Laboratory 10 Hardy-Weinberg Population Genetics
Biology 171L SP18 Lab 10: Hardy-Weinberg Population Genetics Student Learning Outcomes 1. Calculate allele and genotype frequencies 2. Use phenotypes to predict genotype and allele frequencies 3. Observe changes in allele frequencies over several generations in a simulation Relevant Readings 1. Campbell Biology, Chapter 23, especially pp. 483-487 2. A Short Guide to Writing about Biology, Chapter 6, especially pp. 117-120 Homework Synopsis (see page 10-11 & 10-12 for full description) Part I Mastering Biology Part II Science Communication Revising strategies: short answer questions Part III Data Analysis Short answer questions INTRODUCTION Genes are instructions for creating polypeptide chains, the underlying structures of proteins. Unless an organism is haploid (containing just one set of each chromosome), organisms will have two or more copies of each chromosome, which means that they also have two or more copies of the genes, one on each chromosome. Alleles are versions of a specific gene, in which changes to that coded sequence create alternate peptide chains and variations of the resulting protein. Sometimes those changes affect the function of the protein. A gene is simply a recipe, much like a recipe for chocolate chip cookies. There are many different versions of chocolate chip cookie recipes; similarly, there can be many different allelic versions of a gene. These recipes are preserved when they re passed on from generation to generation. A population is a group of individuals from a single species that can interbreed with one another. Over time, from one generation to the next, populations can change dramatically in number, geographic location, and genetic composition. Population genetics is the study of the genetic composition of a population as a whole and how the composition is influenced by natural selection, mutation, gene flow, and genetic drift. Specifically, population geneticists are interested in the frequency and distribution of alleles in an entire population. Allele frequency is calculated in the following manner: Allele frequency = # of copies of a specific allele in a population Total number of all alleles for that specific gene in a population For example, let s consider a population of pea plants growing in the wild. DNA sequencing of all ten (10) of the tall and short pea plants was used to determine the genotypes of each plant. If Biol 171L - SP18 Population Genetics 10-2
you were looking for the allele frequency of the tall allele (T) and short allele (t), knowing their genotypes would make for an easy calculation. Population genotypes (Total=10 plants) TT, Tt, TT, Tt, Tt, Tt, TT, tt, TT, TT Because we are assuming that the pea plant is diploid, the total number of alleles for the height gene is equal to: 2 (ploidy level) * the number of individuals in the population In our example, this is: Total # alleles = 2 * 10 = 20. Similarly, we can find the total number of each allele in the population by counting the alleles of each individual. In our example, there are 14 T (from 9 individuals) and 6 t (from 5 individuals). Using this information, we can now determine the frequency of occurrence of each allele in the population. p represents the allele frequency of the dominant allele q represents the allele frequency of the recessive allele Allele frequency = # of copies of a specific allele in a population Total number of all alleles for that specific gene in a population P = dominant allele = 14 T = 14 = 0.7 or 70% of alleles are dominant T frequency 20 20 q = recessive allele = 6 t = 6 = 0.3 or 30% of alleles are recessive t frequency 20 20 Notice that: p + q = 1. This applies in genes where there are only two alleles. In the early twentieth century, Godfrey Hardy, a British mathematician, and Wilhelm Weinberg, a German physicist, both working independently, expanded upon Mendel s and Darwin s work and developed the field of population genetics research. They studied the effect that heredity had on a population and discovered that evolution occurs when the frequency of alleles in that population changes over time. If evolution is not occurring, then the frequencies of alleles in a population remain constant. Each scientist went further and developed a mathematical equation that can be used to determine the frequencies of alleles within a population. The equation, known as the Hardy-Weinberg Principle, states that p 2 + 2pq + q 2 =1. It is derived from (p + q) 2 = 1 Let s consider what happens when our population of tall and short pea plants breeds with other plants in the population. Below is a Punnett square, but instead of mating two individual plants, let s look at mating the plant population with itself. The population gene pool contains p and q alleles that can be passed on randomly to the next generation. p q p pp pq Biol 171L - SP18 Population Genetics 10-3
