Tuominen_StructuredInquiryCotyledonPhenotyping_v2

Learning Activity Template Life DiscoveryEd Digital Library Portal (http://lifediscoveryed.org/) Title: A Structured Inquiry Approach to Cotyledon Phenotyping Author(s): L.K Tuominen Natural Sciences Department Metropolitan State University St Paul, MN Lindsey.Tuominen@metrostate.edu Abstract: Transmission genetics labs have long been valuable hands-on explorations in undergraduate Introductory Biology and Genetics courses While such labs are strongly analytical, they often present the scientific process as artificially linear and deductive, with a single, straightforward “right answer.” To more accurately represent the roles of inductive logic and uncertainty in the scientific thought process, I have developed a structured inquiry approach to transmission genetics labs grounded in cotyledon color Student lab groups work experimentally to develop a partial answer to the question “How plants inherit cotyledon color?” as it relates to Mendelian inheritance Group members develop their own two-part hypothesis, predicting what phenotypes they expect to see and providing underlying biological reasoning for their prediction based on limited, contextualized information Each group receives its own blinded set of F2 seeds that differ from the seeds some or all other groups receive Group members work together to define the relatively subjective boundaries between colors based on actual observations, then use chi-squared tests to evaluate their initial hypothesis Optional and advanced activities can further enrich scientific thinking in this lab by integrating hypothesis refinement, additional scientific uncertainty, plausible alternative explanations, and designing new experiments Learning objectives: Successful students will be able to:            Distinguish between phenotypes and genotypes Distinguish between characters and traits Use terminology to describe plant breeding experiments involving multiple generations Develop a two-part hypothesis (prediction and underlying reasoning) Make judgements to categorize non-quantitative phenotypes Summarize quantitative data (e.g., tabular data, phenotypic ratios) Carry out a chi-squared analysis Distinguish between statistical and biological hypotheses (e.g., interpret p-values) Assess the validity of a hypothesis based on experimental evidence Propose reasonable phenotypic ratios for monohybrid crosses, incomplete dominance, and/or dihybrid crosses (depending on instructor preference) Optional: Describe the role of plant pigments in producing different leaf colors Learning Activity Template        Life DiscoveryEd Digital Library Portal (http://lifediscoveryed.org/) Optional: Infer possible genotypes based on phenotypes Advanced: Describe the role of biosynthetic pathways in producing pigments Advanced: Identify and record pertinent observations beyond those dictated by the experimental design Advanced: Recognize scientific uncertainty Advanced: Revise or develop a new hypothesis based on experimental results Advanced: Infer F1 and P generation genotypes based on F2 phenotypes Advanced: Propose reasonable phenotypic ratios for epistatic interactions between two genes Timeframe: Instructor preparation time: About two or three hours to gather and prepare materials, assuming that nothing (including seed sources) needs to be ordered from suppliers Preparing labeled, blinded seed packets is the most time-intensive task Instruction and student work: About four hours of class time, distributed unevenly across two or three weeks, depending on the species of seed used Optional or advanced activities will require additional time Observations will be brief (less than twenty minutes) until the majority of seeds have germinated, so other lab activities can be carried out during this time If the instructor takes responsibility for setting up the experiment in advance, students can develop hypotheses and carry out phenotyping on pre-germinated seeds in a single 3-hour lab period List of materials: The classroom will need:  F2 generation seeds from controlled crosses with distinct leaf color traits o While instructors may use any F2 generation seeds to which they have access, commercially available examples include:  Carolina Biological’s Tobacco Seed, Green:Albino 3:1 (#178400)  Carolina Biological’s Tobacco Seed, Green:Yellow Green:Yellow 1:2:1 (#178410)  Carolina Biological’s Tomato Seed, Green:Yellow Green:Yellow 1:2:1 (#178670)  Carolina Biological’s Genetic Corn Seed, Green:Albino (#17730)  Carolina Biological’s Sorghum Seed, Red:Green (#178080)  Wisconsin Fast Plants® F2 Seed, Non-Purple Stem, Yellow-Green Leaf (Carolina Biological Supply, #158888) o I recommend using two or three distinct seed sources, so that each group may or may not be growing the seeds from the same source as neighboring groups Learning Activity Template   Life DiscoveryEd Digital Library Portal (http://lifediscoveryed.org/) o One of the seed sources may be a wild type of the same species for which an F2 cross will be used (advanced option) o One of the seed sources may involve a dihybrid cross with epistasis (advanced option) Instructor lab preparation notebook to record seed source blinding Sunny shelf, growing rack with fluorescent lighting, or greenhouse access Each group will need:         One labeled, blinded packet of seeds prepared from one of the above seed sources (about 100 seeds total; a coin