Biology 156: An Introduction to Mendelian GeneticsDr. Cheryl Blake: Based on the lab from the Desert Vista Lab

Manual

There are no special safety considerations associated with this lab.

BACKGROUND : AN INTRODUCTION TO MENDELIAN GENETICS

Gregor Mendel, an Austrian monk, was the first we know of who completed and

documented a scientific study of the progeny of controlled matings. His work was

unique in that he carefully controlled the inputs (the types of plants that he was using),

replicated his work hundreds of times to ensure the data were not due to random

variation, and recorded the outcomes of much of his work. The process that he followed

is shown in the picture below:

Mendel’s choice of the pea plant for his work was advantageous. Not only is this an organism that has a fast reproductive cycle, and one that generates many offspring in a very short period of time, but the pea plant's flowers have useful qualities as well. Left alone, the flower will self-pollinate (see sec. 9.2B), but these flowers can easily be cross-pollinated as well as you see in the picture. These are the two key options that a geneticist would like to have for organisms. Other famous geneticists have worked with the fruit fly Drosophilia melanogaster and the nematode worm Caenorhabditis elegans (C. elegans), which have many of the same favorable qualities. Imagine doing genetic crosses with elephants: not only would it take years to produce one offspring, but you would have to feed them too!

In this lab, we will work with the organism maize (corn) which was favored by the famous geneticist Barbara McClintock – the discoverer of “jumping genes”. Corncobs are the fruit from one corn plant. Each seed is the product of a cross between the “mother” plant on which the cob was formed and a pollen grain carrying sperm from a “father” plant. An advantage of corn is that the many progeny – the seeds – are aligned for easy data collection.

Mendel began his study of genetics without knowledge of cell structure, mechanisms of cell division, DNA, proteins and other key biological ideas that we take for granted. Some of his contemporaries were describing the cellular nature of life (Schleiden & Schwann and Virchow) while another famous biologist – Charles Darwin – was describing how natural selection could explain the patterns of evolution he had observed. You will begin this lab as Mendel did by observing the outcomes of crosses. You will then develop an understanding of why these outcomes are as they are.

THE NATURE OF THE GENETIC CROSS

A hybrid is the result of the breeding of two individuals. In a monohybrid cross like the one pictured on the previous page, one trait of the organism is the focus of the study (the color of the flowers in this case); other traits are simply ignored. Examining the eye color of the offspring that results from a mating between a blue-eyed person and a brown-eyed person is an example of a monohybrid cross. The parents and offspring clearly have other features such as height, hair color, intelligence etc., but these traits are ignored. Similarly, a dihybrid cross occurs when the experimenter attends to two traits (e.g., eye color and height) in the parents and offspring.

One of the unique features of Mendel’s work was that he always carried his crosses through two generations; you will see why this was so critical as the lab progresses. To simplify recording of information in genetic crosses, the generations are named. The original parents are the P generation. Their offspring comprise the F1 (first filial) generation. Mendel always allowed offspring in the F1 generation to mate with each other; the progeny from this second generation (the “grandchildren” of the original parents) comprise the F2 (second filial) generation.

Another unique feature of Mendel’s crosses was that he began all of his work with true-breeding strains for the trait he intended to study. A true-

breeding or purebred organism is one that produces identical offspring generation after generation when mated to an organism of the same breed (e.g., a purebred collie mated with another purebred collie will have pups that all have collie characteristics). Sec. 9.2D in you text shows diagrams of seven of the traits that Mendel studied as he mated his pea plants. He had, for example, purebred strains of pea plants that always produced purple flowers and strains that always produced white flowers at his disposal. In today’s activity, we will attend to two traits found in corn kernels: the color of the kernel (purple or yellow) and the amount of starch found in the kernel. Starchy kernels are plumper, while sweeter kernels appear to be shriveled in comparison.

EXERCISE A: DETERMINING DOMINANCE & PHENOTYPIC RATIOS IN A MONOHYBRID CROSS

(a) Obtain a bag of corncobs. A different genetic cross has produced each cob.

(a) Take out the corncobs labeled A (all purple kernels) and B (all yellow kernels). Imagine that seeds from each were planted and pollen (sperm) from the plants growing from the yellow seeds were mated with the eggs from the plants that grew from the purple seeds. A cob from this cross would contain many seeds. These seeds would be the generation, while the original purple and yellow seeds would comprise the generation.

(b)The cross described in question 1 was actually done by a biological supply company. The offspring are present on cob C. Describe these offspring.

(c) What are some hypotheses that might explain the appearance of cob C? (Try to think like Mendel: pretend that you don’t know about cells, cell division, DNA or genetics!)

