Lab Instructions_ Genetics and Evolution Act II Mission Memo

Heritability is defined as the proportion of phenotypic variation in a population that stems from additive effects of alleles. In other words, heritability is the proportion of phenotypic variation caused by genes, rather than the environment. Heritability reflects the extent to which the phenotypes of parents determine the phenotypes of their offspring. Thus, heritability reflects a relationship between the phenotypes of parents and the phenotypes of offspring. We can use a linear function to describe this relationship, in which the independent variable is the mean phenotype of parents and the dependent variable is the mean (or expected) phenotype of an offspring. Let’s consider an example. Imagine two traits, which we will call trait 1 and trait 2. Figure 1 shows the linear relationship between the mean parental phenotype and the mean offspring phenotype for each trait. Figure 1. This figure shows the linear relationship between the mean parental phenotype and the mean offspring phenotype for two unique traits, trait 1 and trait 2. The y-axis is a continuous variable - the mean phenotype of offspring. The x-axis is a continuous variable - the mean phenotype of parents. The solid blue line is the linear

A linear model is an extension of a normal probability distribution, in which the mean of the dependent variable depends on the value of the independent variable. This linear model is the same as the one commonly used in algebra courses, where y = ax + b , where y is the value of the dependent variable, x is the value of the independent variable, a is the slope of the linear model, and b is the intercept of the linear model. Rather than calculate the slope and intercept of the model by hand, Microsoft Excel has functions for estimating the slope and intercept of a linear model. To estimate the slope, use the slope function: =slope(dependent observations, independent observations), To estimate the intercept, use the intercept function: =intercept(dependent observations, independent observations), For example, consider the five pairs of observations in the spreadsheet below: Table 1. Values of two variables. Columns are represented with letters “A” and “B”, while rows are represented numerically with 1–5. The first column (column “A”) lists the values of the independent variable. The second column (column “B”) lists the values of the dependent variable. Each row represents a set of observations for the independent and dependent variables. A B 1 Independent variable Dependent variable 2 5 25 3 3 64 4 7 24 5 4 57 6 6 35 For these data, you would calculate the slope and the intercept of the linear relationship by entering the following functions in Excel: Slope =SLOPE(B2:B6, A2:A6) Intercept =INTERCEPT(B2:B6, A2:A6)

7. Estimate the heritability of metal tolerance in this population of sunstalks - 0.41 Appendix 2 Will the mean metal tolerance of sunstalks evolve rapidly enough for the population of stalkleapers to persist? Excellent work. Thanks to your efforts, we know the heritability of metal tolerance in the population of sunstalks. This information is critical for predicting how rapidly the population of sunstalks will adapt to their contaminated environment. Now, we can quantify how quickly the mean metal tolerance of sunstalks will evolve in the population. Evolution by natural selection requires three conditions: 1. Phenotypes vary among organisms in a population, such that the value of a trait differs among some individuals,

Figure 2. This figure shows frequency distributions of measurements of the same phenotype in two distinct populations, population 1 and population 2. In both plots the x-axis represents the measurement of the phenotype. There are 11 bins. The bins are in increments of 10, starting at 0 and ending at 100+. The y-axis is the frequency of (or number of) observations that fall into each of the bins designated on the x-axis. The phenotypes for population 1 ranges from 10 to 90, while the phenotypes for population 2 range from 30-70. Directions: Use Figure 2 to answer question 8. 8. Which population has a greater variance in the trait that was measured in Figure 2? a. Population 1 b. Population 2 As the variation in a trait increases among organisms, the phenotypic variance increases. Conversely, as the variation in a trait decreases among organisms, the phenotypic variance decreases. In Figure 2 the phenotype in population 1 ranges from a value of 10 to 90, while the phenotype in population 2 ranges from 30 to 70. Therefore, the value of the trait varies more in population 1 than it does in population 2.