envelope or small plastic bag will be suitable) One Petri dish (55 mm diameter) Three Whatman filter papers or similar (50 mm diameter) Bottle of distilled water Dissection needle Parafilm Wax pencil or labeling tape with marker Hand lens Procedure and general instructions (for instructor): Course Context: This exercise is ideal for the early weeks of a genetics course or for a genetics module in a general biology course Students should have already been introduced to Mendelian genetics so that they are familiar with relevant terminology (e.g., genotype vs phenotype; P, F1, and F2 generations; monohybrid crosses) Students in a general biology course have been successful when provided minimal initial exposure to this information in lab worksheets These students received more detailed information during the next lecture while developing lab reports outside of class Students in upper level courses should be given more responsibility for the process and should be expected to address one or more of the optional and advanced features described herein Extensions on basic Mendelian principles (e.g., dihybrid cross, codominance and incomplete dominance, Punnett squares, and Chi-squared analyses) and/or more advanced genetics topics (e.g., epistasis, the role of genes in biochemical pathways) can be taught during the lecture portion of class before this activity begins or while the lab is in progress, depending on whether any related optional or advanced activities will take place Preparations Before Lab: Prepare one seed packet per lab group as follows:  For each group, place about 100 seeds from a single seed source into a small plastic bag or coin envelope Learning Activity Template    Life DiscoveryEd Digital Library Portal (http://lifediscoveryed.org/) Mark the packet with a code (e.g., A) before moving on to the next packet Every group’s packet may be marked with a distinct code (e.g., A, B, C, D, E…); alternatively, each group with the same seed source could have the same code marked on the packets (e.g., A, A, B, A, B…) The second approach can allow different groups working with the same seed source to compare results after drawing conclusions (see Student Data for an example of why this is may be pedagogically valuable) Record in the lab preparation notebook the seed source that correlates with each code Next, prepare one tray of experimental materials per group containing one seed packet and each of the other seven items described in the group-specific List of Materials above Finally, set up a growing space large enough for all groups to place their Petri dishes unstacked on a well-lit surface at or above 20oC Natural sunlight and close-set artificial lights will be suitable, while regular classroom lighting will generally be too dim Introducing Students to the Research Question: The lab is introduced to the class as an inquiry-based introduction to the scientific thought process This particular lab is a “structured inquiry” in the sense that the inquiry question and general procedures are pre-assigned, but groups must develop their own hypotheses, make some judgements in recording data, and draw their own conclusions based on that data (Banchi and Bell 2008) Students are informed that the inquiry question for this lab project is, “How plants inherit cotyledon color?” As an introductory exercise, students can discuss within their groups ideas about how inheritance works to refresh their knowledge of transmission genetics from the non-lab portion of the course The instructor can provide brief confirmatory feedback and should then describe to the class that cotyledons are leaf-like structures that develop within a seed during embryogenesis These will be the first “leaves” observed after germination and may look quite different from the first true leaves a plant develops later on If desired, additional seedling anatomy can be described Next, groups can begin thinking about the research question more directly by brainstorming as many potential cotyledon color traits as possible based on what they have previously observed in horticultural or natural settings (as opposed to what colors they speculate might be possible) The instructor can supply reasonable student answers with information on the pigments involved in producing each color For instance, the green color of leaves is due mainly to the pigment chlorophyll, which is required for photosynthesis in terrestrial plants Anthocyanins contribute to red and purple leaf coloration and are produced in response to a variety of biotic and abiotic stressors If chlorophyll is broken down, yellow or orange colors may emerge, which are due to the presence of carotenoids (sometimes in combination with anthocyanin) One group of carotenoids is the xanthophylls, which are yellow and help dissipate excess light energy to prevent damage to the photosynthetic “machinery” in Learning Activity Template Life DiscoveryEd Digital Library Portal (http://lifediscoveryed.org/) cells The other group is the carotenes, which range from red to orange to yellow as concentration decreases Carotenes help transfer light energy to chlorophyll Both xanthophylls and carotenes also have the ability to scavenge oxygen radicals produced during photosynthesis Finally, some plants exhibit leaf patches or entire leaves that are white because they lack pigments Student Hypothesis Development: Students can now begin the hypothesis development