INVESTIGATION 1 - DIHYBRID CROSS IN DROSOPHILA

Transcription

1 INVESTIGATION 1 - DIHYBRID CROSS IN DROSOPHILA Examination of the phenotypic ratio produced from a dihybrid cross involving two mutant phenotypes is the classic means by which the linkage relationships between gene loci is determined. In a cross involving two heterozygous loci, each with complete dominance, the expected phenotypic ratio for unlinked traits that show independent assortment is the classic 9:3:3:1 ratio. In the case of genes that are physically linked on the same chromosome, two loci that are closely linked on the same chromosome will tend to segregate together and distort the expected ratio. Further, in many species, the male and female members have characteristic sex chromosomes, and gene loci that are located on these chromosomes will show differ patterns of segregation in the two genders. You will be provided with either data or culture bottles containing the F 2 generation of two dihybrid Drosophila crosses involving flies with two distinctive mutant phenotypes (e.g., white eyes and/or vestigial wings). Your task is to determine the linkage relationships of the genes responsible for the phenotypes: they may be independently assorting, or they may be linked on autosomal chromosomes, or they may be sex-linked. The F 2 generations you will observe are the result of the parental (P 1 ) cross (Cross A) between: 1( White eyes) X 2 (Vestigial wings) and the reciprocal P 1 cross (Cross B) between: 2 (Vestigial Wings) X 1(White eyes) The two mutant phenotypes you are looking at therefore are: Mutant phenotype 1- White eyes and Mutant 2 - small deformed wings (Vestigial) OBSERVATIONS: Write a description of phenotypes 1 and 2; draw these phenotypes showing or explaining what a normal wing/eye would look like. Count the number of males and females separately to see if the trait is expressed in equalling in both sexes. (Note: The number of males and females may not be equal as females tend to hatch earlier) Count the number of flies with mutant phenotypes 1 & 2, only phenotype 1, only phenotype 2, and neither mutant phenotype (wild). Do not include flies for which you have a problem making an identification:- e.g. body colour - newly hatched flies (identified by an elongated abdomen) will all appear pale as they have not yet developed body pigment. e.g. wing size - newly hatched flies may have wings still folded, so wings appear smaller, darker and irregular in size or shape. Complete Table 2 and add your results to the class list on the board. For this assignment, use the class results. Hint: Fill in tables 3A and B. This will give clues as to whether the mutant genes are on 1

2 the autosomes or sex chromosomes and will help you make a hypothesis. 2

3 REPORT: The results of the experiment will be described in a formal laboratory report. The report should be logically organized, concise and clear. Use the third person, and past tense: The phenotypic ratio was found to be 3:1" not I found a phenotypic ratio of 3:1" or The phenotypic ratio is 3:1". Include the following information, in the following order: PURPOSE: State the purpose of the experiment, which will include observation of the F 2 progeny from a cross of Drosophila involving two mutations. HYPOTHESIS: Draw up a Punnet square of the expected phenotype ratios of the reciprocal crosses in Table 1: Cross A and Cross B. What is your expectation for the linkage relationships between the markers used? Do you expect these two genes to assort independently? Do you think these genes are on the autosomes or sex chromosomes? Having made some assumptions, you will then test whether these are true or not later on. Define the notation you use for each phenotype (see Appendix B), i.e. vg = vestigial wings and the wild type notation = vg +. Also use Appendix B to determine on which chromosomes the loci are located so you can use the correct notation if any are sex-linked. METHODS & MATERIALS : Reference to the specific pages in the lab manual is sufficient. Any changes you made to the given procedure should be described. RESULTS & OBSERVATIONS : Use the past tense: describe what happened during the experiment. 1. Table 2. Results of the dihybrid cross. 2. Description of mutations. Sketches of mutations. 3. Table 3 A and B for the reciprocal crosses to determine the expected F 2 phenotype ratios of the two mutations separately. 4. Based on the pattern of inheritance shown in the results determine if the 2 genes are autosomal or sex-linked; dominant or recessive and independent or linked. 5. Perform Chi Square Tests (Table 4 A and B) for both crosses using class results to test whether the observed results could be the result of a cross between the two mutations you selected, given your hypothesis. DISCUSSION: You should address the following questions in your discussion. Please keep your discussion brief, with only one or two sentences per question. 1. Make a brief statement of the observed results (i.e. ratios) from Crosses A & B. 2. Explain how the results show if the mutations are dominant or recessive. 3. What evidence is there that the two genes involved were independently assorted or linked? 4. Were they autosomal or sex-linked? How do you determine if the mutations were autosomal or sex-linked? Why were reciprocal crosses were performed? What did the results of these crosses tell you? 5. Why was a Chi-Square Test performed? Discuss how well the observed results of the cross of the two mutations fit the expected result from your choice of genes. 6. Are there any sources of error in these experiments? If data was given to you instead of you having to count actual flies, speculate what some sources of error may have been if you had to count the flies. Would these explain any discrepancies or unexpected results? 3

4 Hypothesis: Table 1: Expected Results of Cross A Cross Diagram - Dihybrid P 1 P 1 Genotype* P 1 Gametes produced F 1 F 1 Genotype F 1 Gametes produced * if any of the genes are believed to be sex-linked, use the proper notation when writing the genotype Punnet Square for F 2 using the F1 gametes Expected F 2 phenotype ratio including and excluding sex: (Remember to write in the phenotype (what it looked like) and the number 4

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6 Hypothesis (continued): Table 1: Expected Results of Cross B Cross diagram - Dihybrid P 1 P 1 Genotype* P 1 Gametes produced F 1 F 1 Genotype F 1 Gametes produced * if any of the genes are believed to be sex-linked, use the proper notation when writing the genotype Punnet Square for F 2 using the F1 gametes Expected F 2 phenotype ratio including and excluding sex: 6

7 Table 2: Observations from a dihybrid cross involving two unknown genes. P 1 s Cross A Cross B F 1 s Lab Slot Class # Results Class # Results F 2 s Total Total Total ratio (round to 1 decimal place, don t forget to write phenotypes with numbers) 7

8 Table 3 A: Observed results from examination of 1 Cross A Cross B F 2 No. of No. of Total Ratio No. of No. of Total Ratio Wild Type Total Table 3B: Observed Results from examination of 2 Cross A Cross B F 2 No. of No. of Total Ratio No. of No. of Total Ratio Wild Type Total 8

9 Table 4A: Chi-Square Test for Independent Assortment of the F 2 class results of a cross between the parents P1, a male and a female. Note: round your numbers to 3 decimal places only at the end of your calculations s Observed # of F 2 (O) Expected # (E) Deviation (O-E) (O-E) 2 (O-E) 2 /E (3 d.f.) wild type 1 2 s 1 & 2 Total X 2 = (3 d.f.) Interpretation: i.e. Do you reject or accept your original hypothesis? Why? 9

10 Table 4B: Chi-Square Test for Independent Assortment of the F 2 class results of a cross between the parents P1, a male and a female. s Observed # of F 2 (O) Expected # (E) Deviation (O-E) (O-E) 2 (O-E) 2 /E (3 d.f.) wild type 1 2 s 1 & 2 Total X 2 = (3 d.f.) Interpretation: i.e. Do you reject or accept your original hypothesis? Why? 10