Linkage Mapping in Drosophila

Introduction

Common fruit flies, D. melanogaster, have been found as one of the most useful tools in genetic research. The reason behind that it is a popular experimental organism is due to the high likely hood of producing mutant, visible individuals. Geneticist, Thomas Morgan Hunt, was the first to discover the Drosophila as a model organism to study genetic hereditary. His research showed that the species could randomly obtain genetic mutations that could be visible in the parental generation.

Since fruit flies have a diploid chromosome number of 8 (haploid 4), chromosomal types are easily identified. Various genetic crosses are able to determine that the x-linked gene is found on chromosome 1, while the autosomal genes are located on the 2-4 chromosomes. Mapping of unknown mutations by reciprocal crosses can identify if the alleles are x-linked or autosomal and if these alleles are dominant or recessive. Once the type of allele is determined, other genetic crosses can determine the location of the chromosome that the mutagen is one. For the x-linked mutations, a three factor test cross can induce map order and distance. The physical distance separating two genes on the chromosome (map distance) is directly related the frequency of recombination between markers during meiosis. The greater the map distance of the genes, the greater the probability that there is crossing over when the chromosomes segregate in meiosis.

In this virtual experiment, six mutations (crossveinless, dumpy, ebony, eyeless, sable, & singed) were analyzed with Drosophila. Reciprocal crosses will be performed to decipher the mutant allele. Aside from x-linked mutagens which reside on chromosome 1, other crosses will be executed to discover the chromosomal number of the other mutagens. A three factor test cross will be carried out on for the x-linked alleles to conclude linkage order and map distance. This experiment will test the hypothesis: Reciprocal crosses of D. melanogaster will show that all of the x-linked mutations will not assort independently while autosomal mutations assort independently. Based on analysis of the data, it will conclude if the hypothesis can be rejected or accepted.

Methods

This experiment was performed from the website, http://biologylab.awlonline.com/. The first portion of the experiment determines what type of alleles the unknown mutations were by simple reciprocal crosses. Reciprocal crosses of mutant females and wild type males (and vice versa) are analyzed to see if the progeny of the cross showed the mutant allele as being recessive or dominant as well as autosomal or x-linked. The results of the reciprocal crosses were deciphered by following Table A.

Table A

|If the mutation is: |Offspring of the cross : |Offspring of the cross : |
| |Mutant ♀ x Wild type ♂ |Wild ♂ & Wild ♀ |
|Recessive and autosomal |Wild ♂ & Wild ♀ |Wild ♂ & Wild ♀ |
|Dominant and autosomal |Mutant ♂ & Mutant ♀ |Mutant ♂ & Mutant ♀ |
|Recessive and X-linked |Mutant ♂ & Wild ♀ |Wild ♂ & Wild ♀ |
|Dominant and X-linked |Mutant ♂ & Mutant ♀ |Wild ♂ & Mutant ♀ |

Once the mutation was typed, a three factor test cross was performed on all the x-linked mutations to determine the order and map distance of chromosome 1. In order to perform a three factor test cross, the female must be heterozygous since crossing over in fruit flies only occurs in the female species. On the other hand, the male fruit fly is recessive for all three genes. The number of offspring was recorded and the phenotypes were arranged in pairs to determine if the classes were nonrecombinant or double crossovers. Once the classes are determined, the two classes are compared to find out the gene that was switched in the double crossover. This gene was the gene in the middle for the order. The remaining genes were placed to right and left of this gene. In order to calculated map distance, coefficient of coincidence, and interference, the single crossover classes must be compared to the nonrecombinant classes to determine what gene was switched. Finally, the chromosomal number was determined for the autosomal chromosomes. Several different test crosses were done to determine if the mutation was on chromosome 2 or 3. If the chromosome did not come up positive for chromosome 2 or 3, then it will be located on chromosome 4. All x-linked mutations are found on chromosome 1. From the results of the procedures, the data was recorded and analyzed to reject or approve the above hypothesis.

Results

Table 1: Crossveinless (CV) Mutation of Drosophila

|Offspring |Mutant ♀ x Wild type ♂ |Wild type ♀ x Mutant ♂ |
|Female |Wild 4975 |Wild 4893 |
|Male |Mutant (CV) 5043 |Wild 5054 |
|Results |Recessive X-Linked Trait |

Table 2: Dumpy (DP) Mutation of Drosophila

|Offspring |Mutant ♀ x Wild type ♂ |Wild type ♀ x Mutant ♂ |
|Female |Wild 5020 |Wild 5002 |
|Male |Wild 4904 |Wild 4970 |
|Results |Recessive Autosomal Traits |

Table 3: Ebony (E) Mutation of Drosophila

|Offspring |Mutant ♀ x Wild type ♂ |Wild type ♀ x Mutant ♂ |
|Female |Wild 5005 |Wild 5034 |
|Male |Wild 5029 |Wild 4983 |
|Results |Recessive Autosomal Traits |

Table 4: Eyeless (EY) Mutation of Drosophila

|Offspring |Mutant ♀ x Wild type ♂ |Wild type ♀ x Mutant ♂ |
|Female |Wild 4985 |Wild 4971 |
|Male |Wild 4977 |Wild 5018 |
|Results |Recessive Autosomal Traits |

Table 5: Sable (S) Mutation of Drosophila

|Offspring |Mutant ♀ x Wild type ♂ |Wild type ♀ x Mutant ♂ |
|Female |Wild 4987 |Wild 5002 |
|Male |Mutant (S) 5029 |Wild 4940 |
|Results |Recessive X-Linked Trait |

