Laboratory 9 Mendelian Genetics
Biology 171L FA17 Lab 9: Mendelian Genetics Student Learning Outcomes 1. Predict the phenotypic and genotypic ratios of a monohybrid cross. 2. Determine whether a gene is autosomal or sex-linked, and whether the mutant allele is dominant or recessive Relevant Readings 1. Read Chapters 14 and 15 in Campbell Lab Preparation 1. Read and Print the Lab Manual 2. Print and bring to class the Star Genetics Tutorial, posted on Laulima. 3. Print and bring to class the Fruit fly Exercise 1-1, posted on Laulima. Homework Synopsis (see page 9-14 for full description) Part I Mastering Biology Part II Science Communication Second first draft of photosynthesis paper Part III Data Analysis Star Genetics Fruit Fly Exercise 2-1 INTRODUCTION The concept of Mendelian inheritance includes a set of principles describing how genes are passed from one generation to the next, through gametes, from biological parents to their offspring. Understanding how this happens is important because these genes either directly determine, or at least greatly influence, every aspect of an individual s physical makeup and/or their behavior. The exercises in this lab exemplify some of the classic areas of genetic research that led to an understanding of the principles governing the inheritance of specific traits. Initial experiments were concerned with the transmission of hereditary factors from generation to generation and led to the discovery of Mendel s laws, which define the pattern of inheritance of individual genes. Later experiments identified chromosomes as the physical structures where units of heredity reside and provided firm cytological evidence for the theorems of Mendelian genetics. More recent investigations have addressed the biochemical and molecular basis of gene expression. Modern genetics has as its basis the experimental evidence established by Gregor Mendel in the 1860s. Although Mendel s principles do not explain all of heredity, these basic rules do apply to most genes. In addition, the basic concepts of Mendelian inheritance apply to genes in a wide range of sexually reproducing organisms including humans, plants and insects. Biol 171L -FA17 Mendelian Genetics 9-2
MENDELIAN PRINCIPLES Mendel s first principle is concerned with patterns of inheritance involving one gene (monohybrid cross). The second principle deals with the situation where you want to follow the inheritance of more than one gene (e.g., dihybrid cross). The first Mendelian principle now called the principle of segregation - can be used to make predictions about how the alleles of a gene can be passed from one generation to the next. To appreciate how this works, recall that we each carry two alleles of most of our genes. Gametes formed during meiosis, however, contain only one allele of each gene. The first Mendelian principle simply states that each of the alleles of a gene will segregate into a gamete, and that each allele has an equal likelihood of ending up in a particular gamete. This basically means that half of the gametes will receive one allele of a gene and half will receive the other. How to follow inheritance patterns based on the 1st Mendelian principle. A Punnett square is a graphical method that allows us to predict the outcome of a cross between two individuals. For a monohybrid cross (which we will focus on in this lab), Punnett squares are set up so that the two possible alleles of one gene (at a single locus) from one parent are indicted at the top and the two possible alleles from the second parent are indicated on the side. By following alleles down and across, all possible combinations can be determined. Parent 1 Allele 1 Allele 2 Parent 2 Allele 2 Allele 1 21 22 11 12 Figure 1. A Punnett square showing the combinations possible for the alleles of the same gene from a mating between two individuals Genes on any of the autosomes are simply referred to as autosomal, while genes on the either of the sex chromosomes (the X or the Y) are referred to as sex-linked. For now, we will only consider the possibility that sex-linked genes are on the X chromosome (X-linked). The Y chromosome is important because its presence or absence indicates the sex of the individual. However, very importantly, in most cases it does not carry alleles of genes found on the X chromosome. In addition to the chromosomal location, recall that any of the alleles you are following Biol 171L -FA17 Mendelian Genetics 9-3
may be either dominant (phenotype is expressed with either one or two copies of the allele), or recessive (phenotype is only expressed when two copies of the allele are present). There are several different notation schemes, depending on what organism we are working with, but for the purposes of this lab, we will use capital letters to represent dominant traits, and lowercase letters to represent recessive traits. As an example, let us use flower color in peas. The color purple is dominant to white, which is recessive. If we designate the P the dominant allele, PP is homozygous dominant and will produce purple flowers; Pp is heterozygous and will produce purple flowers; and pp is homozygous recessive and will produce white flowers. Follow these steps to set up a Punnett square for a monohybrid cross: 1. Write down the genotypes of the males and females involved in a mating for the trait you are interested in. Begin with the genotypes representing the parents of a mating. Each allele should be over or beside one of the boxes. This generation is also known as the parental generation or P gen. The genotypes of the parents may often be inferred from the phenotypes of the individuals you have, or from other information given to you. We usually start crosses with true breeding parents, that is, each parent is homozygous for either the dominant trait (e.g., PP) or the recessive trait (e.g., pp). 