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Module 1 Mendelian Genetics Drosophila melanogaster The purpose of this lab is to learn about different aspects of heredity using Drosophila as a model organism. In this lab, students will learn about culturing, mating, and genetics of different Drosophila traits, analysis of test crosses, and Chi squared analysis. Module timeline: · Day 1: Introduction to Drosophila Genetics and A Simple Crosses Make predictions and preparation of starter plates Observations of test crosses and statistical analyses · Day 2: Research Article discussion Design experiment: Choose traits to cross to produce two monohybrid crosses for autosomal traits, one dihybrid cross for autosomal traits, and one sex linked cross- make predictions. · Day 3: Work on traits and crosses. · Day 4: Work on traits and crosses. Record and analyze results. Work on the Graphic summary assignment Module Background Genetics is the study of genes, which carry the information that defines every organism. Genes, with the help of the cells they are in, must do three things: 1) their information must be copied faithfully, 2) their information must be passed precisely to new cells, and 3) their information must result in metabolic reactions and cellular structures. A gene is a sequence of DNA that codes for a protein or sometimes an RNA molecule; the molecule the gene codes for is often called the gene product. A mutation is a change in the sequence of a gene that leads to a different form of the same gene. This new version of the gene is called an allele. Our understanding of the nature of genes at the molecular level came from the concept of allelism; that is, mutations can occur at many places in a gene and there are multiple genes that contribute to the same phenotypic trait. In Mendel’s approach, he utilized a test cross, in which parents exhibited different phenotypic (physical) traits without understanding the genotype, or the alleles for a given gene. Mendel denoted true-breeding parents, which have homozygous alleles for the particular trait, as the P generation. The offspring of the P generation are the F1 generation (first filial) and the offspring of the F1 generation are the F2 generation. In a test cross, there are three important ideas regarding the transmission of the trait between the parents and offspring: 1. Traits can exist in two forms, dominant and recessive. The dominant trait describes the observed trait and the phenotype of the recessive trait is masked by the dominant trait. 2. An individual carries only two genes for the given character, even if multiple genes exist in a population. 3. The two alleles of a gene separate during the process of producing gametes In understanding genetics and the transmission of traits from one generation to the next, a model organism is utilized because they are convenient to study and have specific attributes that make them appropriate for this type of research. Mendel utilized pea plants in his experiments on inheritance and, with modern genetic studies, model organisms tend to have similar characteristics in common: · short generation time and rapid reproduction · large numbers of offspring · small size and simple care requirements In this module, Drosophila melanogaster, a fruit fly will be utilized to study the transmission of traits. This model organism has been utilized in genetics research since the early 1900s because it can be raised in the lab easily and inexpensively, mating can be easily controlled, the life cycle is complete within two weeks, and females lay large quantities of eggs. These traits, along with a large variation in traits, make fruit flies an easy subject to test hypotheses about how traits are passed from parents to offspring. Given the time frame of the lab over the summer session and, that we are working from home, we will be using an online “fly lab”, the VCISE: Drosophila program to perform our test crosses. This program can be found at https://www.sciencecourseware.org/vcise/drosophila/index.html Statistical Analysis for crosses (info provided by sciencecourseware.org) A chi-square analysis is a statistical test that measures how well a suggested numerical hypothesis compares to the observed results. In other words, do the observed results reflect acceptable differences from the expected values? The actual offspring numbers that result from the mating are compared to expected values based on the student’s hypotheses for the ratios among the different types of offspring. For example, based on some genetic mechanism that you are proposing, you believe that there should be a 4 to 1 ratio of wild type flies to flies with paisley eyes. Of course, the ratio won't be exactly 4 to 1; one has to allow for random error. The question is: do the results differ "significantly" from a 4 to 1 ratio? To put it differently, if the 4 to 1 ratio is true, what is the probability that you would get deviations from a 4 to 1 ratio that are as large (or larger) than the deviations which you observe in the data? Statisticians call this probability the "level of significance." So how do you calculate the level of significance? Statisticians have derived a test statistic called "chi-square" that can be used compute the level of significance. The chi-square test statistic measures the deviations of