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Lab 2: Mendelian Genetics Why do you share traits similar to the ones that your parents have? Why is it that a certain trait that your father has was passed to your siblings, but not to you? These questions were asked for hundreds of years. Traits are simply different forms of a character. For instance, AB and O are two different traits for the character blood type. Black hair and blonde are two different traits for the character hair color. Heredity is the passing of traits from the parents to the offspring, but it was not understood until a meticulous monk grew some peas.

Gregor Mendel was born to peasant parents in 1822. He was educated in a monastery and eventually became a monk. He was sent to the University of Vienna to study science and mathematics and had aspirations to become a teacher, but he failed to pass the certification exams. He returned to the monastery and eventually became an abbot. He was active in an informal science club. The club’s members were expected to perform some type of scientific investigation and report it back to the club. Mendel chose to try to repeat earlier experiments using pea plants, but he intended to keep better track of the results of the crosses. (Notebooks and detailed note taking are essential in science!) It was his training in science

and mathematics that influenced his empirical (mathematical) approach to inheritance. Mendel prepared an experimental design and set out to determine how these traits were passed from one generation to the next. By raising thousands of pea plants over many generations, strictly controlling how pollen moved between plants (by doing it himself), and keeping amazingly meticulous notes, Mendel was able to propose a theory of heredity. He did all this with knowing less about DNA than you do. Mendel presented his paper, Versuche über Pflanzenhybriden (Experiments on Plant Hybridization), at two meetings in 1865 and received some notice. When Mendel's paper was published in 1866, it was seen as essentially about hybridization rather than inheritance and had little impact and was cited about three times over the next thirty-five years. It was not until it was rediscovered and corroborated by experimentation in the 1900’s that it was seen as a critical tome on genetics, which today is one of the most important and famous theories in the history of science. Mendel’s theory is composed of five hypotheses: 1. Parents do not transmit traits directly to their offspring. Instead, Mendel said, “factors” (we now

call them genes) are transmitted to the offspring which allow for the expression of these traits.

2. Alternative forms of a given factor lead to alternative traits. Alternate forms of a gene are called alleles. An individual’s appearance, which is due to the combination of its alleles, is called its phenotype (physical appearance) and we call the actual combination of alleles (at the DNA level) its genotype (AA, Aa, aa).

3. In diploid species, each parent contains two copies (alleles) of the factor (gene) governing each trait. The copies may or may not be the same. If they are the same, the individual is said to be homozygous (same, e.g. AA or aa). If the copies are different, then the individual is said to be heterozygous (different, e.g. Aa).

4. The presence of an allele does not ensure that a trait will be expressed in the individual that carries it. Alleles can be dominant (upper case letter, e.g., A) or recessive (lower case letter, e.g, a). Dominant alleles will usually mask the phenotype coded by recessive alleles, but the recessive alleles will still be present and may still be passed to the offspring.

5. The two alleles that an individual possesses at each gene do not affect each other’s inheritance.    

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How did he test these hypotheses? He measured several traits of thousands of pea plants. The picture below shows the characters that Mendel studied. Each character is on the left and its traits are labeled under their picture.

And he made crosses. That means he mated plants of certain traits in controlled combinations. Whenever he mated purebred plants with purple flowers to purebred plants with white flowers (the parental or P generation) all the offspring (the filial or F1 generation) had purple flowers. It seemed that the white trait disappeared. We can visualize what Mendel saw and reasoned by using a Punnett Square. A Punnett Square is a simple tool to predict the outcome of a cross because it shows how alleles from the parents separate in gametes (the sex cells: egg and sperm) and randomly recombine in the zygote after fertilization. MONOHYBRID CROSSES Since we are following two alleles from one gene, this is a monohybrid cross. To start, we have to arrange the alleles so that the alleles from one parent are across the top and the alleles from the other parent are down the side. The letters that are written at the top and along the side are a reflection of the allele within the gamete. Remember there is only one allele in each gamete since gametes are haploid. The squares allow you to randomly put one allele with another (simulating fertilization).

Phenotype Genotype

Purple flowers

RR or Rr

White flowers

rr

Female R R

Male r

r

1

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These squares simulate how the alleles segregate into gametes and then combine into offspring. The male only has the recessive r, so he will pass his recessive r into each of four sperm. Likewise, the female will contribute her R to all of her eggs.

All of the offspring Mendel grew from this cross were purple.

The phenotypic ratio is 4:0 purple to white, or 100% purple flowers. The genotypic ratio is 0:1:0. They are all heterozygous. But when he crossed two plants from the F1 generation, he found their offspring (the F2 generation) had a phenotypic ratio of 3:1 purple to white. The white trait had reappeared. Here’s that cross.

The phenotypic ratio is 3:1 purple to white, or 75% purple flowers. The genotypic ratio is 1:2:1. This means that on average, 25% of the F2 generation are RR, 50% are Rr, and 25% are rr. A single offspring has a 25% chance of being white. This is a probability, not a guarantee. Mendel reasoned that the factor controlling white flowers had not disappeared, but was hidden. He called this a recessive condition. The purple trait was dominant. He found similar results in the six other characters pictured above. The only way to get this 3:1 ratio of phenotypes is if the alleles of one gene separate from each other during meiosis (when sperm or eggs are formed) and put together during fertilization randomly. You can draw out the alternatives to prove it to yourself.

