INTRODUCTION TO GENETICS USING TOBACCO (Nicotiana tabacum) SEEDLINGS

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1 INTRODUCTION TO GENETICS USING TOBACCO (Nicotiana tabacum) SEEDLINGS By Dr. Susan Petro Based on a lab by Dr. Elaine Winshell Nicotiana tabacum Objectives To apply Mendel s Law of Segregation To use Punnett squares to predict the genotypic and phenotypic outcomes of a cross To review simple dominance, incomplete dominance and polygenic inheritance To determine the method of inheritance for chlorophyll production in two strains of tobacco To learn about null and alternate hypotheses To use the Chi-square (X 2 ) test to evaluate a hypothesis Introduction The genes of most organisms exist in pairs with one gene occupying each of the homologous chromosomes (except for the genes on sex chromosomes that are not homologous). The genes may be identical or not. In fact, there may be many forms of a gene in a population. These forms are called alleles. If the organism has identical alleles of a gene on each of its homologous chromosomes, it is called homozygous; if the alleles are different it is called heterozygous. When an organism produces its gametes by meiosis, the alleles separate. This is the explanation for Mendel s First Law - the Law of Segregation. If the organism is heterozygotic, it produces two kinds of gametes; if homozygotic, it produces only one kind. The male and female gametes combine at fertilization. A random process unites the gametes in different combinations. If one gene with two alleles affects a trait, you can determine the F 2 generation of a cross between two F 1 heterozygotes by use of the Punnett square. For example, suppose you have a heterozygote with the genotype Aa and you mate it with a second heterozygote also Aa. (This is called a monohybrid cross.) The Punnett square that shows this cross is diagrammed below: female gametes Genotype Frequencies: A a AA = 0.25 male gametes A AA Aa Aa = 0.50 a Aa aa aa =

2 If you had two genes A and B each with two alleles affecting one trait (polygenic inheritance) you would use a Punnett square to do a dihybrid cross to determine the F 2 generation. See below. Male gametes AB Ab ab ab Female gametes AB Ab ab ab AABB AABb AaBB AaBb AABb AAbb AaBb Aabb AaBB AaBb aabb aabb AaBb Aabb aabb aabb If you had three genes each with two alleles affecting a trait (also polygenic inheritance) then you would do a Punnett square for a trihybrid cross with A, B and C genes. Purpose In this experiment, you will try to determine the inheritance pattern for chlorophyll production in two strains of tobacco. You have learned about a number of types of inheritance in lecture simple dominance, incomplete dominance, and polygenic inheritance. (Codominance will be investigated using human blood types in this lab period so it will not be a possibility for the tobacco inheritance.) Your task is to decide which, if any, of these types of inheritance fits your data for Strain1 and Strain 2. You will do this by determining the expected phenotypic and genotypic ratios of the F 2 generation for the possible methods and comparing them to your data using the Chi square test. Read Comparing Count Data: Dealing with Variability in your lab manual in Appendix C beginning at the bottom of the second column on page 449 and ending on page 452. This will explain the null and alternate hypotheses and the chi square test. Procedure One week prior to the laboratory each table will plant a packet of seeds of the F 2 generation of Strain1 on a black agar plate. The seed packets are labeled Strain1. Sprinkle the seeds carefully so you get an even distribution over the entire plate. Each table will also plant on another black agar plate a packet of seeds of the F 2 generation of Strain 2. These seed packets are labeled Strain 2. Label your plates with your lab class and table number (e.g. Tuesday PM Table 3) and which seeds are in each plate Strain1 cross or Strain 2 cross. The laboratory staff will germinate them for a week in the light at a temperature between C. If the week preceding this lab is spring break then the laboratory staff will plant the tobacco seeds for you. 2

3 The answers to the questions below are to be typed including the Punnett squares and all tables. Handwritten work will not be accepted. Question 1: What would your null and alternate hypotheses be for each of the three possible types of inheritance for the two strains of tobacco? Question 2: In order to make predictions based on your hypotheses you must determine the expected phenotypic ratios of offspring in the F 2 generation. In order to do this begin by doing a Punnett square for each of the three types of inheritance. You may choose whatever letters of the alphabet you like for the alleles. See lecture notes for conventional way to write these i.e. when does one use capital letters and when does one use lower case letters. Question 3: After you have completed the three F 2 Punnett squares above use the examples given in lecture to help you determine what might be expected (e.g. how many possible colors for polygenic inheritance involving two genes each with two alleles like the wheat grain color example or how many possible colors in a case of incomplete dominance (one gene with two alleles) like the four o clock (Mirabilis jalapa) example or how many colors in a case of simple dominance (one gene with two alleles) such as with Mendel s pea blossoms. Number of colors (not the names of the colors) expected in: Simple dominance Incomplete dominance Polygenic inheritance Question 4: Now what would the expected ratios (again the numbers not the names) of the colors be in the F 2 generation of these types of inheritance? Simple dominance Incomplete dominance Polygenic inheritance 3

4 Step One After making your null and alternate hypotheses and predictions for F 2 generations for each of the three types of inheritance above you will use the dissecting microscope to classify the seedlings by color. Question 5 - How many colors of seedlings do you see in Strain 1? How many colors in Strain 2? Question 6 - What terms would you use to describe the colors that you see e.g. dark green, yellow, etc? In Strain #1? In Strain #2? Step Two Once you have reached a consensus at your table list your colors in the table below and then remove each seedling with a forceps as it is counted to prevent counting the same seedling twice. Enter the experimental data in the tables below. Results There are five rows in the tables below, but you may not have observed five different colors, you may find you have observed only two or three. You will definitely not have observed more than five colors. Table 1 Experimental data for F 2 offspring of Strain 1 cross Phenotype Number observed Total Table 2 Experimental data for F 2 offspring of Strain 2 cross Phenotype Number observed Total 4

5 Question 7: Based on the number of colors you found can you eliminate any of the three possible types of inheritance for Strain 1? Which type(s) were you able to eliminate? What about Strain 2? To what type(s) of inheritance have you narrowed down your choice(s) for Strain 1? Strain 2? Write the null hypothesis (hypotheses) for your choice(s) for each strain Null hypothesis (hypotheses) for Strain 1 Null hypothesis (hypotheses) Strain 2 Question 8: For the null hypothesis (hypotheses) you just made for Strain 1 and Strain 2 what number of each phenotype should you have expected to get based on the total number of seedlings you counted using the ratios you determined with your Punnett squares? Strain 1 Strain 2 Use these answers to go ahead with the calculation of the Chi-square test of your results. 5

6 CALCULATION OF CHI-SQUARE (χ 2 ) Strain 1 Cross - Table data Phenotype Observed (o) Expected (e) (o-e) (o-e) 2 (o-e) 2 e Total = χ 2 Degrees of Freedom - Confidence level - Critical Value - Strain 2 Cross - Table data Phenotype Observed (o) Expected (e) (o-e) (o-e) 2 (o-e) 2 e Total = χ 2 Degrees of Freedom - Confidence Level - Critical Value - Question 9: Based on your Chi square data above should your final null hypothesis be rejected or accepted for the method of inheritance of Strain 1? Why? Question 10: Based on your Chi square data above should your final null hypothesis be rejected or accepted for the method of inheritance of Strain 2? Why? Last revised April 8,

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