Table 6: Singed (SN) Mutation of Drosophila

|Offspring |Mutant ♀ x Wild type ♂ |Wild type ♀ x Mutant ♂ |
|Female |Wild 4982 |Wild 5118 |
|Male |Mutant (CV) 5002 |Wild 4946 |
|Results |Recessive X-Linked Trait |

Table 7: Results from the Three Factor Cross of X-Linked Mutations

|Phenotype |Number |
|Wild |3762 |
|SN |63 |
|CV |311 |
|SN; CV |856 |
|S |837 |
|SN; S |286 |
|CV; S |74 |
|SN; CV; S |3882 |
|Total |10071 |

Analysis

From the results obtained from the mapping of Drosophila linkage, map distance, linkage order, coefficient of coincidence, and interference can be determined. In order to define these results a three factor test cross was performed with the x-linked mutations: crossveinless, sable, & singed.

|Phenotype |Number |
|Wild |3762 |
|SN; CV; S |3882 |
|SN |63 |
|CV; S |74 |
|SN; CV |856 |
|S |837 |
|CV |311 |
|SN; S |286 |
|Total |10071 |

Once the test was completed, analysis was performed to determine which mutations were nonrecombinant or double crossovers.

Linkage Order: CV-SN-S

To determine the linkage order, the nonrecombinant class was compared with the double crossover class to decipher the one gene that was switched in the double crossover. This gene will be the middle gene. The remaining genes were placed to the left and right of this gene. The remaining genes that were not grouped as recombinant or double crossovers are known as single crossover classes. These single crossovers were then analyzed against the nonrecombinant class to clarify which gene was switched. This analysis determines the SN-S crossovers and the CV-SN crossovers. Once the data from this inquiry was collected, calculations for map distance, double crossover frequency, coefficient of coincidence, and interference for the x-linked mutations.

|Calculation for Map Distance |
|Genetic Map = [Recombinants/(Recombinants + Parents)] * 100 |
|SN-S |[(856+837+63+74)/10071] * 100 = 18.17 |
|CV-SN |[(311+286+63+74)/10071] * 100 = 7.28 |
| |
|Calculation for Double Crossover Frequency (Expected & Obtained) |
|Expected Double Crossover Frequency |(0.1817 * 0.0728) = 0.0132 |
|Obtained Double Crossover Frequency |(63+74)/10071 = 0.0136 |
| |
|Calculation for Coefficient of Coincidence & Interference |
|Coefficient of Coincidence |0.0136/0.0132 = 1.03 |
|Interference |1-1.03 = -0.03 |

Determination of Chromosomal Location of Autosomal Genes

|Dumpy (DP x PR) |Chi Squared Value = 422.0328 df = 2 |Chromosome 2 |
|Ebony (E x RI) |Chi Squared Value = 456.17 df = 2 |Chromosome 3 |
| | | |
| | | |
| | | |
| | | |
|Eyeless (EY x AP) |Chi Squared Value = 5438.00 df = 3 |Chromosome 4 |
|Eyeless (EY x SS) |& | |
| |Chi Squared Value = 5920.00 df = 3 | |
|Sable (S) |N/A |Chromosome 1 |
|Singed (SN) |N/A |Chromosome 1 |
|Crossveinless (CV) |N/A |Chromosome 1 |

In order to reveal the chromosomal location of each mutation, additional crosses were performed. The non x-linked mutations went through various crosses to establish if they were located on chromosome 2, 3, or 4. From the results obtained from Chi-Squared analysis, the dumpy mutation was located on chromosome 2, the ebony mutation was located on chromosome 3, and the eyeless mutation was located on chromosome 4.

Conclusion

Genetic mutations are a permanent change of DNA sequencing. Sometimes mutations can have a lethal affect on the organism while other times, it has promoted evolutionary change through natural selection. In this study, D. melanogaster were crossed based on various mutations. These mutations were established in one parental sex and crossed with the wild type of the other parental sex. These genetic and reciprocal crosses helped determine if the mutations were x-linked or recessive autosomal traits. As a result of the crosses, the dumpy, ebony, and eyeless mutations were all recessive autosomal traits. On the other hand, the crossveinless, sable, and singed mutations were all x-linked since the original and reciprocal cross was unbalanced for male progeny.

Once the x-linked mutations were determined, a three factor test cross was performed to determine the order of linkage and map distance between these genes. The results of this test determined that the linkage order was CV-SN-S. The physical distance separating two genes on the chromosome (map distance) is directly related the frequency of recombination between markers during meiosis. The greater the map distance of the genes, the greater the probability that there is crossing over when the chromosomes segregate in meiosis. The map distance between SN-S was 18.17 mu and CV-SN was 7.28 mu. By calculating map distance for D. melanogaster, it narrows down the approximate location of the gene mutation.

Since all the x-linked genes are found on chromosome 1, additional crosses were performed to determine the location of the other mutations. The non x-linked mutations went through various crosses to establish if they were located on chromosome 2, 3, or 4. From the results obtained from Chi-Squared analysis, the dumpy mutation was located on chromosome 2, the ebony mutation was located on chromosome 3, and the eyeless mutation was located on chromosome 4.

In conclusion, the hypothesis established, that reciprocal crosses of D. melanogaster will show that all of the x-linked mutations will not assort independently while autosomal mutations assort independently. This hypothesis was accepted from the data obtained from the reciprocal and three factor crosses. Based on this experiment, one can determine the type of mutation based on the reciprocal crosses. If reciprocal crosses are performed where both progeny are wild, the mutation is determined to be recessive and autosomal. If reciprocal crosses are performed, where the results show a mutant male and wild female in the first cross and both wild males and females in the second cross, the mutation is said to be recessive and x-linked.

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NC

DC

SN-S crossover

CV-SN crossover