Excellent work, yet again. Thanks to your efforts, we now know the phenotypic variance ( V p ), the heritability ( h ), and the selection gradient ( β ). You're now ready to predict the rate at which metal tolerance will evolve in the population of sunstalks that we replanted in the contaminated area. This rate will enable us to answer the question “Will metal tolerance evolve rapidly enough in the sunstalk population to ensure the stability of the population?” Directions: For question 13, use your estimates of the heritability of metal tolerance ( h ), the phenotypic variance of metal tolerance of offspring that were replanted in the contaminated area ( V p ), and the selection gradient of metal tolerance ( β ) to calculate how much the mean metal tolerance of the replanted population will change over a single generation ( Δx̅ ). Note that the percentages in all of the data used to estimate h , V p , and β were already converted to numbers between 0 and 100; therefore, your answer should be expressed as a number instead of a percentage. Use Excel for calculations. Round all calculated values to the nearest hundredth of a decimal place. For example, if you calculate the value as 3.8218%, round to 3.82%. 13. How much will the mean metal tolerance change over one generation? Express your answer in units of change in the mean metal tolerance per generation. A positive value indicates that the mean value of metal tolerance will increase by that amount during each generation. A negative value indicates that the mean value of metal tolerance will decrease by that amount during each generation. Change in the mean metal tolerance during one generation = 0.66 Step 3: Weigh the evidence and conclude whether metal tolerance of sunstalks will evolve rapidly enough for the population of stalkleapers to persist. Because of your careful planning in Step 1 and your quantitative analyses in Step 2, you can now conclude how many generations will be required for the population of sunstalks to evolve complete tolerance of heavy metals. Recall that we are measuring metal tolerance in terms of the percentage of healthy tissue when a sunstalk is grown in contaminated soil. Therefore, complete tolerance of heavy metals is defined by a sunstalk that has 100% healthy tissue when grown in contaminated soil. The mean metal tolerance in the population of sunstalks will evolve by natural selection until it reaches the maximum of 100%. The question that remains is how many

Figure 4. This figure shows the structure of a strand of DNA (deoxyribonucleic acid) on Earth, including base-pairing between the nucleotides. This molecule consists of two interwoven chains, each chain consisting of four types of nucleotides: adenine (A), thymine (T), guanine (G), and cytosine (C). Each nucleotide consists of a phosphate group, a 5-carbon sugar ring, and a nitrogenous base. The phosphate-sugar complex covalently bonds with the phosphate-sugar complexes of adjacent nucleotides to form each strand of DNA. The nitrogenous bases of one strand form hydrogen bonds with the nitrogenous bases of the complementary strand, forming a double-stranded molecule. The complementary bases in DNA are as follows: adenine (A) of one strand forms hydrogen bonds with thymine (T) of the complementary strand, and guanine (G) of one strand forms hydrogen bonds with cytosine (C) of the complementary strand. Properties of DNA from Sarcannus Organisms on Sarcannus have DNA, but the nucleotides in this DNA contain different nitrogenous bases. A molecule of DNA from Sarcannus comprises the following bases: narithov (N), ornithov (O), panithov (P) and zylathov (Z). These bases form hydrogen bonds similar to those that connect complementary strands of DNA on Earth. Specifically,

narithov (N) forms hydrogen bonds with ornithov (O), whereas panithov (P) forms hydrogen bonds with zylathov (Z). Thanks to data collected by GUS and IRMA, we know that the gene that affects the function of the azure pods has three alleles, P 1 , P 2 , and P 3 . Each allele differs from the others by just a few nucleotides. But these differences are sufficient to affect the phenotype. The three alleles and key sections of their DNA are shown below. Notice that each of these alleles differs from the other alleles by only one nucleotide. When reading the nucleotides from left to right, allele P 1 and allele P 2 have a different nucleotide in the 11th position. Likewise, allele P 1 and P 2 P 3 have a different nucleotide in the 17th position as compared to allele P 3 . The relevant nucleotides have been bolded. Allele 1 (P 1 ): N N P Z N O O O P P N N N N P Z Z O Z P Z N N O P N Z Z O O O O Z P O N N N Z Z O O O O Z P P N P Z P O O N Z O P P N N Allele 2 (P 2 ): N N P Z N O O O P P P N N N P Z Z O Z P Z N N O P N Z Z O O O O Z P O N N N Z Z Z O O O Z P P N P Z P O O N Z O P P N N Allele 3 (P 3 ): N N P Z N O O O P P N N N N P Z Z Z P Z N N O P N Z Z O O O O Z P O N N N Z Z O O O O Z P P P Z P O O N Z O P P N N Of the three alleles for this gene, two alleles (P 1 and P 2 ) code for a functional protein that binds a chemical released by stalkleapers, triggering an azure pod to close. The third allele (P 3 ) fails to produce a functional protein. Unfortunately, the P 3 allele is linked to an allele for another gene that increases metal tolerance. As natural selection increases the frequency of metal tolerance in the population, it will also increase the frequency of the P 3 allele in the population. We cannot let this happen.

