AP Lab #7 - Mendelian Corn Genetics Introduction A gene is a unit of heredity on a chromosome and can have alternate forms called alleles. Alleles for a particular gene occur in pairs and in sexually reproducing organisms each parent contributes one allele to their offspring thus ensuring generic variability. Alleles however can be expressed as dominant, and are usually designated by a capital letter (for example, "B"). Alleles whose expression is masked by dominant alleles are recessive, and are designated by a lower case letter (for example, "b"). The genotype of an organism whether dominant (BB) hybrid/heterozygous (Bb) or recessive (bb) includes all the alleles present in the cell; and the manifestation for that particular trait is called the phenotype. Corn is an excellent choice for Mendelian inheritance studies because kernel color and texture is controlled by a single pair of alleles and the gene for blue and smooth kernels is dominant over the yellow and wrinkled phenotype. In this exercise, you will examine an ear of corn and determine the type of cross and genes responsible for the coloration and texture of the corn kernels like the one show below. There are four grain phenotypes in the ear. Purple and smooth (A), Purple and wrinkled (B), Yellow and Smooth (C), Yellow and Wrinkled (D); two conditions will be examined: A monohybrid cross and a dihybrid cross. Monohybrid Cross of Corn - A monohybrid cross begins with experimental breeding between two parents that breed true for different forms of a single trait. The trait you will investigate in this problem is kernel color. The two forms of kernel color we will look at are Purple (RR) and Yellow (rr). You will also be looking at kernel texture; smooth (DD) and wrinkled (dd) Dihybrid Cross of Corn - A dihybrid cross begins with experimental breeding between two parents that breed true for two different traits. The trait you will investigate in this problem is kernel color and texture. The two forms of kernel color we will look at are Purple wrinkled (RRDD), purple smooth (RRdd), yellow wrinkled (and Yellow, smooth and wrinkled. We start with a plant homozygous for purple kernels and cross it with a plant homozygous for yellow kernels. The offspring that result from this cross are called hybrids and are the F1 generation. When two individuals from the F1 generation are crossed the offspring is called the F2 generation. Background In these activities, you will investigate several phenotypes of corn that are expressed in the seed. You will be given ears of corn for your investigation. Each seed on an ear results from a separate fertilization event. Further, the seeds on the ear are the F2 from a cross that began with two parental varieties of corn with contrasting phenotypes. Materials: • F1 generation Ear of corn (purple: yellow) • F1 generation Ear of corn (smooth: wrinkled) • F2 generation Ear of corn (purple: yellow, smooth: wrinkled) • • • Corn Parental Cross Card A Corn Parental Cross Card B Transparency marker Exercise A: The parental (P) and first filial (F1) generation Your group will be given two Corn Parental Cross Cards, A and B, that show a cross of two parental varieties of corn with contrasting phenotypes (as explained in the Introduction) as well as the F1 corn resulting from the cross. Two of you will 1 work with the cross shown on card A and two will work with the cross shown on card B. All the P corn varieties are homozygous for all the genotypes you will be studying. Examine the cross that is shown on your Corn Parental Cross Card. Collect information and complete steps 1-5 below. 1. Will you treat this as a monohybrid cross or a dihybrid cross? Explain your answer. _______________________________________________________________________________________________ _______________________________________________________________________________________________ 2. Provide on a one-word description of the phenotype of each of your P corn varieties (you may use the ones provided in the introduction section). Record the descriptions in the blanks below. Phenotype of A-1 ___________________________ B-1 - ___________________________ Phenotype of A-2 ___________________________ B-2 - ___________________________ 3. Provide the symbols to represent the alleles by which these phenotypes are inherited (you may use the ones provided in the introduction section). Record the symbols in the blanks below. Genotype of A-1 ___________________________ B-1 - ___________________________ Genotype of A-2 ___________________________ B-2 - ___________________________ 4. Which allele is dominant? Explain how you know. _______________________________________________________________________________________________ _______________________________________________________________________________________________ Exercise B: F1 and F2 Cross Using the Punnett square below and information you recorded in Exercise A along with your knowledge of genetics; record the results of the crossing between the F1 generation individuals from card “A” Genotypes and expected ratios for the F2: Below the Punnett square, give the phenotypes and their expected ratios for the F2 in card “A” Phenotype Ratio __________________________ __________________________ __________________________ __________________________ __________________________ __________________________ Once again record the results of the crossing between the F1 generation individuals from card “B” Genotypes and expected ratios for the F2: 2 Below the Punnett square, give the phenotypes and their expected ratios for the F2 in card “B” Phenotype __________________________ __________________________ __________________________ Ratio __________________________ __________________________ __________________________ 5. What is the genotype of the F1? _______________________________________________________________________________________________ _______________________________________________________________________________________________ To simulate the F2 generation; obtain ear of corn with the two contrasting phenotypes, purple: yellow and smooth: wrinkled. Working in pairs; count and record in Table 1 and 2 (below) the number of grains of each phenotype; one person should call out the phenotypes while the other records them in the table. To make the count, begin with one row of kernels and using a transparency marker lightly mark the kernel while recording the appropriate phenotype of each kernel in that row; for ease, place “tic” marks in the wide space in the table below. Continue counting to ensure that you count for at least five rows. When finished counting, total your results by providing the average from at least three group members. Table 1: Observed phenotype count for F2 in card “A” PURPLE Phenotypes: YELLOW Team Count Total: Total: Total for all the phenotypes counted: Table 2: Observed phenotype count for F2 in card “B” Phenotypes: SMOOTH WRINKLED Team Count Total: Total: Total for all the phenotypes counted: 6. What evidence do you have that the traits you are investigating are actually following Mendelian genetics expectations? Could they simply be the result of environmental factors? ___________________________________________________________________________________________ ____________________________________________________________________________________ ____________________________________________________________________________________ 3 7. If the phenotypes are under genetic control, are they inherited through a single gene, a few genes, or many genes? How do you know? ___________________________________________________________________________________________ ____________________________________________________________________________________ 8. Using the data from Table 1 & 2 and information from your Punnett square, calculate the expected counts for the F2 and record them below. Table 1 Phenotype _______________ Expected count _______________ Phenotype _______________ Expected count _______________ Table 2 Phenotype _______________ Expected count _______________ Phenotype _______________ Expected count _______________ Are the deviations from the expected results for the phenotypic ratio of the F2 generation within the limits expected by chance? To answer this question, statistically analyze the data using Chi-square analysis. Refer to the Chi- Square (x2) Test section on Table 4 - Chi-Square Values and Probabilities. In the space below, calculate the Chi-square statistic for the F2 generation (use it as an example for the rest of the calculations for this lab). Refer to Table 4 to determine the probability that is associated with your x2 statistic. X2 = Ʃ Table 1 Kernel Purple Yellow Totals 9. Chi Square Value = 10. Degrees of Freedom = Total Observed (O) X2 = Ʃ 12. Chi Square Value = 13. Degrees of Freedom = Total Expected (E) (O-E)2/E _______ _______ 11. Were the results obtained by chance alone? Table 2 Kernel Purple Yellow Totals (𝐸𝐸𝐸𝐸𝐸𝐸𝐸𝐸 −𝑂𝑂𝑂𝑂𝑂𝑂𝑂𝑂)2 𝐸𝐸𝐸𝐸𝐸𝐸𝐸𝐸 Total Observed (O) Yes No (𝐸𝐸𝐸𝐸𝐸𝐸𝐸𝐸 −𝑂𝑂𝑂𝑂𝑂𝑂𝑂𝑂)2 𝐸𝐸𝐸𝐸𝐸𝐸𝐸𝐸 Total Expected (E) _______ _______ 14. Were the results obtained by chance alone? Yes 4 No (O-E)2/E Exercise C: A Different Cross As a team, look again at the Corn Parental Cross Cards, “A” and “B”. Remember that all of the P seeds are homozygous. Suppose you performed a new cross using, as your P corn varieties A-1 and B-1 only; working as a team, collect the following information about this cross as you complete the steps below. 