenotypes are the non-crossover phenotypes because they have the highest
frequencies.
After the frequencies are determined, χ2 are used to determine if there is linkage
occurring. If the phenotypes stray from the 1:1:1:1 expected ratio, then the genes are
linked on the X-chromosome. Table 3 clearly depicts this relationship for each individual
trait.
Table 3: χ2 Value for Individual Traits in F2
(Observed (Expected)
)
E
O
(O-E)2
(O-E)2/E
χ2
Trait 1: (wild-type vs. white eyes)
Total=352
=4.08
P<.05 Reject Null Hypothesis
+ phenotype
196
176
400
2.04
w phenotype
156
176
400
2.04
Trait 2: (wild-type vs. forked
bristles)
Total=270
=26.14
P<.05 Reject Null Hypothesis
χ2
+ phenotype
177
135
1764
13.07
f phenotype
93
135
1764
13.07
Trait 3: (wild-type vs. miniature
wings)
Total=352
=4.08
P<.05 Reject Null Hypothesis
+ phenotype
172
176
400
2.04
m phenotype
180
176
400
2.04
χ2
Each of the values is outside of the critical values is greater than what is allowed
at the .05 level of significance, therefore the null hypothesis the independent assortment
is occurring is rejected. There is linkage present, because there is a deviation from the 1:1
ratio (Total Number/2=Expected).
The value of traits is also determined for when they are assorting independently.
The expected ratio is 1:1:1:1. However, the null hypothesis is rejected once more because
the value the χ2 is 84.409, which is greater than the critical value. Therefore, the genes
are linked. It is important to note that the degrees of freedom is the amount of classes into
which data can be grouped minus one. There are 8 expected phenotypes of classes. The
observed values are very different from the expected, therefore linkage is present due to
large deviations. They are clearly depicted in Table 4:
Table 4: χ2 Values for Traits Assorting Independently in F2 Generation
Class
(Phenotype)
(Observe
d)
O
(O-E)2
(Expected)
E
(O-E)2/E
+++
91
44
2209
50.20
wfm
60
44
256
5.82
++m
46
44
4
.090
wf+
17
44
729
16.57
+fm
33
44
121
2.75
w++
38
44
36
.818
w+w
41
44
9
.201
+f+
26
44
324
7.36
Total=352
χ2 =84.409
P<.05 Reject Null
Hypothesis
Once it is clear that linkage is happening, it is possible to make a genetic map of
the X-Chromosome. The map distances are determined by calculating the number of
recombinants divided by the total number. Figure 1 demonstrates the map distance and
gene order present on the chromosome.
Figure 1:Genetic Map of X-Chromosome in Drosophila
I (X) Chromosome
I
I
I
I
(.29 m.u.)
(.28 m.u.)
w_______ m______________________ f
I
I
(.57 m.u.)
Figure 1: Legend
f locus
bristle morphology (f)
57 m.u.
w locus
eye color (w)
0 m.u.
m locus
wing length (m)
29 m.u.
The locus for bristle morphology and eye color are 57 map units apart. However,
it is important to note that interference can occur. In this situation a crossover in one
region prevents a second crossover event in a nearby region. The coefficient of
interference is used to calculate the amount of interference. It is the observed double
crossovers divided by the expected double crossovers. In this cross it is 43/44=.97. The
interference is 1-.97=.03. This means there is positive interference.
Discussion:
It was found that the three traits are X-linked. This was clear because the F1 males
had the mutant phenotype. The gene order was found to be eye coloration, wing length,
and then bristle morphology (wmf). The distance between the bristle and eye coloration
was 57 m.u. The distance between bristle and eye color was 57 m.u. The distance
between eye color and wing length was 29 m.u. The data supported the genetic map,
though it important to understand that there may be rounding error that would effect the
accuracy of the predictions. There are many errors throughout the experiment that could
have led to discrepancies between data collected and established map. Firstly, it was often
hard to distinguish or label between certain sexes and phenotypes, which may have to
lead to an inaccurate frequency for a particular phenotype or sex, which would greatly
affect the calculation for map distance. The experimental gene map was similar to the
actual gene map. The large difference was at the second loci for wing length (miniature of
normal length). Though, this may be also due to counting error. The loci for eye
coloration and bristle morphology were accurately predicted. The null hypothesis when
calculating the χ2 was rejected because the observed value deviated from the expected
ratio of 1:1:1:1, which means that independent assortment is not occurring.
Works Cited
Carlini, David. Three-Point Mapping in Drosophila Handout. American University,
2013.Print.
Carlini, David. Working with Drosophila Lab Handout. American University, 2013. Print.
Klug, William S. Concepts of Genetics. Boston: Pearson, 2011. Print.