INTRODUCTION TO GENETICS USING TOBACCO (Nicotiana tabacum) SEEDLINGS

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1 INTRODUCTION TO GENETICS USING TOBACCO (Nicotiana tabacum) SEEDLINGS By Dr. Elaine Winshell Updated and Revised by Dr. Susan Petro 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 use the Chi-square (X 2 ) test to evaluate a hypothesis Nicotiana tabacum 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 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 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, codominance and polygenic inheritance. (Codominance will be investigated using human blood types in this lab period so it will not be considered as a possibility for the tobacco inheritance.) Your task is to decide which if any of the other three types of inheritance fits your data for Strain1 and Strain 2. You will do this by determining the expected phenotoypic 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 top of page 442. This will explain the null and alternate hypotheses and the chi square test. Procedure Approximately one week prior to the laboratory the laboratory staff will plant two black agar plates, one with seeds of the F 2 generation of Strain1 and the other with seeds of the F 2 generation of Strain 2. The plates will be incubated for a week in the light at a temperature between C. 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? 2

3 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 your lecture notes for the 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 flower example or how many colors in a case of simple dominance (one gene with two alleles) such as seen in Mendel s peas. 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 three types of inheritance? Simple dominance Incomplete dominance Polygenic inheritance 3

4 Step One Now classify the seedlings by color. You may use a dissecting microscope if you find it difficult to distinguish the seedling colors. 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 in Strains 1 and 2 e.g. dark green, green, light green, yellow, etc? 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 only two or three. You will definitely not have more than five colors. Table 1 Experimental data for F 2 offspring of Strain 1 cross Phenotype Number Total Table 2 Experimental data for F 2 offspring of Strain 2 cross Phenotype Number 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 Strain1? Which type(s) were you able to eliminate? Why were you able to eliminate them? To what type(s) of inheritance have you narrowed down your choice(s) for Strain1? Write the null hypothesis (hypotheses) for your choice(s) for each strain Null hypothesis (hypotheses) for Strain1 Null hypothesis (hypotheses) for 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 the null hypotheses be rejected or accepted for the method of inheritance of Strain 1? Why? Question 10: Based on your Chi square data above should the null hypotheses be rejected or accepted for the method of inheritance of Strain 2? Why? Last revised August 29,