phase, which typically takes about an hour when combined with the introductory exercises Groups should be informed that they will be receiving unknown seeds and some details about these seeds I typically note the species of the seeds, that the seeds in any given packet are all from the same cross, that different packets may come from different crosses, and (for an introductory course) that both grandparents in every cross were true breeding for cotyledon color Groups then work separately to generate a hypothesis about the phenotypic ratios for cotyledon color they would expect to see in their own seeds The hypothesis should include both a prediction and some underlying biological reasoning The prediction should be exclusively based on observation, while the biological reasoning should link in some way to their knowledge of both leaf color traits discussed while brainstorming and transmission genetics The hypothesis generation step is intentionally both guided and based on limited information It would often require a lucky guess for a group’s predictions (and therefore hypothesis) to be fully supported after the data analysis phase This intentional limitation of a priori knowledge is intended to help students move away from the desire to get the “correct” answer on the first try and towards the use of the hypothesis as a tool to help identify what new information has been learned through observation The former is a pedagogy to which most students have been thoroughly enculturated, while the latter better represents the practice of scientific thinking I recommend instructor feedback after groups have developed their hypotheses I have provided feedback both as a formal exercise in an upper level genetics course (i.e., instructor review of lab notebooks documenting the thought process and grading based on a rubric) and as an informal one in a general biology course (i.e., instructor conferral with groups after they have developed their hypothesis, with approval required before the group can begin the experimental phase) Setting Up the Experiment: This stage can be carried out immediately after hypothesis generation, although it may also be delayed until after students read and reflect on formative feedback related to their group’s hypothesis Students will place three filter papers in the bottom of a Petri dish and wet the papers with water All layers of filter paper should be thoroughly soaked, but excess water should be poured off Seeds can then be transferred from the packet to the top layer of filter Learning Activity Template Life DiscoveryEd Digital Library Portal (http://lifediscoveryed.org/) paper If desired, students can spread out their seeds across the filter paper using a dissection needle The Petri dish can then be closed and sealed with Parafilm Each group should label its own dish so that they can identify it later, then transfer the dish to a well-lit area This portion of the work typically takes about 20 of class time when groups are sharing some resources As a time-saving alternative, the instructor can set up individual petri dishes several days prior to the lab The length of advance planning will depend on the species (e.g., tobacco will take about 7-10 days to germinate, while Fast Plants® will take about 3-5 days) This would remove student responsibility for experimental setup and interim observations to determine if germination has proceeded in a large enough proportion of the population to complete phenotyping, so it is most suitable for scenarios in which the exercise needs to be restricted to a single lab period When I have implemented this option in an introductory course, students were responsible for documenting the environmental conditions where the seedlings had been germinated Phenotype Observations: The data collection phase may require parts of 1-3 lab periods, depending on the germination times of the species used Full data collection is not necessary until a majority of the seeds have germinated Observations before this stage should be qualitative, focusing on what cotyledon colors are observed and which appear to be more abundant or less abundant This is also a good time for group discussions to determine how each cotyledon color trait will be delimited This typically takes less than 20 minutes of class time each day Final data collection should consist of actual counts of each color trait It is often helpful to encourage students to make additional observations related to seeds that cannot be phenotyped (e.g., seed did not germinate, seedling germinated but died, mold was present on seed or seedling), as this can help groups more explicitly consider sources of error Final data collection typically takes about 30 minutes of class time Analysis of Qualitative and Quantitative Data: Each group uses its experimental data to assess its initial prediction based on both qualitative analysis and a chi-squared test Each group should record its work and findings in the project lab notebook This phase and the Drawing Conclusions phase together typically require about 30-45 minutes; one or both can be carried out immediately after data collection if desired Additional details are provided in the student handout Qualitative analysis focuses on the categories of cotyledon color and their relative abundance Note that “unexpected” relative abundances can be produced by including a wild type seed line or a seed line with a dominant allele that