We can use tools for gene editing to restore the function of azure pods. By editing the DNA of a sunstalk, we can convert the pod closure gene from one allele to another one. On Earth, we use a molecular tool known as CRISPR-Cas9 to edit genes. CRISPR is an abbreviation for “Clustered Regularly Interspaced Short Palindromic Repeats”, and Cas9 refers to the “CRISPR-associated protein 9”. Much easier to say "CRISPR-Cas9", isn't it? CRISPR-Cas9 was discovered in bacteria on Earth, which use this molecular system to detect and attack the DNA of viruses. Scientists have used CRISPR-Cas9 to develop a fast and effective tool for editing genes in living organisms. The molecular complex that forms CRISPR-Cas9 includes an RNA strand and a protein that acts as an enzyme. The RNA guides the complex to a specific region of DNA, by recognizing a complementary sequence of nucleotides. When this guide RNA binds to DNA, the Cas9 enzyme cuts the double stranded DNA at specific locations, enabling this segment of DNA to be removed from the chromosome. The cell then uses its own molecules to repair the DNA. If left to chance, the cell will repair the DNA with a random sequence of nucleotides. However, by including a piece of DNA with the CRISPR-Cas9, we can ensure that the cell replaces the excised DNA with a specific sequence of nucleotides. In this way, scientists can edit any gene to convert one allele into another allele. Using CRISPR-Cas9, we plan to edit the gene for pod closure, inserting an allele that enables the azure pods to fully close. However, I need your help in determining the best allele to insert into the sunstalk pod closure gene. We’ll follow two steps to determine the best allele to insert into the sunstalk genome. Step 1: Determine which allele would restore the function of azure pods. Analyze the genotypes and phenotypes associated with the pod closure gene in sunstalks. With this information, you will be able to determine which of the three pod closure alleles would restore the function of azure pods in Step 2. Step 2: Determine which allele(s) should be added or removed from the sunstalk population. Using the results of your analysis in Step 1, recommend which allele(s) should be removed or inserted into the sunstalk population that was replanted in the contaminated area of the sanctuary. Step 1: Determine which allele would restore the function of azure pods.

As with most animals on Earth, the sunstalks of Sarcannus are diploid. Each cell of a diploid organism contains two copies of each gene, one copy from each parent. Therefore, a sunstalk will have two copies of the gene that affects the azure pods, each of which could be a unique allele. We must consider how each allele combines with other alleles to affect the phenotype of a sunstalk. This information will enable us to decide how we should edit the gene to restore the function of the azure pods. Our goal is to identify which allele enables the azure pod to close properly. We'd prefer that this allele were dominant to other alleles, such that only one copy is needed to ensure that the azure pods will function properly. To assist in our analysis, GUS and IRMA have determined the phenotype associated with each genotype for the trait pod closure. Here is what they found: Genotype(s) Phenotype P 1 P 1 , P 1 P 2 , P 2 P 2 , and P 1 P 3 Pod fully closes P 2 P 3 Pod partially closes P 3 P 3 Pod does not close Using this information, I need you to determine the nature of the relationship between each allele for the pod closure gene. Directions: Use the information provided by GUS and IRMA to answer questions 16-21. 16. The P 1 allele is ____ to the P 2 allele. a. dominant b. recessive c. incompletely dominant d. neither allele is dominant or recessive to the other 17. Explain how the information that GUS and IRMA provided supports your answer to the previous question. - As stated above, with presence of the P1 allele, the pod will close, and without it, the pod only partially closes at best. Although both P1P2 and P2P2 alleles also close the pod, yet, if the P2 allele were to be paired with a P3 allele, it will only

