dihybrid cross instruction PDF

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Practical 3: Drosophila Dihybrid Cross Introduction The fruit fly, Drosophila melanogaster, is an excellent organism for investigating heredity. It was first used as a model by T.H. Morgan. Drosophila are commonlyoccurring wild flies and are harmless to humans as is the medium used for their culture. Drosophila is a diploid organism with the low chromosome number of 2n=8. It has a short generation time, produces large families of offspring, is easy to handle, and has many mutant characters with which to work. The crosses require a total of 4 weeks to perform and we will only look at the F 2 generation. However, for completeness, the whole of the investigation is described in this schedule. This practical complements the lectures on Mendelian inheritance. The cross can be performed using the autosomal mutants vestigial wing (vg) and ebony body (e). The two mutants can be introduced into the cross from two parents such as by crossing vestigial wing, ebony body flies x wild type (i.e. flies with normal wings and normal body colour) or, one mutant can be introduced from each parent (vestigial wing, wild type body x wild type wing, ebony body): the latter cross is the one that has been performed. The results for these two crosses will be the same because we are dealing with unlinked genes and the F1 is simply heterozygous at both gene loci. All the F 1 flies will be wild type since both mutations are recessive. The F1 flies are self-crossed to produce the F2 generation. The F1 flies produce four types of gametes in equal frequencies by independent assortment, (1/4VG/E; 1/4VG/e; 1/4vg/E; 1/4vg/e). The gametes then combine at random in pairs to give four phenotypic classes in the F2 in a ratio of 9:3:3:1. Your actual results will differ from the theoretical ratio and it is possible to see whether your values are significantly different from the theoretical values by performing a chi-squared test, an example of which is shown in the appendix. Procedure  You are provided with vials containing the F2 generation  Take up one of the vials and etherise the flies as follows:  Dip the cotton bud provided into the ether and transfer it to the glass tube provided  Replace the funnel  Tap the side of the culture bottle or tube sharply but lightly on the bench as shown until all the flies adhering to the plug have been shaken off it. Do not knock the bottle too sharply as this may loosen the culture medium causing it to fall out when the bottle is inverted  Quickly remove the plug from the culture bottle and place the mouth of the bottle over funnel of the etheriser  Invert the culture bottle and etheriser and gently tap the culture bottle to encourage the flies to pass into the etherizer. Flies are quickly anaesthetised and are soon ready for examination.

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 If preferred, the flies may be etherised until dead.  At the end of this time, quickly remove the culture tube, plug it, then remove the funnel from the etheriser and immediately empty out the flies onto a white card.  Check the bottle for remaining flies.  Familiarise yourself with the pertinent phenotypic characteristics of the wild type and mutant flies  Using a hand lens or stereomicroscope, count the various phenotypes, after consulting the diagrams of the various markers, and incorporate the values into the table Sexing flies In mature adult flies, the main differences between the sexes are obvious, even to the naked eye. The female has a larger and more pointed abdomen than the male, particularly when she is distended with eggs. She also has several bands of dark markings on her abdomen, whereas the male has fewer bands and his abdomen is short and blunt with a much darker 'tail end'. The male is also characterized by the presence of a sex comb, consisting of about 10 dark bristles on the uppermost tarsal joint of the foreleg. This sex comb is a diagnostic feature of the male and can be used to distinguish the sexes in the first 2 hours after hatching, when the form and pigmentation of the body are not fully developed. To see the sex comb it is necessary to use a stereomicroscope or a high-power hand lens. Caution is needed in relying on the absence of the comb for identifying females (you may simply not be seeing it!), especially when these are to be used to set up a cross. For absolute certainty, students should distinguish the two sexes according to differences in their genitalia.

Size

Abdomen

Sex comb

Wild type

Ebony body

Vestigial wing

Ebony body Vestigial wing

Questions/items to include in your report 1. We could have used Parental crosses EE/VGVG x ee/vgvg to generate Ee/VGvg flies in the F1 generation. Why is it better to use the cross EE/vgvg x ee/VGVG? 2. Draw a Punnett square depicting the cross between the F1 flies – show the gametes. 3. What phenotypic ratio are you expecting to see in the F2 generation? Phenotype VG/E VG/e vg/E vg/e

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4. Use the chi-square test to determine whether this data conforms to the ratio that you are expecting from the Punnett square.

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5. If the data does not give you the expected result, give some explanations as to why this may be the case?

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