generates a cotyledon color other than green The latter can create opportunities for later discussion about different ways mutations influence gene function Learning Activity Template Life DiscoveryEd Digital Library Portal (http://lifediscoveryed.org/) Quantitative analysis is based on the chi-squared test The “expected” cases are based on calculating expected counts of each phenotype category from the specific numerical ratios the group initially predicted, while the “observed” cases are based on the phenotypic categories and counts within each category the group actually measured Undergraduate genetics textbooks often provide a step-by-step procedure and a table for determining 2 and its associated p-value, respectively (e.g., pp 101-104 in Hartl 2014) Detailed descriptions of the chi-squared method and software tools carrying out calculations are also available online (e.g., McDonald 2014) Students often need guidance in recognizing that they should base the calculation of expected seed numbers of each trait on the number of seeds actually phenotyped for cotyledon color, rather than the total number of seeds initially “sown” on the Petri dish Some students may need a reminder of how many degrees of freedom are involved in the test; this is simply the number of phenotypic categories minus one Finally, students usually need guidance is in interpreting the meaning of p-values This may be the case even where students have completed other statistics or lab-based courses, due to the particular way in which chi-squared analysis is used in genetic inheritance The statistical (null) hypothesis for the chi-squared test is based directly on the biological prediction: if the p-value is less than 0.05, we have demonstrated that “the ratio does not fit” and must test another hypothesis about the inheritance ratio In contrast, most biological experiments use the statistical hypothesis as a quantitatively testable straw man argument: if the p-value is less than 0.05, we have demonstrated that “a difference does exist” and the biological prediction (i.e., reason for carrying out the experiment) is supported Because of these challenges, I recommend groups work on the quantitative analysis at least partly during lab time This allows time for students to raise questions within their group and while the instructor is present Groups can also work outside of class on this phase if additional time is needed Instructor feedback may be helpful at this phase Drawing Conclusions: After qualitative and quantitative analysis, each group should have enough information to more thoroughly evaluate its initial hypothesis and sources of error This phase typically requires no more than 45 minutes, including both time for group discussion and developing a summary paragraph describing the group’s conclusions If additional time is available, other activities can be implemented at this phase Some of these are described in Advanced Options I recommend a summative assessment of the lab notebook or of a lab report when this phase is completed Once each group has turned in its lab notebook or lab report for the project, all groups may check their conclusions against the seed packet “key” to find out the actual type of seed they have grown Learning Activity Template Life DiscoveryEd Digital Library Portal (http://lifediscoveryed.org/) Procedure and general instructions (for students): Note to instructors: A project overview and description of each project phase are shown beginning on the next page This document represents the way I have typically taught this module using tobacco seeds in a twice-weekly genetics course with integrated lecture and lab For an upper-level course, this module is designed to be the first lab activity of the semester The version presented here does not directly address most of the optional or advanced activities, but it is compatible with some of them, such as Developing and Testing New Hypotheses (see Advanced options) I recommend providing students with instructions like this after the introductory refresher and brainstorming activities It may be possible to follow up with activities such as Inferring F1 and P generation Genotypes and Phenotypes from F2 Individuals as a class or homework exercise after groups turn in their lab notebooks or a lab report Finally, the instructor will need to replace the [###-###] and [###] in Phase IV with page numbers from a relevant textbook Alternatively, an online source such as that previously described could be used (McDonald 2014) Learning Activity Template Life DiscoveryEd Digital Library Portal (http://lifediscoveryed.org/) Lab Project: Cotyledon Phenotypes and Genotypes In this project, we will explore the inquiry question: “How plants inherit cotyledon color?” using standard methods in transmission genetics Today your group will receive a packet of “unknown” seeds All seeds in the packet are from the F2 generation of the same cross The two grandparents (P generation) were each true-breeding for some cotyledon color, although the two P generation individuals may have had different cotyledon colors The expected project timeline is shown in the table below The phases are described in more detail afterwards Week A Week B Week C Tuesday Thursday [Lab work] Introduction, Phase I, Phase II [For next class] Turn in lab notebook for instructor review of hypothesis Reflect on instructor feedback, refine hypothesis if needed, water