GENETICS the study of hereditary transmission and variation. In exercises conducted in the last lab, you learned about the movement of chromosomes within a cell during both mitosis and meiosis. However, you didn’t learn how cells get the particular chromosomes they have, or how those particular chromosomes produce different “traits” in different organisms. This is what you will work on in the current lab. Gregor Mendel is considered the “father of genetics”, yet he had no idea of chromosomes, genes or DNA. He only knew that parents passed on traits to their offspring, and did so in predictable patterns. Thus, you are going to begin your study of genetics with much more information than Mendel could have hoped for in the late 1800s. We now know, for example, that hereditary information is encoded in GENES, sequences of DNA which code for a single protein, and that CHROMOSOMES are very long strands of DNA containing hundreds or thousands of genes. We also know that mitosis produces exact copies of diploid cells, while meiosis reduces diploid cells to haploid cells and results in the production of gametes. Each diploid cell has two copies of every chromosome, one from the mother and one from the father; these chromosomes are called HOMOLOGOUS CHROMOSOMES, and the different forms of a gene are called ALLELES. Even without this wealth of information, Mendel was able to formulate two basic laws concerning chromosomal arrangements in gametes. The first is the LAW OF SEGREGATION, which states that during the production of gametes the two variations (alleles) of one gene segregate so that offspring acquire one allele from each parent during meiosis (Anaphase I), allowing for predictable ratios of traits in the offspring. The second is the LAW OF INDEPENDENT ASSORTMENT, which states that when two or more genes (traits) are inherited, the alleles of those different genes assort independently into the gametes. In other words, the allele a gamete receives for one gene does not influence the allele received for another gene. Using these two laws, one can determine 1) the ratio of different gametes any given genotype can produce, and 2) the ratio of offspring genotypes and phenotypes produced by the cross of any two individuals. These will be illustrated in the following exercises. Watch the following video to gain a better understanding of the relationship between genes and alleles https://youtu.be/pv3Kj0UjiLE Watch the following video to learn how to use Punnett Squares to predict genotype and phenotype probabilities of offspring https://youtu.be/i-0rSv6oxSY MENDELIAN GENETICS IN CORN Corn is an excellent species to use for analysis of patterns of inheritance because a single ear of corn is actually a collection of hundreds of offspring (kernels). Thus, if the genotypes of the parents are known, it is possible to easily observe the ratios of phenotypes in a large number of offspring relatively quickly. The corn we will use for the following exercises is similar to that which you eat. Several visible (phenotypic) differences exist and we will use these differences to examine patterns of inheritance. The two phenotypic traits we will observe involve the color and shape of the kernels. Corn has a protein layer in the seed coat (the aleurone layer), which can be either yellow or purple. There is a single gene which codes for the aleurone protein, with the purple (R) allele dominant to the yellow (r) allele. Corn also has the capacity to store carbohydrates as starch or simple sugars, and this capacity is also determined by a single gene with two alleles. Kernels which store carbohydrates as starch have smooth (S) surfaces, while those which store carbohydrates as simple sugars have a wrinkled (??) surface. The S allele is dominant to the ??? allele. In the following exercises you will be provided with ears of corn that represent the F2 generation. The P generation (the parent-generation) is a cross of one double homozygous dominant individual (RRSS) and one double homozygous recessive individual (????????), ???????? ?? ????????. You will need to determine the genotype of the F1 generation (offspring of the P generation cross), and the F2 generation (offspring of the F1 generation cross). *Note, in cases where the letter designation for dominant and recessive alleles look similar, we draw a line over the recessive allele, as seen with the alleles for smooth vs wrinkled, S = dominant; ??? recessive. These traits are easy to examine because they are controlled by a single gene, have only two alleles for each gene, and exhibit a model of COMPLETE DOMINANCE. In the model of complete dominance, individuals with heterozygous genotypes express the phenotype of the dominant allele only. There are two other models of dominance with which you should be familiar: in INCOMPLETE DOMINANCE heterozygous individuals express a phenotype that represents a mixture of the two alleles, while in CO-DOMINANCE heterozygous individuals express a phenotype that maintains both alleles in their original form. As an example, imagine a gene that controls petal color in flowers with two alleles, W for white petal and ?? for red petals. A heterozygous (????) individual would have white flowers if the alleles exhibited complete dominance, pink flowers if the alleles exhibited incomplete dominance, and white flowers with red spots if the alleles exhibited co-dominance. As an aside, many traits are controlled by more than one gene, or have more than two alleles for each gene, making them more difficult to study. 