Female R R

Male r r r

r r r

Female R R

Male r Rr Rr

r Rr Rr

2 3

Female R R

Male r Rr Rr

r Rr Rr

4

Female R r

Male R RR Rr

r Rr rr

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This led him to describe what is now known as Mendel’s first law: The Law of Segregation

Mendel’s first law states that alleles in a pair segregate during gamete formation. In other words, the two alleles of a gene in the parent (one from both of its parents) separate randomly during meiosis. Therefore each sex cell only carries one of the alleles from each pair of alleles. Which allele it carries (grandmom’s or granddad’s) is random.

Because of this law, we can predict the possible outcomes of any monohybrid cross, if we know the parent’s genotypes. We can also use the ratios to determine an unknown genotype or inheritance pattern. While crosses cannot be used to accurately predict the alleles that will be passed to a particular offspring (because it is random), all the possible outcomes can be determined, along with the probabilities that the outcome will occur. A simple Punnett Square is a useful tool for determining the genotypes and phenotypes that could occur, as well as their probabilities. Taking two individuals from the F1 generation (any two, since the all individuals are genetically identical in regard to flower color), we can cross them and get the F2 generation. Try this on your own now.

What is the genotypic ratio of the F2 generation? What is the phenotypic ratio of the F2 generation?

The above situation is an example of Complete Dominance. There is a lot more to inheritance of most traits than what is described above, but typically, Mendel’s Laws apply. We will learn in class a number of extensions to Mendel’s laws.

Female

Male

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DIHYBRID CROSSES Mendel also had the wherewithal to follow two characters (two genes) simultaneously. This is now known as a dihybrid cross. Mendel crossed plants with yellow and round seeds (two dominant traits) with plants that had green and wrinkled seeds (two recessive traits). He hypothesized that these two pairs of alleles would be inherited in one of two ways. Either…

(1) The alleles are always inherited together, such that all the offspring would be either (Dependent assortment)

a. Yellow with round, or b. Green with wrinkled

Or

(2) The alleles are inherited independently, meaning that you could see all possible combinations (Independent assortment)

a. Yellow with round b. Yellow with wrinkled c. Green with round d. Green with wrinkled

Keep in mind that each gamete (sperm and egg) gets one allele from both genes. Yellow and green are encoded by the same gene but are different alleles, just as round and wrinkled are encoded by the same gene but are different alleles. So in this instance he was looking at 2 different genes that had 2 different alleles a piece. Thus in our Punnett square we will use 2 different letters to designate the different genes and upper and lower case to designate the different alleles. The figure below shows the possible outcomes of either hypothesis and his results.

Whenever he made this cross, he found that the offspring had a phenotypic ratio of 9:3:3:1. Notice how his results showed that these traits are inherited independently of each other and that the table on the right is a useful way to set this up to make predictions.

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This led him to describe what is now known as Mendel’s second law: B. The Law of Independent Assortment

The second law states that alleles of different genes assort independently of one another during gamete formation. This means that alleles from one gene do not affect the inheritance of alleles from another gene. An individual that is heterozygous in two different genes (e.g., Aa and Bb) will pass on those genes in random combinations in equal proportions (e.g., AB = Ab = aB = ab). This law comes into play when doing dihybrid crosses (see below).

Using Mendel’s method, predict the outcome of a dihybrid cross between a homozygous tall pea plant with green pods and dwarf pea plant with yellow pods. Remember that you have to represent each possible gamete and each gamete will have both genes (but no more than one allele from each gene). Use the following symbols: Gene 1 alleles:

Tall = T Dwarf = t Gene 2 alleles:

Green pods = G Yellow pods = g

What is the genotypic ratio of the F1 generation? What are the possible phenotypes and their ratios in the F1 generation?

Possible g

amete

s

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Additional Practice Problems (Given: A = normal skin color, a = albino, B = bald, b = furry) 1.  Cross  Aa  with  aa  

What is the genotype ratio? What is the phenotype ratio? 2.  Cross    Bb  with  Bb                                

What is the genotype ratio? What is the phenotype ratio?

3.  Cross AABb with aaBb  

What is the genotype ratio? What are the possible phenotypes and their ratios in the F1 generation?