seeds if needed, Phase III (qualitative) if possible Water seeds if needed, Phase III (qualitative) N/A N/A Phase III, Phase IV; Phase V if null hypothesis is rejected Phase V, Phase VI, and Wrap-Up Complete chi-squared testing and interpretation Turn in lab notebook for instructor review of project work and findings Phase I – Hypothesis Development: Work within your group to generate a hypothesis about what sort of phenotypic ratios for cotyledon color you would expect to see in your seeds The hypothesis should include both a prediction and some underlying biological reasoning The prediction describes what you think you will observe after your seeds have germinated What cotyledon colors (i.e., phenotypes) will you see? In what ratios you expect to see them? The biological reasoning is an explanation of why you think you will observe the things you have predicted you will see For example, what pigment is responsible for producing each color? How many genes you think are involved in the inheritance of these colors? For any given Learning Activity Template Life DiscoveryEd Digital Library Portal (http://lifediscoveryed.org/) gene, how many alleles you think might exist? How does that relate to the ratio of cotyledon colors you expect to see? Record your group’s thought process and hypothesis in your lab notebook Phase II – Setting Up the Experiment: The goal of this step is to create an environment where your seeds can germinate and cotyledon development can occur        Place the three filter papers into the base of the Petri dish Soak the papers with distilled water No excess water should be present in the dish Transfer your seeds from the package into the Petri dish With a fingertip or dissection needle, distribute your seeds evenly across the wet filter paper Cap the Petri dish and wrap it with Parafilm to maintain high humidity in the dish Label you group’s Petri dish with a wax pencil or labeling tape and marker so that you can identify it later, even if it gets moved Move the sealed dish to the growing area Phase III – Phenotype Observations: During early germination, few seeds will have visible cotyledons You may take qualitative notes at this time and should discuss with your group members how many leaf color phenotypes are present and what those phenotypes are If helpful, use a hand lens to make judgments about which colors are the same and which are different You should record your decisions on color phenotypes in the lab notebook, but you not need to take quantitative data until over half of the seeds have germinated When most seeds have germinated, your group should record quantitative data First, identify the distinct cotyledon color phenotypes, then count the number of seedlings with each phenotype Again, a hand lens can be helpful You may also want to record the number of seedlings that have died due to drought or infection and the number that have not yet germinated on the day of data collection These “no cotyledon color recorded” categories can be helpful for thinking about data quality and potential sources of experimental error Phase IV –Analysis of Qualitative and Quantitative Data: The goal of this phase is to use your experimental data to assess your group’s initial predictions about cotyledon phenotypes and the ratios in which they would occur As before, you should record your group’s thinking in your lab notebook Qualitative analysis: Did you see the cotyledon color traits you predicted? If not, what other traits did you see instead? You may also want to think about whether particular leaf traits were more abundant or less so than you predicted, without necessarily thinking about specific numbers or ratios Learning Activity Template Life DiscoveryEd Digital Library Portal (http://lifediscoveryed.org/) Quantitative analysis: Use a chi-squared test to determine if your group’s data are consistent with what you predicted you would see The procedure for carrying out this test is provided in pages [###-###] of your textbook, and the table for determining the p-value from the 2 value and degrees of freedom is found on page [###] You will need to carry out the test on raw data (i.e., the numbers of expected and observed seedlings for each trait), instead of summary data (i.e., the expected and observed phenotypic ratios) This test involves multiple steps, so feel free to ask questions if you get stuck along the way! Phase V – Revising the Hypothesis: When a hypothesis does not align well with observations, it’s time to develop and test a new one! If your group’s seeds had different cotyledon colors than you expected, or if those seeds showed up in different relative proportions than you expected, or if you rejected your null hypothesis in chi-squared analysis (i.e., the phenotypic ratio was different from your prediction), spend some time as a group revisiting Phase I This time, you come to hypothesis development with much more information about your seeds than you had earlier Feel free to use those observations to rethink both the predicted phenotypic ratio and the underlying inheritance that would be involved in producing that ratio Once you have created a revised hypothesis (prediction + underlying biological reasoning), go back to Phase IV to show how your new prediction aligns with your qualitative data Carry out a new chi-squared test to determine whether or not you can reject the new null hypothesis Phase VI – Drawing Conclusions: The goal of this phase is to re-evaluate whether and how your ideas about cotyledon color inheritance have changed through the course of the experiment Discuss the following questions with your group members:         Do you think your data are reliable enough to evaluate your initial hypothesis? How did you decide whether or not your data were reliable? What possible sources of error you think might have influenced your experiment? If you repeated this experiment to improve data quality, what factor(s) would you change and why? Were your qualitative and quantitative results consistent with your group’s initial prediction? Why or why not? What are the implications for your group’s initial biological reasoning? Was any alternative hypothesis more compatible with your observations? Why you think so? How has your thinking changed as you moved through the experimental process? You may want to take notes on your group discussion, but it is not absolutely necessary When you’ve completed the discussion, write a paragraph to summarize your answers to these questions Once you turn in your lab notebook, I will post the seed packet key so you can validate your results Learning Activity Template Life DiscoveryEd Digital Library Portal (http://lifediscoveryed.org/) Suggestions for assessing student learning: In upper level courses, I typically require that all groups turn in a lab notebook documenting the group’s thought processes and initial experimental setup (i.e., after completion of Phases I and II) Providing formative feedback on each group’s initial hypothesis and level of procedural detail can be valuable early in the course so that students familiarize themselves with any lab notebook grading rubric and to help the instructor assess students’ comfort level with hypothesis development in a lower-stakes situation I have previously based summative assessment of the overall project in upper level courses on the lab notebook after the completion of the conclusions phase Lab Notebook Rubric shown on the next two pages was used for assessment in this case The Participation category is used to separately grade each member of the group Since individual group members rotated the note-taking role in this course, “Credit” for each member’s contributions is also included for that role as a necessary criterion to facilitate Participation grading When implementing this lab in introductory biology, I have expected students to keep a lab notebook but based summative assessment on a group lab report The Lab Report Rubric, shown after the first rubric, was applied to this and all other group lab reports throughout the semester Note that the Lab Notebook Rubric and Lab Report Rubric are not intended for use together on the same project Learning Activity Template Life DiscoveryEd Digital Library Portal (http://lifediscoveryed.org/) Lab Notebook Rubric (Upper Level Genetics): Points Participation No evidence of participation, rudimentary notes Student has clearly participated Credit (Note-Taker) Contributions are not described Some contributions are described Thought Process Unclear why the work is being carried out Student “leads” the rest of the group, taking on most of the work Documents own contributions, but not those of other group members Work is evenly divided, but student only takes on specific types of tasks All group members participate evenly in terms of effort and tasks, clearly benefitting from each other’s help Clearly documents contributions of self and others, including credit for ideas Clearly documents own work and ideas with some credit to others Some evidence of a hypothesis as rationale for the experiment or justification for work Hypothesis and conclusions are clear and consistent; conclusions basically make sense given the data Hypotheses, specific predictions, and conclusions are clear; support for conclusions are clearly based on evidence, potentially including the use of statistics; reasons for work "detours" are explained Hypotheses, specific predictions, and conclusions based on evidence are present, plus some mention of “side ideas” reflections on what new has been learned, changes in ideas midexperiment based on observation, etc (Continued on next page) Points Learning Activity Template Procedures Data Unclear and not repeatable by a reader Many parts of the procedure are mentioned It is unclear whether or not any observations have been carried out Some results are mentioned, but they are incomplete, vague or use incorrect scaling, units, etc The main features of the procedure are provided, but deviations and trouble-shooting are not mentioned Numerical data (where applicable) is recorded without any qualitative observations; any sketches of gels are not interpretable Life DiscoveryEd Digital Library Portal (http://lifediscoveryed.org/) The main features of the procedure are provided with some indication of tweaks, accidents, or troubleshooting The main features of the procedure, any deviations, and trouble-shooting steps are documented so that a reader could repeat the work without further assistance from the student Both numerical data (where applicable) and qualitative observations are recorded; any subjective data categories are clearly defined; any sketches of gels are interpretable Both numerical data (where applicable) and qualitative observations are recorded with clear descriptions of any subjective categories; observations/ data clearly