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Lab Report-The Study of a Dihybrid Cross in the Drosophilia melanogaster

Updated on April 15, 2016

Abstract

Transmission genetics, or Mendelian genetics, looks at the transmission of genes and traits through successive generations. The transmission follows two laws, namely the laws of segregation and independent assortment. This experiment aims to investigate recessive alleles, and the law of independent assortment. We set out to determine if the 9:3:3:1 ratio was applicable to the fruit fly. A dihybrid cross was carried out between the Dump Sepia Mutant male and the wild type female Drosophilia melanogaster also known as the fruit fly. The progeny was then crossed an additional time to form the F2 generation thereby creating a circumstance wherein we could test for Mendel’s Law of Independent Assortment. After five weeks of study, the results were compared to the expected results based on Mendel’s laws. Based on the chi square results, Mendel’s law of Independent Assortment is not applicable to the fruit fly of the dumpy sepia mutant.

Introduction

Transmission genetics is the study of how genes and traits are transmitted from generation to generation. It provides scientifically accepted theories of the inheritance of genes by offspring, and how specific inheritance patterns determine the genotypes and phenotypes of individuals. These combine in the offspring to produce the phenotypes displayed by the offspring. Alleles are inherited according to a set of rules, whereby it is possible to predict the frequency of a certain trait occurring within a population.

Transmission genetics may be termed Mendelian genetics, as a monk by the name Gregor Mendel was the first to propose the modern theory of inheritance. Mendel carried out crosses on the Pisum sativum or garden pea plant. Based on his results, he formulated a theory regarding the transmission of traits through generations. Mendel outlined two laws as the foundation of his theory; the Law of Segregation and the Law of Independent assortment. The Law of Segregation states that each organism inherits one allele of each gene from each parent. The Law of Independent Assortment states that these alleles segregate independently of one another.

This experiment uses a dihybrid cross in order to show that the heterozygotes are not differentiable from homozygotes of the dominant genotype. This experiment also shows the Law of Independent Assortment, as two traits are being analysed. It is possible to see how these traints are inherited independently of each other. The organism used is the Drosophilia melanogaster, commonly known as the fruit fly. It meets all the necessary requirements for experimentation; as it has a short generation time of 10 days, it produces large amounts of offspring at a time, thereby providing a large sample for analysis, it thrives under laboratory conditions and the genetic mutations are easily visible in phenotypic make up.

Methods and Materials

The experiment was carried out over a 5 week period, the weekly steps taken are outlined here under.

Week 1: Female virgin flies were used for this cross, since the female fruit fly has the ability to store sperm and use it later. Male and female flies were transferred into a large culture bottle, containing a culture medium of agar, mealie meal, syrup, molasses, water and live yeast, in order to support the growth of the flies and their offspring. The culture bottle was then incubated, at room temperature, for one week in dim light conditions

Week 2: an etherising jar was prepared by moistening the lid of a jar with ether. Adult flies were discarded from the culture bottle by shaking them from the culture bottle into the etherising jar. In this way, adult flies were prevented from mating with their larval offspring, after hatching. The culture bottle was then incubated, at room temperature, for one week in dim light conditions (6).

Week 3: Hatched flies in the culture bottle were etherised, then removed from the culture bottle. Ensuring they were still alive, 2 males and 5 females were picked. These flies were returned to the culture bottle, which was incubated, at room temperature, for one week in dim light conditions. An etherising jar was prepared by moistening the lid of a jar with ether. All flies (other than selected ones) were discarded by etherisation.

Week 4: The same procedure as week 2 is followed, in order to prevent cross generation mating.

Week 5: An etherising jar was prepared once more. All the flies were transferred to this jar. Once the flies stopped moving, they were removed from the jar. Each fly was classified phenotypically, and the numbers of each phenotype variant were counted. All the flies are then discarded of, by placing them back in the etherising jar.

Results

P1: Wild type female x Dumpy sepia male

dp+/dp+ ; se+/se+ x dp/dp ; se/se


dp/se

dp/se

dp+/se+

dp+/dp; se+/se

dp+/dp; se+/se

dp+/se+

dp+/dp; se+/se

dp+/dp; se+/se

F1: dp+/dp; se+/se

P2: Female x Male

dp+/dp; se+/se x dp+/dp; se+/se

Gametes: dp+/se+ ; dp+/se ; dp/se+ ; dp/se

dp+/se+

dp+/se

dp/se+

dp/se

dp+/se+

dp+/dp+; se+/se+

dp+/ dp+ ; se+/ se

dp+/dp ; se+/ se+

sp+/dp ; se+/ se

dp+/se

dp+/dp+; se+/se

dp+/ dp+ ; se/ se

dp+/dp ; se+/ se

dp+/dp ; se/ se

dp/se+

dp+/dp ; se+/ se+

dp+/dp ; se+/ se

dp/dp ; se+/ se+

dp/dp ; se+/ se

dp/se

dp+/dp ; se+/se

dp+/dp ; se/ se

dp/dp ; se+/ se

dp/dp ; se/se

F2: 9 normal wings, red eyes : 3 normal wings, black brown eyes: 3 short wings, red eyes: 1 short wings, black brown eyes