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Corn Genetics In this activity, we are going to determine the genotypes of various corn plants. Each ear of corn consists of hundreds of corn grains (aka kernel or seed). Each seed is an individual offspring, i.e., the product of a cross between two parents; and each seed was created independently of the other seeds. In effect, the ear of corn is made of hundreds of babies. Imagine you perform a monohybrid cross between two corn plants. The first plant has purple grains (kernels or seeds), and the second plant has yellow grains. This cross resulted in corn ears with all purple grains. You hypothesize that this seed color trait is an example of complete dominance, with purple being the dominant trait. You cross two of the heterozygous F1 generation corn plants and predict their offspring, the F2 generation, will yield a 3 purple to 1 yellow ratio (your hypothesis). You decide to perform a chi-square analysis to make certain the ratio you expect (3:1) is what is observed. In the following cross, you observe (count) 765 purple grains and observe (count) 345 yellow seeds. Refer to Lab 1 for instructions for chi square. Your Hypothesis or the Expected Ratio is: 3:1 = ¾ : ¼ = 0.75 : 0.25 In order to determine the expected number, you first must add together all the observed individuals to determine the total number of individuals. Then, for each group, multiply the total number observed by the chosen ratio. For example, the purple grains would be: 1020 (total observed) X 0.75 (expected ratio) = 765. This means that you expected to see 765 purple grains, but you observed 745 purple grains. We would like to know if what we observed is statistically different from what we expected. In other words, is your hypothesis that purple is dominant to yellow supported by the evidence produced by this cross? You can use the chi square statistic to compare the observed and expected ratios. Chi-Square Formula χ2 = Σ (O - E)2 E Observed Expected (O-E) (O-E)2 (O-E)2/E Purple 745 765 -20 400 400/765 =0.52 Yellow 275 255 20 400 400/255 =1.78 Total = 1020 1020 χ2 = 2.3 After performing this calculation, determine the degrees of freedom, and use the Chi-square table on the in Lab 1 to determine the p values. The degrees of freedom equal the number of phenotypes - 1. How many degrees of freedom? What is the p-value?

What is the χ2 value? What do these results mean?

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BIOL 1108K Lab 2 Genetics Postlab Name______________________________ 1. Define the following terms:

a. Genotype-

b. Phenotype-

2. Fill in the genotypes of the following Punnett Square. You may have to complete the genotypes of the parents and the offspring.

3. Below, indicate the phenotype of each individual.

4. What is a testcross and what is it used for?

5. Could Mendel have deduced the law of independent assortment using a monohybrid cross? Explain your answer.

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Experiment #1. Mystery Monohybrid Cross Your lab assistant Clumsy Clyde has mixed up several corn plants, and you are unsure of the parents of an important ear of corn. You must use your genetics skills to determine who the parents are. You were fortunate enough find out from a second research assistant, Reliable Ricky, that the female parent used in the cross was heterozygous (Aa). Thus, based on the observation of recessive and dominant phenotypes in the grains of the “mystery corn”, it was deduced that the male parent could be either Aa or aa. Possibility 1: Cross Aa with Aa 6. Use a Punnett Square to determine the outcome of this possibility:

7. What are the possible genotypes and their

expected ratios?

8. What are the possible phenotypes and their expected ratios?

9. Use Chi-Square to test this expectation. Observed # Expected # (O-E) (O-E)2 (O-E)2/E Purple

Yellow

Total = χ2 =

p =

10. Using the results of your chi-square, could these be the parents of your mystery corn? Explain.    

A = purple a = yellow

Use the expected NUMBER of offspring, not the ratio, here.

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Possibility 2: Cross Aa with aa 11. Use a Punnett Square to determine the outcome of this possibility:

12. What are the possible genotypes and their expected ratios?

13. What are the possible phenotypes and their expected ratios?

14. Use Chi-Square to test this expectation. Observed # Expected # (O-E) (O-E)2 (O-E)2/E Purple

Yellow

Total = χ2 =

p =

Using the results of your chi-square, could these be the parents of your mystery corn? Explain.

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Experiment #2. Mystery Dihybrid Cross Your lab assistant, Clumsy Clyde, is at it again. He has mixed up several additional corn plants, and you are unsure of the parents of an important ear of corn. You must use your genetics skills to determine who the parents are. Your second research assistant, remembers that the female parent used in the cross was heterozygous (AaBb). Thus, based on the observation of recessive and dominant phenotypes in the grains of the “mystery corn”, you have deduced that the male parent could either be AaBb or aabb. Possibility 1: Cross AaBb with AaBb 15. If the AaBb parent was crossed with an AaBb individual, what would be the possible genotypes?

16. What are the possible phenotypes and their expected ratios?

17. Chi-square test using the expected values from the AaBb with AaBb cross Observed # Expected # (O-E) (O-E)2 (O-E)2/E Purple, Smooth

Purple, Wrinkled

Yellow, Smooth

Yellow, Wrinkled

Total = χ2 = p = 18. Using the results of your chi-square test, could these genotypes have been the parents of your mystery

corn? Explain.

A = purple a = yellow B = smooth b = wrinkled

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Possibility 2: Cross AaBb with aabb 19. If the AaBb parent was crossed with an aabb individual, what would be the possible genotypes?

20. What are the possible phenotypes and their expected ratios?

21. Chi-square test using the expected values from the AaBb with AaBb cross Observed # Expected # (O-E) (O-E)2 (O-E)2/E Purple, Smooth

Purple, Wrinkled

Yellow, Smooth

Yellow, Wrinkled

Total = χ2 = p = 22. Using the results of your chi-square test, could these genotypes have been the parents of your mystery

corn? Explain.