partially close, hence it can be seen that the P2 allele is not dominant towards the P3 allele. Since the P1 allele is dominant against the P3 allele, as the P1P3 allele will close the pod in comparison, it can be concluded that the P1 allele is also doiminant towards the P2 allele. 18. The P 1 allele is ____ to the P 3 allele. a. dominant b. recessive c. incompletely dominant d. neither allele is dominant or recessive to the other 19. Explain how the information that GUS and IRMA provided supports your answer to the previous question. - The point of difference is when P1P3 allele is present, the pod fully closes, however with the P2P3 variant does not fully close. Hence, it can be concluded that P1 is the dominant allele. 20. The P 2 allele is ____ to the P 3 allele. a. dominant b. recessive c. incompletely dominant d. neither allele is dominant or recessive to the other 21. Explain how the information that GUS and IRMA provided supports your answer to the previous question. - As shown above, P2P2 allele will result in a full pod closure, while a P2P3 allele results in a partial closure. This means that the P3 allele is affecting the P2 allele is some way. If P2 was dominant against the P3 allele, the P2P3 allele should fully close. Yet, it only partially closes, and if the P2 allele was recessive to the P3 allele, the P2P3 allele should not close at all as a P3P3 allele does not close at all. hence it can be inferred that the P2 allele is incompletely dominant against the P3 allele.

Step 2: Determine which allele(s) should be added or removed from the sunstalk population. Recall that the goal is to use CRISPR-Cas9 to edit the gene for pod closure in the surviving sunstalk population, inserting - ideally - a single allele that enables the azure pods to fully close. This allele would ideally be dominant to other alleles, such that only one copy is needed to ensure that the azure pods will function properly. Also recall that of the three alleles for this gene, two alleles (P 1 and P 2 ) code for a functional protein that binds a chemical released by stalkleapers, triggering an azure pod to close. The third allele (P 3 ) fails to produce a functional protein. Unfortunately, the P 3 allele is linked to an allele for another gene that increases metal tolerance. As natural selection increases the frequency of metal tolerance in the population, it will also increase the frequency of the P 3 allele in the population. To prevent this, we must remove the P 3 allele as well as any other allele(s) that would allow for the emergence of either partial or no pod closure for sunstalks. Ultimately, we would ideally want all the sunstalks pods to close after this intervention. Using your answers to Step 1 of this appendix, please recommend which of the two remaining alleles - P 1 or P 2 - we should remove from the population as well as which of the two remaining alleles we should insert into the population. Directions: Use your answers to questions 16-21 to answer questions 22-25. 22. Which allele - P 1 or P 2 - should be removed from the sunstalk population? a. P 1 should be removed b. P 2 should be removed 23. Explain how the information that GUS and IRMA provided supports the claim you made in the previous question. - As determined above, the P1 allele is dominant over both the P2 allele and P3 allele, whereas the P2 allele is incompletely dominant against the P3 allele, which causes only a partial closure. Hence the P2 allele should be removed in order for the P1 allele to be more prevalent and produce offspring that can fully close no matter if it is paired with a P2 or P3 allele.

24. Which allele - P 1 or P 2 - should be added to the sunstalk population? a. P 1 should be added b. P 2 should be added 25. Explain how the information that GUS and IRMA provided supports the claim you made in the previous question. - As determined above, the P1 allele is dominant over both the P2 and P3 allele, hence it should be added in order to produce offspring with the phenotype that can fully close the pod, as both P1P2 alleles and P1P3 alles can fully close.