Chi-Square Test for group results

  1. H0 = The cross between the drosophila flies followed Mendel’s law of independent assortment ratio of 9:3:3:1

H1= The cross between the drosophila flies did not follow Mendel’s law of independent assortment ration of 9:3:3:1

  1. Reject H0 if x2calc > x2(3;0.05) = 7.81

i.) Phenotypes

ii.) Observed

iii.) Expected

iv.) (O-E)2/E

i.) Wild Type

ii.) 254

iii.) 282.375

iv.) 2.851

i.) Dumpy Mutant

ii.) 110

iii.) 94.125

iv.) 2.678

i.) Sepia Mutant

ii.) 94

iii.) 94.125

iv.) 0.00017

i.) Dumpy Sepia

ii.) 44

iii.) 31.375

iv.)4.726

i.) Total

ii.) 502

iii.) 502

iv.) 10.26

X2cal = 10.26

  1. Since X2cal > 7.81, we reject H0
  2. Therefore, it can be concluded that the cross between the drosophila flies did not follow Mendel’s law of independent assortment ratio of 9:3:3:1 at a 5% level of significance.

Chi-Square test for class results

  1. H0 = The cross between the drosophila flies followed Mendel’s law of independent assortment ratio of 9:3:3:1

H1= The cross between the drosophila flies did not follow Mendel’s law of independent assortment ration of 9:3:3:1

  1. Reject H0 if x2calc > x2(3;0.05) = 7.81

i.) Phenotypes

ii.) Observed

iii.) Expected

iv.) (O-E)2/E

i.) Wild Type

ii.)863

iii.) 958.5

iv.) 9.52

i.) Dumpy Mutant

ii.) 410

iii.) 319.5

iv.) 25.63

i.) Sepia Mutant

ii.) 287

iii.) 319.5

iv.) 3.31

i.) Dumpy Sepia

ii.) 144

iii.) 106.5

iv.) 13.20

i.) Total

ii.) 1704

iii.) 1704

iv.) 51.66

X2calc = 51.66

  1. Since X2cal > 7.81, we fail to reject H0
  2. Therefore, it can be concluded that the cross between the drosophila flies does not follow Mendel’s law of independent assortment ratio of 9:3:3:1 at a 5% level of significance.

Discussion

As confirmed by the experiment, the dumpy sepia (double mutant) alleles are recessive, due to the observation that they are only expressed in the homozygous form

As anticipated, one phenotype was observed in the F1 generation, while a variety of phenotypes were observed in the F2 generation. This corresponds with the predictions made in the Punnet squares. As anticipated, one phenotype was observed in the F1 generation, while a variety of phenotypes were observed in the F2 generation. This corresponds with the predictions made in the Punnet squares. The F1 generation was Dumpy mutant with truncated wings, throughout. The F2 generation had some flies showing the wild phenotype of normal wings and red eyes, some flies showed the mutant phenotype of truncated wings and red eyes, some displayed normal wings with sepia type eyes, and some had a mixture of the two phenotypes.

In this experiment, dumpy sepia male flies were crossed with wild type female flies. Since the Chi-square test proved that the cross did not in fact follow the 9:3:3:1 ratio, the cross contradicted Mendel’s law of independent assortment. Overall, the general trend should have obeyed Mendel’s Law of Independent Assortment but there could have been a number of factors as to why this did not happen. These factors include human errors, errors in counting and calculations, errors in the lab, or possible contamination. Other factors include incorrect breeding conditions, failing to securely shut the culture bottle, over etherising to the point of death. Alternatively, our results were correct and the dumpy sepia alleles do not in fact obey Mendel’s Law of Independent Assortment. Based on the chi- squared test, the null hypothesis was rejected. Thus, there is contradiction with Mendel’s laws and one could further improve the ratio, and attain correct results, by repeating these steps with an even greater sample size, more accuracy and less errors.

Using a bigger sample, grouping the data obtained from the same experiment performed by different groups. Overall, we see that the trend is now even further from the ratio of 9:3:3:1. This could be more accurate if one was to increase the sample size and repeating the experiment.

Based on the chi-squared test, the null hypothesis is now rejected, there is a significant difference between the observed and expected numbers of flies of each phenotype. The results prove to be contradictory to Mendel’s laws. This could be due to a variety of factors, including but not limited to, incorrect breeding conditions, failing to securely shut the culture bottle, over etherising to the point of death.

In conclusion, recessive alleles were observed in this experiment, as the heterozygote alleles were indistinguishable from the homozygote. Both genotypes were expressed as the wild type, phenotypically. It must also be concluded that the Drosophilia melanogaster does not follow Mendel’s Law of independent assortment, as the observed ratio greatly differed to 9:3:3:1 ratio, which is in contradiction with this law.

References:

Campbell, N., Reece, J. B., Urry, L.A., Cain, M. L., Wasserman, S. A., Minorsky, P. V. and Jackson, R. B. (2011) Campbell Biology(9), Pearson

Williams, J. A., Scott, I. M., Atkin,A. L., Brook, W. J., Russel, M. A. and Bell, J. B. (1990) Genetic and molecular analysis of vgU and vgW: two dominant vg alleles assosciated with gene fusions in Drosophilia, Genetics 125(4), 833-844

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