q pq qq The population has the alleles p and q in the gene pool and individuals in the population mate with other organisms drawing from the same gene pool. The genotypes that arise are: pp or p 2 = homozygous dominant = These organisms have inherited two dominant alleles from the gene pool. Each parent possessed at least one dominant allele that was passed on to these organisms. qq or q 2 = homozygous recessive = These organisms have inherited two recessive alleles from the gene pool. Each parent possessed at least one recessive allele that was passed on to these organisms. 2 qp = heterozygous These organisms have inherited a dominant allele and a recessive allele from the gene pool. It is multiplied by two, because these organisms can result from two different scenarios. The Hardy-Weinberg principle restated is (and the common way of presenting the equation): p 2 + 2 pq + q 2 = 1.0 Note that our Punnett square example shows the possible alleles that are used to create the next generation, but the ratio of dominant to recessive alleles is not 50:50 as the Punnett square shows in that form. Below we enter the allele frequencies for both alleles for a more accurate prediction of the ratio of genotypes in the next generation. 0.7 p 0.3 q 0.7 0.3 p q P 2 pq (0.49) (0.21) pq q 2 (0.21) (0.09) The predicted percentages of the following genotypes are: p 2 homozygous dominants = 0.49-49% of population will most likely be homozygous dominant 2pq heterozygotes = 0.42-42% of population will most likely be heterozygotes q 2 homozygous recessives = 0.09-9% of population will most likely be homozygous recessive Predicting frequencies from phenotype observations: Biol 171L - SP18 Population Genetics 10-4
Obtaining actual genotypes for all members is unrealistic for most populations, but predictions can be made by deducing one genotype frequency and then calculating the remaining of the genotype and allele frequencies. To apply the principle, at least one of the allele frequencies must be known. Let s use an example of a genetically inherited trait: coat color in cats. A completely white coat is caused by an allele that is dominant (W) over an allele that causes a coat to have some color (w). Which of the two cats are you able to absolutely identify their genotype by looking at their phenotype? Cat A Cat B The correct answer is Cat B. Cat B is orange and white, and this phenotype can only occur when the individual is homozygous recessive for the coat color gene. The genotype must be ww for you to be able to see the expression of the recessive allele. You are unable to definitively identify Cat A s genotype because there are two possibilities. Cat A could be either homozygous WW or heterozygous Ww, where the dominant W masks the presence of the recessive w. When trying to calculate the frequency of genes in a population, start with the frequency of individuals exhibiting the recessive trait. This allows you to calculate the frequency of homozygous recessive individuals. It is important to note that dominant does not mean that the allele is the most numerous allele in a population, nor that it is the best allele in the population. Likewise, recessive does not imply rare or deleterious. In a population of 1,000 cats, you observe that 950 have some color and 50 are pure white. q 2 = genotype frequency of homozygous recessive = 950/1000 = 0.95 We expect that ~95% of the population is homozygous recessive (ww). Given q 2, you can calculate q by taking the square root. q = 0.975 ~97.5% of the alleles in the population gene pool are probably the recessive allele (w). Given q, we can calculate p. p + q = 1 p = 1 0.975 p = 0.025 ~2.5% of the alleles in the population gene pool are probably the dominant allele (W). Biol 171L - SP18 Population Genetics 10-5
We can also calculate a prediction of the genotype frequencies. p 2 = 0.025 x 0.025 = 0.000625 We expect that ~0.0625% of the population is homozygous dominant (WW). 2pq = 2 x 0.025 x 0.975 = 0.04875 We expect that ~4.875% of the population is heterozygous (Ww). These calculations allow us to predict the frequencies in a population, based on knowledge of one frequency. However, it is important to understand that frequencies are rarely what will actually be observed. The Hardy-Weinberg Principle makes a number of assumptions about the factors that allow a population to remain static and predicts equilibrium of unchanging frequencies in a population under ideal conditions, where adaption and evolution are not occurring. The conditions that keep a population from evolving include: 1) random mating within a population; 2) a population of infinite size; 3) no immigration or emigration; and 4) no genotypes subject to selective pressure. The reality is that one of more of these assumptions is being broken in any given population all the time. Consequently, the Hardy-Weinberg equation allows us to get an idea of what the frequencies of alleles in a population are at a given point in time. Those frequencies provide a baseline for looking at change over time. Once frequencies are known for a specific generation in a population, we would expect to see those same frequencies in later generations, provided there are no mitigating factors that have caused changes to the gene pool. Genes That Taste Not So Good Our sense of taste is being investigated as being a product of our genetic make-up. Scientists are finding that dietary preference may be linked to each individual s perception of taste, which may have a greater genetic basis than we knew. One of the best examples is a chemical phenylthiocarbamide, better known as PTC. In 1931, an accident in the lab dispersed this harmless powder into the air and the scientists present discovered that some of them detected a bitter taste, while others could not taste anything at all. This ability for some individuals to taste particular flavors while others cannot is not restricted to humans. Recent research has shown that similar abilities exist in Japanese macaques (see: http://blogs.discovermagazine.com/inkfish/2015/09/22/taste-mutation-helps-monkeys-enjoyhuman-food/#.waqssdzatpc). Despite the fact that several genes encode for proteins for bitter taste receptors, ensuing experiments determined that the ability to specifically taste the bitterness of PTC is determined mostly by a single gene. This gene is located on chromosome 7. Sequencing of the gene from various individuals revealed that there are three versions, or alleles, that differ slightly. The change in the DNA code creates a protein that is abnormally shaped. This protein receptor can t interact with the PTC molecule and the individual will not be able to detect PTC, or it s bitter taste. Biol 171L - SP18 Population Genetics 10-6