2 EXERCISE 1: Monohybrid Cross in Corn – Coat Color In this exercise, you will observe the phenotypic frequencies of corn kernels with respect to coat color. Remember that R is the allele for purple seeds and is dominant to the r allele for yellow seeds. Materials: Procedure: 1. Use the given P generation genotypes to fill in the P generation cross: ???? ?? ???? 2. Use a Punnett square to determine the possible F1 genotypes. The gametes produced by one parent are shown in blue on the left while the gametes produced by the other parent are shown in yellow above. The genotypes of the offspring (F1 generation) formed by combining gametes from both parents are shown in the boxes. r r R R The predicted genotype ratio among F1 individuals can be determined by comparing the numbers of each F1 genotype in the Punnett square. In this case, there is only one genotype, so the genotype ratio for the F1 generation is “all Rr” or “100% Rr”. The predicted phenotype ratio among F1 individuals can be determined by assigning phenotypes to each genotype in the Punnett square and comparing the numbers of each F1 phenotype. Again in this case, there is only one phenotype, so the phenotype ratio for the F1 generation is “all purple” or “100% purple”. Because each parent can contribute only one type of gamete, all of the offspring are expected to be the same. A cross between a homozygous dominant individual and a homozygous recessive individual will always yield predicted genotype and phenotype ratios of 100%. 3 3. The F2 generation is formed by crossing two individuals from the F1 generation. Use the data obtained in the Punnett square in #2 above to determine the F1 generation cross: ________ X _________ 4. Use a Punnett square to determine the possible F2 genotypes. R r R r As above, the predicted genotype ratio among F2 individuals can be determined by comparing the numbers of each F2 genotype in the Punnett square. The ratio should always be written with the numbers associated with each genotype. 4a. The predicted genotype ratio for the F2 generation is _____RR : _____ Rr : _____ rr. The predicted phenotype ratio among F2 individuals can be determined by assigning phenotypes to each genotype in the Punnett square and comparing the numbers of each F2 phenotype. Since both RR and Rr genotypes produce purple phenotypes, there are three purple phenotypes and one yellow phenotype represented in the Punnett square. The ratio should always be written with the numbers associated with each phenotype. 4b. The predicted phenotype ratio for the F2 generation is _____ purple : _____ yellow. Because each parent can contribute more than one type of gamete, all of the offspring are not expected to be the same. A cross between two heterozygous individuals (with identical genotypes) will always yield a predicted genotype ratio of 1 (homozygous dominant) : 2 (heterozygous) : 1 (homozygous recessive), and a phenotype ratio of 3 (dominant) : 1 (recessive). Read #5-6 completely before continuing. If you do not understand, ask your instructor to explain. 5. Test your predicted phenotype ratio (essentially, a hypothesis) by counting 100 corn kernels at random (several adjacent rows). In order to avoid counting 100 kernels three different times, we will count only once (for the dihybrid cross) and extract the relevant data for each exercise. Your instructor will explain the appearance and variation within the different phenotypes. Make sure you understand what you are doing before you start so that you have to count kernels only once. When you are finished, record the number of kernels of each phenotype in the left-hand column of the table at the bottom of page 69. The total should add up to 100. Now combine your data with the class, recording the class total for each phenotype in the center column in the table on page 69. This will serve to increase your sample size and reduce the likelihood that you will count more of one phenotype by chance (this is known as sampling error). If this does not seem to make sense, think about flipping a coin. You have a 50/50 chance of flipping heads. If you flip the coin four times, then you are predicted to get heads twice and tails twice but you may actually get four heads instead. If you flip that same coin 1,000,000 times, you’re more likely to get 50% heads. You should be a little freaked out if you flip heads a million times in a row! If there are six groups in your class, the total of all four phenotypes should add up to 600. 