15. Give the genotypes and phenotypes for the P and F1 of this cross. Parental A-1 Genotype B-1 Genotype x Phenotype Phenotype F1 Genotype Phenotype 16. In the space below, construct a Punnett square showing the results of crossing your F1 to obtain an F2. Below the Punnett square, give the phenotypes and their expected ratios for the F2 of this cross. 17. Phenotypes and expected ratios for the F2: Phenotype Ratio _________________ __________________ _________________ __________________ Phenotype __________________ __________________ Ratio __________________ __________________ Obtain an F2 ear of corn with the purple: yellow smooth: wrinkled phenotypes; for this crossing exercise; count and record phenotypes in Table 3 below (for this exercises you must count the entire ear of corn). Use the wide space in the table to make “tic” marks and provide the average from at least three group members. 18. Table 3: F2 Phenotype Count for Purple/smooth and Yellow/wrinkled Phenotypes Purple/Smooth Purple/Wrinkled Yellow/Smooth Yellow/Wrinkled Team Count Total: Total: Total for all phenotypes counted: Total: 5 Total: 19. Calculate the expected counts for the F2 and record them below. Kernel Total Observed (O) Total Expected (E) Purple Yellow Sweet Starchy Totals (O-E)2/E 20. x2 = ____________ 21. Were the results obtained by chance alone? Yes No 22. On the basis of the data recorded in Table 3, what new inference can you make about the inheritance patterns of the phenotypes? (Are they linked? Why or why not?) __________________________________________________________________________________________________ __________________________________________________________________________________________________ Table 4 - Chi-Square Values and Probabilities (5% or Less is Considered Significant) In this table, note the column titled, "Degrees of Freedom." The degree of freedom is always one less than the number of different phenotypes possible. For the monohybrid F1 in this experiment we have two possible phenotypes so there are 2 - 1 = 1 degree of freedom. For a dihybrid F2, there are four possible phenotype combinations and 3 degrees of freedom. The numbers to the right of the Degrees of Freedom column in the table are X2 values. Degrees of Freedom 1 2 3 4 5 6 7 8 9 10 p=99% 95% 80% 50% 30% 20% 10% 5% 1% 0.000157 0.02 0.11 0.29 0.55 0.87 1.23 1.64 2.08 2.55 0.00393 0.10 0.35 0.71 1.14 1.63 2.16 2.73 3.32 3.94 0.064 0.44 1.01 1.64 2.34 3.07 3.82 4.59 5.38 6.17 0.45 1.38 2.36 3.35 4.35 5.34 6.34 7.34 8.34 9.34 1.07 2.41 3.66 4.88 6.06 7.23 8.38 9.52 10.6 11.7 1 .64 3.21 4.64 5.98 7.28 8.55 9.80 11.1 12.2 13.4 2.71 4.60 6.25 7.78 9.24 10.6 12.0 13.4 14.7 15.9 3.84 5.99 7.81 9.48 11.1 12.6 14.1 15.5 16.9 18.3 6.63 9.21 11.3 13.2 15.0 16.8 18.5 20.1 21.6 23.2 Results obtained not by chance (reject null hypothesis) By chance The percentages given at the top of each column represent the probability that the variation of the observed results from the expected results is due to chance. If the probability value is greater than 5%, we accept the null hypothesis; that is, our data was obtained by chance alone and did not meet or provide evidence of Mendel’s laws of probabilities. Example: Image that Chi-Square X2 = 2.43 and from the table for 3 degrees of freedom; we can see that 2.43 is between 30% and 50%. By statistical convention, we use the 0.05 probability level as our critical value. If the calculated chi-square value is less than the 0.05 value, we accept the null hypothesis (that our results were obtained by chance). If the value is greater than the critical value, we reject the null hypothesis. Therefore, because the calculated chi-square value is greater than the critical value of 5%, we accept that the data fits Mendel’s dihybrid 9:3:3:1 ratio. 6
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