2. Using this information, follow the alleles in the Punnett square to see how these gametes may be combined at fertilization to form diploid zygotes. These zygotes establish the genotypes of the individuals found in the next generation, which is called the first filial or F1 generation. P Parent 1 P p Pp Pp Parent 2 p Pp Pp Figure 2. Punnett square showing the results of a cross between two true breeding parents. Parent 1 is homozygous for the dominant trait, PP. Parent 2 is homozygous for the recessive trait, pp. Biol 171L -FA17 Mendelian Genetics 9-4
Female ( ) X A X A Male ( ) Y X a X A Y X a X A X A Y X a X A Figure 3. Punnett square showing a cross between a female with a sex-linked dominant trait and a male with a sex-linked recessive trait. The predicted progeny of this cross are 50% dominant trait males (X A Y) and 50% dominant trait females (X a X A ). Punnett squares can be used to follow traits on autosomes, or on either of the sex chromosomes. The principle of segregation and the use of the Punnett squares apply to both cases, but they must be set up and interpreted slightly differently. When following sex-linked traits using Punnett squares, females are, by convention, placed at the top. To follow the alleles from one generation to the next, consider these different scenarios based on possible chromosomal locations and dominant/recessive relationships of the alternative allele: Autosomal recessive Autosomal dominant X-linked recessive X-linked dominant A Punnett square should be constructed for each of these different scenarios. For each case, a reciprocal cross, where the phenotypes are reversed for each of the sexes, should also be performed. For example, in the first Punnett square, a male having the dominant trait would be crossed with a female having the recessive trait. The reciprocal would be a male having the recessive trait crossed with a female having the dominant trait. All together, this means that you will have a total of eight Punnett squares to consider. A further test, the test cross, can be conducted, when we are trying to determine whether a phenotype is homozygous or heterozygous. In this case, we consider the offspring produced from a cross between the dominant phenotype and a homozygous recessive individual. The ratio of progeny that results from this cross allow us to determine whether the parent was homozygous or heterozygous. Biol 171L -FA17 Mendelian Genetics 9-5
a) Parent 1 b) Parent 1 B B B b b b bb b Parent 2 Parent 2 b bb Figure 4. a) Punnett square showing a cross between a homozygous recessive individual (bb) and a phenotypically dominant individual with the genotype, BB. The phenotypic ratio from this cross is 4:0 (dominant ():recessive (bb)), or 100% dominant (). b) Punnett square showing a cross between a homozygous recessive individual (bb) and a phenotypically dominant individual with the genotype,. The phenotypic ratio from this cross is 1:1 (dominant ():recessive (bb)), or 50% dominant () and 50% recessive (bb). Drosophila melanogaster While Mendel chose the common garden pea for his experiments, Thomas Hunt Morgan, in 1907, turned to the vinegar fly, Drosophila melanogaster, to answer some of the fundamental genetic questions being asked at the time. It was a good choice since several factors make Drosophila an ideal organism for genetic experiments. These factors include: 1. They can be easily cultured in small vials containing only a nutrient medium. 2. At 25 C they have a short life cycle which is completed in about 10 days. 3. They are prolific breeders, with each mated female capable of producing several hundred offspring. 4. An enormous number of spontaneous and inducible mutations have been found and studied. The haploid (n) number of chromosomes is 4, and the chromosomes are designated X(1), 2, 3, and 4. The 2, 3, and 4 chromosomes are the same in both sexes and are referred to as autosomes to distinguish them from the X and Y sex chromosomes. Drosophila females are characterized by two X chromosomes, while Drosophila males have an X and Y chromosome. Chromosome 4 and the Y chromosome contain so few genes that for all practical purposes they can be ignored. Thus, almost the entire genetic content of the Drosophila genome resides on only three chromosomes: X, 2, and 3. The larvae possess giant polytene chromosomes, which can be used for genetic mapping. It is easy to determine the sex of the pupa and adults. Because Drosophila are well characterized, they are good organisms for investigating sex-linked traits. These are the organisms we will be investigating in this week s exercises. Biol 171L -FA17 Mendelian Genetics 9-6