connect to any modifications to procedure or changes in thinking; any sketches of gels are interpretable Learning Activity Template Life DiscoveryEd Digital Library Portal (http://lifediscoveryed.org/) Lab Report Rubric (Introductory Level General Biology): Points Introduction Hypothesis Methods Results Discussion Fewer than three sentences of background information are provided At least a paragraph of background information is provided in a separate section of the report In addition, at least one reference is cited in this section and listed in a separate section at the end of the report (The text and lab handouts are valid references.) Some justification for why the work was done is presented Research question and two-part hypothesis are present in a separate section of the report In addition, research question aligns with that presented in class; prediction and underlying reasoning in hypothesis are clear and not conflict with established knowledge discussed in class A bulleted list of work is presented instead of a narrative and/or key features of the procedure are not mentioned The main features of the procedure are provided in a narrative format in a separate section of the report, but some details are missing In addition, narrative includes a description of any changes or accidents in the procedure Some results are mentioned, but they are incomplete or vague Qualitative observations are described and (where applicable) numerical data are presented in a separate section of the report In addition, subjective categories are clearly defined, and sketches and graphs are interpretable Some interpretation of the meaning of the results is included Discussion of whether the data are reliable and what the results tell us about the hypothesis are presented in a separate section of the report In addition, the conclusions not conflict with the information provided in the Methods and the Results and link back to the “broader world” of biology (from the introduction) Learning Activity Template Life DiscoveryEd Digital Library Portal (http://lifediscoveryed.org/) Student data: Asking students to make their own judgements about seedling phenotypes can potentially have rich pedagogical benefits When teaching this module with tobacco green:yellow green:yellow 1:2:1 seed, I have noticed that student groups differ in their tendencies to “lump” or “split” ambiguous phenotypes such as yellow green and green For example, during an upper-level Genetics course, one group that received these seeds analyzed only two distinct categories despite recording three categories during their observation phase as follows : Named Phenotype Initial Observations For Chi-Squared Dark Green Light Green 21 26 Yellow 11 11 This presents a “teachable moment” related to the difficulty scientists may face in recording qualitative data, to changes that occur during seedling development, and to the similarities and differences between genotypes and phenotypes between two modes of inheritance (Mendelian dominance and incomplete dominance) One way of helping the entire class recognize scientific uncertainty through this lab module is to allow members of different groups to compare their observational categories and analytical strategies after each group draws its conclusions Advanced options: Developing and Testing New Hypotheses: Due to the relatively broad nature of the hypothesis generation phase, some groups may reject their null hypothesis during qualitative and/or chi-squared analysis More engaged students may be left with a lack of closure: “if the hypothesis was incorrect, what was the real inheritance ratio?” Furthermore, less engaged students that fail to reject the null hypothesis during chi-squared analysis may be left with an overly confident sense of the certainty of their hypothesis: “my answer was the real one, I not need to consider alternative hypotheses.” One way to further engage both types of students is to require that groups develop and use the chi-squared method to test at least one new hypothesis after completing the initial one Groups that reject their initial hypothesis are able to engage in a realistic hypothesis refinement process These students will experience the iterative nature of science in the sense that they need not start “from scratch” in their thinking, but can instead base their new predicted inheritance ratios on actual observations Alternatively, testing secondary hypotheses can highlight the need to consider multiple possibilities before settling on a single explanation as the correct one, particularly in cases where more than one hypothesis cannot be rejected (e.g., epistatic traits may sometimes be difficult to distinguish from characters determined by incomplete dominance or single-locus dominance and recessiveness) Learning Activity Template Life DiscoveryEd Digital Library Portal (http://lifediscoveryed.org/) In cases where multiple hypotheses about inheritance cannot be rejected, the entire class can gain a sense of closure by designing (but not necessarily carrying out) one or more experimental crosses By (hypothetically) growing the F2 seeds to maturity, plants with specific cotyledon colors can be selected for crossing to distinguish between the two hypotheses Inferring Multigenerational Genotypes and Phenotypes from F2 Individuals: An optional activity to conclude the project, and one that will likely provide a direct connection to transmission