4 6. Since we are only dealing with the color phenotype in this exercise, we need to extract the appropriate information from the data in the table on page 69. This can be done by compiling the data for the two phenotypes that have purple kernels, and separately for the two phenotypes that contain yellow kernels, and recording them in the table on the following page. The totals for your data and the class data should still add up to 100 and 600, respectively. Phenotype Number of Kernels (Your Data) Number of Kernels (Class Data) Mendel’s Expected Phenotype Ratio Class Actual Phenotype Ratio Purple 3 # of purple kernels/ # of yellow kernels Yellow 1 # of yellow kernels/ # of yellow kernels Total 100 (Record below) You can now determine the actual phenotype ratio of the kernels (remember that each kernel is an F2 individual resulting from an F1 cross). In order to determine the ratio from the class data, find the phenotype with the smallest number of individuals (kernels), then divide both numbers by that smallest number. If you have done it correctly, you should end up with a ratio of something to one (x:1). Record the actual F2 phenotype ratio below (round ratios to the nearest tenth; 0.1). Remember to indicate the phenotypes (in this case, the colors), not just the numbers. ____________________ : ____________________ EXERCISE 2: Monohybrid Cross in Corn – Coat Texture In this exercise, you will observe the phenotypic frequencies of corn kernels with respect to coat texture. Remember that S is the allele for smooth seeds and is dominant to the ??? allele for wrinkled seeds. Refer back to Exercise 1B or ask your instructor for help if you have trouble with this exercise. Materials: Ear of F2 corn Procedure: 1. Use the given P generation genotypes to fill in the P generation cross. __________ X __________ 2. Use a Punnett square to determine the possible F1 genotypes. 5 a. What is the predicted genotype ratio for the F1 generation? b. What is the predicted phenotype ratio for the F1 generation? c. Are all F1 individuals expected to be the same? 3. The F2 generation is formed by crossing two individuals from the F1 generation. Use the data obtained to determine the F1 generation cross: __________ X __________ 4. Use a Punnett square to determine the possible F2 genotypes. a. What is the predicted genotype ratio for the F2 generation? b. What is the predicted phenotype ratio for the F2 generation? c. Are all F2 individuals expected to be the same? 5. Test your predicted phenotype ratio (i.e., your hypothesis). Extract the appropriate information from the data in the table on page 69 and record it in the table below. Refer to instructions #5-6 in Exercise 1B. Phenotype Number of Kernels (Your Data) Number of Kernels (Class Data) Mendel’s Expected Phenotype Ratio Class Actual Phenotype Ratio Smooth 3 # of smooth kernels/ # of wrinkled kernels Wrinkled 1 # of wrinkled kernels/ # of wrinkled kernels Total 100 (Record below) Record the actual F2 phenotype ratio from the class data below (round ratios to the nearest tenth; 0.1). Remember to indicate the phenotypes, not just the numbers. ____________________ : ____________________ 6 EXERCISE 3: Dihybrid Cross in Corn In a dihybrid cross we are following the patterns of inheritance of two different traits (genes). In the following exercise, you will determine how the previous traits, coat color and coat texture, combine in the offspring. To the uninformed, it might seem like the dominants should always travel together, so that when you have purple seeds you will also have smooth seeds. This isn’t necessarily the case, however. As long as the two genes reside on different chromosomes (i.e., are not linked), they should assort (combine) independently of one another in the offspring (Mendel’s second law). We will test this assumption. Materials: Ear of F2 corn Procedure: 1. Use the given P generation genotypes for a dihybrid cross to fill in the P generation (pg 63, paragraph 4). __________ X __________ 2. Use a Punnett square to determine the possible F1 genotypes. Remember that each gamete will have two alleles, one for color and one for texture. (Technically, this Punnett square should have 16 squares. Why do we only have 4 squares?) Hint: Ask your lab instructor. a. What is the predicted genotype ratio for the F1 generation? b. What is the predicted phenotype ratio for the F1 generation? c. Are all F1 individuals expected to be the same? 3. Use the data obtained to determine the F1 generation cross. __________ X __________ 7 4. Use a Punnett square to determine the possible F2 genotypes. Again, remember that each gamete will have two alleles, one for color and one for texture. Compare the results from your Punnett square to the predicted genotype ratio below: 1 RRSS : 2RRSs : 1 RRss : 2 RrSS : 4 RrSs : 2 Rrss : 1 rrSS : 2 rrSs : 1 rrss Assign phenotypes to each of the genotypes in the Punnett square and calculate the predicted phenotype ratio for the F2 generation. Record it below, and have your instructor verify that your Punnett square and expected phenotype ratio are correct for this exercise. _____________________ : _____________________ : _____________________ : _____________________ A dihybrid cross between two doubly heterozygous (i.e., heterozygous for both traits) individuals with identical genotypes will always yield these predicted genotype and phenotype ratios. 5. 6. Test your predicted phenotype ratio (hypothesis) above using the data from the table below. Record the actual phenotype ratio from the class data below (round to the nearest tenth; 0.1). Phenotype Number of Kernels (Your Data) Number of Kernels (Class Data) Mendel’s Expected Phenotype Ratio Class Actual Phenotype Ratio Purple, Smooth 9 PS/YW Purple, Wrinkled 3 PW/YW Yellow, Smooth 3 YS/YW Yellow, Wrinkled 1 YW/YW Total 100 (Record Below) _____________________ : _____________________ : _____________________ : _____________________ Color each unique genotype with a different color (same genotype, same color) and see if you can find the pattern. 