Because we have two alleles (one on each chromosome) for the bitter taste receptor gene, there are varying levels of PTC tasting. TT two normal alleles creating normal receptors resulting in strong bitter taste Tt only one normal allele creating normal receptors, while the recessive allele is creating receptors that will not be able to detect PTC. Heterozygous individuals can taste PTC, but may have different views of its bitterness. tt- no normal allele and thus no receptors capable of detecting PTC. Homozygous recessive individuals will not identify PTC with a bitter taste. There are multiple receptor proteins involved with taste. You will also be testing your ability to taste thiourea and sodium benzoate. Sodium benzoate is used as an additive and preservative because of its ability to kill yeast and bacteria. It is naturally present in small amounts in some fruits and spices. Those able to taste sodium benzoate describe the taste differently. Sodium benzoate tastes bitter or salty to some, or slightly sweet to others. Children have a higher number of taste buds and may find flavors more intense and thus more intensely unappealing. Our number of taste buds decreases as we age and our sense of taste changes as we age. You might want to go try some broccoli; it may have become your new favorite food! Preparation for Lab 1. Read through Introduction 2. Read Campbell, pp. 479-486 3. Research topics and terms that you are not familiar with or do not fully understand 4. Read through Experiment Procedure PROCEDURE: Part A: Calculating the allele and genotype frequencies of Taste Genes in a Class Population Every student will be observing his or her own taste abilities. Make sure you are not chewing any candy or gum (which you shouldn t be doing in a lab). Obtain a piece of control paper. Place it on your tongue. You should not detect any distinguishable taste beyond the paper. Use this as a comparison when testing the sample papers. Dispose of paper in a trash can (do not swallow). Obtain a piece of PTC test paper. Place it on your tongue. Record whether the PTC paper tastes bitter or has no taste. Record the results for the thiourea paper and the sodium benzoate paper. Biol 171L - SP18 Population Genetics 10-7
In all three tests, use T, t to represent those genes and the ability to taste each test paper is a dominant trait. Determine your possible genotype(s) based on your physical ability in each test. Treating the class as a population, collect class data for population analysis. Test Paper Your Data Your possible genotype(s) Class Data Taster? Non-taster? Use (T,t) alleles # Tasters # Non-tasters Control Paper Sodium Benzoate Paper Thiourea Paper PTC Paper Part B: Investigating Allele Frequency Changes over Multiple Generations For the last part of this lab, you will be observing the change in allele frequencies over five generations, utilizing your class as a hypothetical population. Each member of your section will be mating randomly with another member of your section to produce a new generation. Each pair of students will mate twice to replace their original genotype, through 5 successive generations. You will then use the data you collect to compare how allele frequencies change under different conditions. 1) Populations in Equilibrium In this scenario, you will be examining allele frequencies when there is no selection pressure, mating is random, there is no immigration or emigration, and there is no mutation. 1. Each student selects two cards from the gene pool this is your starting genotype. 2. Randomly pair off with another student. For this part, choose your partner randomly - gender does not matter. 3. Turn your cards upside down (or place them behind your back), so you and your partner cannot see which cards are which. Pick a card from your pile. 4. Your partner will also shuffle and randomly pick a card from his/her pile. 5. These cards represent the random selection of chromosomes to be passed on through gametes to form the next generation. Your card and your partner s card should be paired together and this will be the genotype of your first offspring. 6. Record your first offspring s genotype on your data sheet. Biol 171L - SP18 Population Genetics 10-8