genetics homework problems, is to ask each group to infer the possible genotypes of its F2 seeds of each phenotype The most straightforward approach in this case is to allow groups to first compare their conclusions about their F2 seeds’ inheritance patterns against the “key.” With greater certainty about the phenotypic ratio, the group can directly determine the F2 genotypes of their seeds This activity can be further elaborated by encouraging students to determine the genotypes and phenotypes of the two previous generations of seed In cases where students have been informed that the P generation plants are both true-breeding, determining the F1 and P generation genotypes should be compatible with textbook and homework problems with the assumption that cotyledon color is determined by a single locus Once genotypes are assigned, determining phenotypes should also be straightforward: all possible phenotypes would be visible (and already associated with specific genotypes) in the F2 seeds This task becomes more complex for phenotypes involving two loci (see also Dihybrid Crosses or Epistatic Traits) In this case, it would be more valuable to have students consider the possible P generation genotypes and phenotypes instead of focusing on getting a single “correct” set While the above approach is straightforward, scientific realism can be added in classes where students have strong grounding in scientific thinking skills (e.g., an upper level Genetics course) In this case, each group can determine the F2 genotypes based exclusively on the group’s own experimental conclusions, without reference to the “key.” Determination of F1 and P generation genotypes and phenotypes can then be carried out as previously This approach allows students to deduce new information without reference to any new external information, but technical or logical errors made during analysis or while drawing conclusions can be propagated at this stage For this reason, any summative assessment of this work should be structured to avoid penalizing students multiple times for an early error Addressing the Possibility of Lethal Alleles: If students have learned about genetic lethality prior to the hypothesis generation phase, it may be helpful to tell students that seeds may not germinate, or may die after germination, due to overwatering, underwatering, fungal or bacterial growth, or other factors Lethal effects in seeds most often occur during seed development, prior to germination; as long as the seeds Learning Activity Template Life DiscoveryEd Digital Library Portal (http://lifediscoveryed.org/) not appear to be “deflated” or off-color when they are first placed on the Petri dish, it is fairly unlikely that lethal alleles are involved The multiple ways in which a seed or seedling can die represents a source of scientific uncertainty Thus, eliminating lethal genetic effects as a possibility can help students focus on other possible explanations for the observed phenotypic ratios In contrast, leaving the possibility of lethal alleles open can encourage upper-level students to record data beyond cotyledon color alone and to consider whether lethal alleles may be involved in cotyledon color inheritance Including Wild-Type Seeds: One way to guarantee unexpected F2 results is to include one or more packets of wild-type seeds in the class Ultimately, the knowledge that both individuals in the P generation were true-breeding should help most students narrow down the possibilities to focus on two homozygous wild-type parents One exception is that introducing this module after teaching about epistasis and/or complementation analysis allows an epistatic interpretation of F2 seeds that all share the same phenotype I have included wild type seeds for at least one group every time I have taught this lab project The primary challenge for the groups that receive these seeds is confusion over how to work with predicted phenotype categories that had no observations in the chi-squared analysis Once students are reminded that they expected a non-zero number of seeds in these categories and observed zero seeds with such phenotypes, they are able to complete the analysis successfully Dihybrid Crosses: If students have already learned about dihybrid crosses at the hypothesis generation stage, it would be reasonable to include at least one set of F2 seeds that show variation in a second character in addition to cotyledon color In this case, asking that groups take notes on their seeds beyond just cotyledon color will help demonstrate the scientific value of making broad observations, rather than observations exclusively focused on the hypothesis After completing chi-squared analysis based only on cotyledon color traits, groups could be asked to refine the initial hypothesis to incorporate phenotypes for the second character Testing this hypothesis would involve a more comprehensive chi-squared analysis Epistatic Interactions and Pigment Biosynthesis Pathways: If students learn about two-gene epistasis prior to or during this lab module, it would be reasonable to include at least one set of F2 seeds exhibiting epistatic interactions in cotyledon color and to require that students develop underlying reasoning for their predicted phenotypic ratios that explicitly references pigment biosynthesis as a biological process requiring one or two enzymes operating within a biochemical pathway Note that relatively low (