8 EXERCISE 4: Visible Phenotypes in Humans In this exercise, you will examine some traits possessed by you and your classmates. Notice as you proceed through the list that just because a trait is common in a population does not necessarily mean that it is controlled by the dominant allele. Procedure: 1. Read the descriptions of each trait and fill in your phenotype in the table below. 2. List your genotype in the table. For example, if you have the recessive trait for gene N, your genotype is homozygous recessive nn. If you have the dominant trait for gene N, your genotype is not fully known, so fill in N_ on the chart. If you know that one of your parents is recessive for that trait, you must have received a recessive allele (n) from him/her, so your genotype is Nn. 3. Give your phenotype to your instructor so he/she can provide you with the phenotypic results for the class. Use the in the chart to analyze phenotypic results for a group. Widow’s peak The W allele for a widow’s peak (pointed hairline) is dominant to the ?? allele for a straight hairline. Bent little finger The B allele for a pointed little finger is dominant to the b allele for a straight little finger. You have the bent little finger if you lay your hand flat on the bench and the last joint of your little finger bends toward your fourth finger. Pigmented iris The P allele produces pigment in the front layer of the iris, resulting in green, hazel, brown, or black eyes. This is dominant to the ?? allele, which does not produce pigment in the front layer, and results is blue or gray eyes (you are seeing the color of the back layer of iris). Hitchhiker’s thumb The H allele for non-hitchhiker’s thumb is dominant to the h allele for the hitchhiker’s thumb (you can bend your thumb back at an angle of 60° or more at the last joint. Interlacing fingers Fold your hands together with your fingers interlaced. If your left thumb crosses over the right, you have the dominant allele C. The ?? allele is recessive, with the right thumb crossing over the left. Red Hair The G allele for any hair color other than red is dominant to the g allele for red hair. Acondroplasia The D allele for acondroplasia, extremities disproportional (smaller) to body size, is dominant to the d allele for average height and proportioned extremities. Characteristic Your Phenotype Your genotype # of Dominants in class # of Recessives in class Widow’s peak 3 21 Bent little finger 17 7 Pigmented iris 18 6 Hitchhiker’s thumb 20 4 Interlacing fingers 5 19 Red Hair 0 24 Achondroplasia 0 24 5. Considering the class results in the table on the previous page, which traits have more individuals with the recessive phenotype than the dominant phenotype? 6. Individuals with the dominant phenotype have an ambiguous genotype, in that we know they have at least one dominant allele (e.g., “G_”). Without analyzing the individual’s genes or DNA, what additional information could we use that might tell us whether the second allele is dominant (homozygous) or recessive (heterozygous). 9 Watch the following video to see how some genes follow different inheritance models https://youtu.be/YJHGfbW55l0 Photo of Gregor Mendel (1880). Mendel is considered to be the founder of the science of genetics. His experiments on pea plants in the 1850s-1860s led to his discovery of many of the rules of heredity that we now refer to as the laws of Mendelian inheritance. While he postulated that traits were inherited as discrete units, he did not know what those were. It was not until the1900s that scientists tied together Darwinian evolution, Mendelian genetics, and the discovery of DNA to figure out that genes were the mechanism by which traits are passed from generation to generation. Public domain photograph from the frontispiece of Mendel’s Principles of Heredity: A Defence (1909) taken from Wikimedia Commons 10 Name__________________________________________ LAB 7 QUESTIONS: 1. Coat color in corn kernels exhibits the complete dominance model, resulting in either purple or yellow kernels. What additional phenotype might we expect if coat color exhibited a model of co-dominance? What additional phenotype might we expect if coat color exhibited a model of incomplete dominance? (1 points) Co-dominance: Incomplete dominance: 2. If you are given the phenotype of an F2 kernel (purple/smooth, purple/wrinkled, yellow/smooth, or yellow/wrinkled), can you know the genotype of that kernel? If so, which ones can be determined, and which can’t? Explain. (2 points) 3. Examine the actual phenotype ratio from Exercise 3 and compare it to the predicted phenotype ratio. Do the predicted and actual ratios match exactly? Provide a possible explanation for why the actual ratio didn’t match the predicted ratio exactly. (2 points) 4. Which of Mendel’s laws are you exploiting in the analysis of a dihybrid cross? Explain. (2 points) 5. Are dominant characteristics always more frequent in a population than recessive characteristics? (1 points) 6. Two heterozygous parents mate. What is the chance of them having an offspring that is homozygous recessive? (1 point) 7. You meet someone with a straight little finger. Can you determine their genotype? Why or why not? 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