the observed values from the "expected values" that you would get if your hypothesis is true. The formula for calculating the chi-square () test statistic is: In this formula, you take observed number for each phenotype, Oi, subtract the expected number, Ei, square the difference, and divide the squared difference by the expected number. You sum the chi-squared terms for all of the phenotypes to obtain your test statistic. If the squared deviations between the observed and expected values are small (i.e., the observed and expected values are similar), the test statistic will be small. Thus, the data support the hypothesis. On the other hand, if the squared deviations between the observed and expected values are large, the test statistic will be large and, thus, there is a smaller probability that the hypothesis is true. This will lead to small values for the level of significance, and a hypothesis that should be rejected. The test statistic can be compared with a theoretical probability distribution to obtain the level of significance. This probability distribution depends on the "degrees of freedom" which equals number of phenotypic groups used in the calculation minus one. The Drosophila program automatically calculates the level of significance. If the level of significance is large, there is a good chance (high probability) that the deviations from your hypothesis are simply due to random error. In other words, there is no evidence to reject your hypothesis. The hypothesis fits the data. On the other hand, if the level of significance is small (less than 0.05), it is unlikely (low probability) that the deviations from your hypothesis are due to random error alone. Therefore, your hypothesis is probably wrong. In other words, if there is a less than a 5% chance that the deviations from your hypothesis are due to random error, then you should reject your hypothesis. Your hypothesis is inconsistent with the data. A new ratio based on a different genetic hypothesis should be entered. Day 1- Example test crosses 1. Monohybrid Cross A monohybrid cross only considers one trait for transmission between the parents and offspring for an autosomal (non-sex chromosome) To work through an example monohybrid cross, in the Drosophila program begin by adding a adding a wildtype female (denoted by WW below) and a black body male (denoted by bb below) to the “cart” and “check out”. The shipping speed should be like Amazon Prime. Using the box below, complete the Punnett square to predict the F1 generation genotype and phenotype. Parental Generation FemaleMale WW bb F1 Generation 1.1 Based on this test cross, what is the predicted genotype and phenotype of the F1? 1.2 After performing the test cross, do the actual results match your Punnett square? How many individual flies had a wildtype phenotype? How many had a black body phenotype? Next, select a male and a female from the F1 generation and add them to a new mating jar to perform another test cross to produce the F2 generation. F2 Generation Using the box above, complete the Punnett square to predict the F2 generation genotype and phenotype. 1.3 Based on this test cross, what is the predicted genotype and phenotype of the F2? 1.4 After performing the test cross (ensure that the “Ignore sex” box is checked while interpreting the results), do the actual results match your Punnett square? How many individual flies had a wildtype phenotype? How many had a black body phenotype? Based on these numbers, do the observed numbers match the predicted numbers? Was there a significant variance between the predicted number and observed number? How do you know? 2. Dihybrid Cross A dihybrid cross examines two different phenotypic traits for transmission between the parents and offspring. In this example, we will select two different traits for the fruit flies. To begin this example, begin shopping in the Drosophila program by adding a wildtype female (denoted by WWEE below) and an apterous wing male that is eyeless (denoted by bb below). Parental Generation FemaleMale WWEE aaee F1 Generation 2.1 Based on this test cross, what is the predicted genotype and phenotype of the F1? 2.2 After performing the test cross, do the actual results match your Punnett square? How many individual flies had a wildtype phenotype? How many had a black body phenotype? Next, select a male and a female from the F1 generation and add them to a new mating jar to perform another test cross to produce the F2 generation. F2 Generation Using the box above, complete the Punnett square to predict the F2 generation genotype and phenotype. 2.3 Based on this test cross, what is the predicted genotype and phenotype of the F2? 2.4 After performing the test cross (ensure that the “Ignore sex” box is checked while interpreting the results), do the actual results match your Punnett square? How many individuals were produced with each genotype/phenotype? Was there a significant variance between the predicted number and observed number? How do you know? 3 Sex-linked cross Another example of linkage involves gene on the sex chromosomes (generally X and Y) and rereferred as sex-linked. A gene found on the X chromosome is described as X-linkage and a gene found on the Y chromosome is described as Y linkage. The hypothesis of X-linkage describes how a female has two copies of a gene