IN-CLASS EXERCISES: Laboratory Exercise 1 Goal: Using the Mendelian principle of segregation and the Punnett Square, make predictions on paper describing what you might expect to see in a genetic cross, given different inheritance scenarios in true-breeding parents. True breeding individuals are those that are homozygous for any given trait, i.e., homozygous dominant or homozygous recessive, irrespective of whether the trait is wild type or mutant. Procedure: 1. Given a P gen cross between mutant and wild type, true-breeding individuals, make predictions on paper of inheritance patterns for a mutant allele according to each of the possible scenarios described. Assign genotypes in the P gen, and follow genotypes and phenotypes from the parental generation (P) to the F1 using the Mendelian principle of segregation and Punnett squares. It is not necessary to go beyond the F1 right now. 2. For each of the eight scenarios, make sure that both genotypes and phenotypes are clearly indicated for individuals in both the P and F1 generations. Use the notation AA, Aa, aa (or any other letter) to designate homozygous dominant, heterozygous and homozygous recessive traits, respectively for the autosomal cases. Use X F X F, X F X f, X f X f, (female possibilities) X F Y and X f Y (male possibilities) for the sex-linked genotypes (or other letters). Use the designation wild to indicate the dominant trait and mutant to indicate the recessive trait. Punnett Square Predictions: For each of the four possible modes of inheritance, use the boxes to diagram the F1 progeny possible from the two possible P gen crosses. In this first exercise, all Pgen are homozygous. Note: in Punnett squares, females are always represented on the top and males on the side. Laboratory Exercise 2 What do you notice about Scenarios 1, 2 and 6? What about Scenarios 3, 4 and 7? In order to establish conclusively whether a trait is autosomal or sex-linked, it is often necessary to cross the F1 progeny. Goal: Using the Mendelian principle of segregation and the Punnett square, make predictions on paper describing what you might expect to see in a genetic cross given different inheritance scenarios. Procedure: 1. Given an F1 cross between mutant and wild type individuals, make predictions on paper of inheritance patterns for a mutant allele according to each of the possible scenarios described. Assign genotypes in the F1, and follow genotypes and phenotypes from the F1 Biol 171L -FA17 Mendelian Genetics 9-7
to the F2 using the Mendelian principle of segregation and Punnett squares. 2. For each of the eight scenarios, make sure that both genotypes and phenotypes are clearly indicated for individuals in both the F1 and F2 generations. Use the notation listed for scenarios 1-8 to follow the inheritance from F1 to F2. Laboratory Exercise 3 Now that we have a method of establishing whether a trait is dominant or recessive and whether it is autosomal or recessive, we can extend our investigation to individuals that are not truebreeding, i.e., heterozygous individuals. As mentioned earlier, the test cross allows us a means of determining whether the trait expressed by an individual is dominant or recessive and whether it is autosomal or sex-linked. We can also determine if the individual selected for mating is truebreeding or heterozygous. Goal: Using the Mendelian principle of segregation and the Punnett square, make predictions on paper describing what you might expect to see in a genetic cross given different inheritance scenarios. Procedure: 1. Print and follow the instructions given in the Star Genetics Tutorial, posted on Laulima. 2. Print and follow the instructions given for Fruit fly Exercise 1-1, posted on Laulima. Biol 171L -FA17 Mendelian Genetics 9-8
Lab 9 Homework Due Week of October 30, 2017 Part 1 Mastering Biology (51 points): A. Answer the questions in the assignment entitled 10. Population Genetics on the Mastering Biology site. You have until the night before lab at 11:59pm to complete these questions. Part 2 Science Communication (20 points): A. Preparing Your 2nd Draft Now that your first draft has been reviewed, it s time to prepare the second draft. Revising is an important part of the writing process. Revising gives you the opportunity to strengthen your arguments, improve organization and clean up shaky grammar. Having an outside party review your rough draft is a crucial step in the writing process. Reviewers often see things that we, as writers, miss and they can help us to spot the areas that need improvement. With your peer s comments in hand, you now have the opportunity to improve your report, before your TA sees it for the first time. Using Chapter 6 of Pechenik, 2013 (A Short Guide to Writing about Biology), revise your lab report for the photosynthesis experiment. Use your peer s comments to help guide you in your revisions. Consider each of your reviewer s comments carefully. You may disagree with the reviewer on some points. And, you may not choose to incorporate every suggestion, but you must be prepared to explain why you are choosing not to follow the reviewer s recommendations. As for the first draft, your second draft must be typed, 12- pt font, Times New Roman font, double- spaced with 1 margins. Format your paper following Council of Science Editors guidelines (see http://guides.library.manoa.hawaii.edu/biolabs for more information). This draft is not optional. The draft is worth 20 points, so that your TA can provide substantial feedback without penalizing you for any errors. Your TA s feedback will help you improve your final draft (due week of Nov 28, 2017). Part 3 Data Analysis (30 points) Complete Fruit Fly Exercise 2-1, questions 1-4 (please use version in the Lab 9 folder on Laulima). Biol 171L -FA17 Mendelian Genetics 9-9