7. Take your card back and reshuffle your four cards. 8. You and your partner should once again randomly select one chromosome card each to create a second offspring. Record that offspring s genotype on your partner s datasheet. 9. Now begin the second generation by taking the genotype of your offspring. Your partner will play the part of one offspring and you will play the part of the other offspring. 10. Reevaluate your index cards to make sure you have the correct chromosomes for your offspring (mimicking meiosis). E.g., for a homozygous TT offspring, you should have two (2) T cards; for a heterozygous Tt offspring, you should have one (1) T and one (1) t cards. Return extra or get extra new cards from the gene pool. 11. Find a new partner, and generate two more offspring (3 rd generation) using the steps 13-19 above. Record the results. 12. Repeat for a total of five generations. 13. Collect class data for all generations (TA will have master list). 14. Calculate the allele frequencies of T and t after five generations. 2) Populations under Directional Selection In this scenario, you will be examining the change in allele and genotype frequencies when there is selection against homozygous-recessive offspring. In this scenario, if an individual is homozygous-recessive, they will not live long enough to reproduce. 1. Follow the same procedure as Part B, however if an offspring is produced with the genotype tt, it will not survive. Reproduce again with the same partner until you have two surviving offspring and use those offspring as the second generation to generate a third offspring. 2. Repeat until you have five generations of offspring and data. 3. Combine the class results and calculate the allele frequencies. 3) Populations under Stabilizing Selection In Scenario 2, you should have noticed that alleles that are deleterious, that is, are harmful or injurious, are removed from the population over time. Sometimes however, alleles that are harmful may also provide an advantage under specific conditions. In this scenario, recessive individuals will die before reproducing (like in Scenario 2), but some homozygous dominant individuals may also die (see: Figure 23.17, Campbell). 1. Follow the mating procedure in the same way as for Scenario 2, that is, if you get tt, continue reproducing until you get TT or Tt. Additionally, each time you produce a TT, flip a coin to see if TT will die. If the TT dies, continue reproducing until you have surviving offspring. 2. Continue for a total of five generations. Record the genotypes after every generation. Biol 171L - SP18 Population Genetics 10-9
3. Combine your group s fifth generation results with those of the other populations in the class and calculate the new allele frequencies. How did allele frequencies change over time? Biol 171L - SP18 Population Genetics 10-10
Data sheets for Allele Frequency Exercises (Individual Student Data): Population 1 - No Selection Student Counts Generation TT Tt tt Initial 1 2 3 4 5 "T" Alleles "t" Alleles Population 2 - Selection against 'tt' Student Counts Generation TT Tt tt Initial 1 2 3 4 5 "T" Alleles "t" Alleles Population 3 Stabilizing Selection Student Counts Generation TT Tt tt Initial 1 2 3 4 5 "T" Alleles "t" Alleles Biol 171L - SP18 Population Genetics 10-11
Lab 10 Homework Due Week of April 9, 2018 Part 1 Mastering Biology (43 points): A. Answer the questions entitled 11. Phylogenetics on the Mastering Biology site. You have until the night before lab at 11:59PM to complete these questions. Part 2 Science Communication (10 points): Revising is an extremely important part of writing. In Chapter 6, Revising, Pechenik (2009) outlines eight areas (e.g., content, clarity, completeness, etc.) that writers can focus on when revising their drafts. Think about your peer s comments regarding your first draft, and the ways you utilized their comments to help you revise. a. Using Pechenik s headings (e.g., content, clarity, completeness, etc.), describe the top three areas that you need to work on in your final draft. You may have fixed some things; you may include these in this description. Use examples from your peer reviewer s comments to support your statement (e.g., my reviewer pointed out that I was using titles instead of figure captions). (5 pts) b. Using the suggestions provided by Pechenik (2009), outline the strategies you will take to review your TA s comments and incorporate them into your final draft. (5 pts) Part 3 Data Analysis (20 points) Discussion Questions (must be typed for full credit): Part A Genetics of Taste Refer to the following spreadsheet: https://docs.google.com/spreadsheets/d/1- mauydu56gf1tosfyq1wmhy3su7hblvyryx999--aa8/edit?usp=sharing 1. How do your individual section results for taste compare with the entire class population? Do your section or class data match the Hardy-Weinberg predictions? Why or why not? (3 pts.) 2. Analyze your section as an ideal or not ideal candidate for Hardy-Weinberg studies. (Hint: how many students are in your section? What are the assumptions of H-W?) (2 pts) Biol 171L - SP18 Population Genetics 10-12
3. Consider the assumptions necessary for Hardy-Weinberg equilibrium to persist in a population (p.10-6 in the lab manual). For each assumption, list 2-3 ways these assumptions could be violated in real populations. (8 pts) Part B Changes in Allele Frequency Over Time 1. What do the allele frequency changes over five generations tell us in each part of this lab (i.e., populations 1, 2 and 3), that is, how do allele frequencies change under the different scenarios: no selection, selection against a lethal condition, stabilizing selection? (3 pts.) 2. What factor might allow deleterious alleles to persist in the population? (Hint: see pp. 492-493 in your text book) (2 pts) 3. What problems result from having a very small population of organisms? (2 pts.) Biol 171L - SP18 Population Genetics 10-13