(d)The same biological company allowed kernels like those on cob C to grow into mature plants, and allowed them to pollinate each other. Cob D, the generation, clearly consists of both purple and yellow kernels. Did this second cross disprove any of your hypotheses from question 3? Explain.

(e) Working with your partner, identify the color of 100 kernels on your D cob. Record the number of your purple and yellow kernels here:

Purple Yellow(f) Add the data for all the D cobs in the class below:

Purple Yellow Total

(g)What was the ratio of purple to yellow kernels for the class as a whole? In other words, what percentage of the kernels in this second generation were purple and what percentage were yellow?

%Purple (# purple/total) %Yellow (# purple/total)

Ratio of purple to yellow (divide the % of purple by the % of yellow) (h)Complete the data table* of Mendel’s work below. Then comment on

the similarities and differences within his data and between these data and yours.

P trait A X P trait B # trait A # trait B in Total Ratio in F2

in F2 F2 F2Spherical

seedsWrinkled 5474 1850 7324 2.96 : 1

Yellow seeds Green 6022 2001Purple flowers

White 705 224

Inflated pods Constricted 882 299Green pods Yellow 428 152

Axial flowers

Terminal 651 207

Tall stems Dwarf 787 277*Adapted from Table 10.1 in Purves et. al., LIFE, The Science of Biology

5 th Ed., 1998

BACKGROUND CONT. : THE PUNNETT SQUARE - PREDICTING OUTCOMES IN MONOHYBRID CROSSES

Now, consider what you know about genetics, cell structure and cell division. As you studied the process of meiosis in the previous lab, you learned that the daughter cells from a meiotic division have half the number of chromosomes found in the parent cell. You also learned about the nature of the gene: genes, of course, are simply the directions for making cellular proteins. Finally, you learned that homologous chromosomes have the same genes in the same order, but the specific directions on the two

homologous chromosomes might vary.

The genetics term "allele" refers to the possible versions that a particular gene might have. The gene locus is the position on a particular chromosome in which we find the gene. In this figure, we see a pair of homologous chromosomes. The locus for flower color is identified, and two versions of the gene - the allele for purple flowers and the allele for white flowers are shown.

In our corn kernels, there are two alleles at a particular locus that controls the color of the kernel. One allele codes for an enzyme (a protein) that catalyzes the production of a purple pigment. The alternative allele at that locus does not code for a functional enzyme, and kernels are the “default” color yellow (i.e., they are “purple lacking”).

When Mendel crossed pea plants with purple flowers with purebred plants with white flowers, all the offspring in the first generation had purple flowers. Similarly, the F1 generation seeds (peas) resulting from a cross of tall and short plants were all tall. In all cases that Mendel recorded, one trait appeared to overpower the other trait in the F1 generation. He termed this trait the dominant trait. Interestingly, the trait that had been masked – the recessive trait – reappeared in the second generation (F2) as you see in the figure.

Mendel’s careful work over two generations demonstrated that the recessive trait had not truly been "overcome"; it was not gone, but simply being masked in the first generation. His identification of a dominant trait in the F1 generation also showed that, contrary to popular belief, these traits did not simply blend to produce a hybrid mixture. For example, purple-flowering X white-flowering plants did not produce plants of medium or blended color - they were all purple; tall plants X short plants were not of medium height - they were all tall.

Mendel described a particulate hypothesis to explain his observations. He suggested that each parent had two particles that provided the information for a specific trait, and that one of each of these particles must be present in the egg or sperm produced by the parent. Purebred parents would have only one type of particle (but two copies). Mendel developed the habit of using a capital letter to identify the dominant trait. He used the small letter to identify the alternative, recessive trait. For example, once he had identified that tall trait dominated the short trait in the F1 generation, he identified the tall-producing particle (remember that he did not yet know what genes were) as “T” while short-producing particles were assigned “t”. Thus a purebred tall parent would have two T particles, while a purebred short parent would have two t particles. The tall parent would make eggs (or sperm) with one T particle, while the short parents sperm (or

eggs) would contain one t particle. Fertilization would result in an offspring with one of each particle (one T and one t), and the observed, dominant, tall appearance.(i) Which trait is dominant for kernel color in corn?

Examine the other cobs in your bag (you’ll need the key to identify which is which) and determine which trait is dominant: sweet (shriveled) or starchy (plump).

Return your attention to the structure of a chromosome and the concept of a gene. We know that each cell has two homologous chromosomes with loci for many traits. The diagram below shows three possibilities for chromosome combinations in a situation such as you saw corn. If you work as Mendel did, your identification of purple as the dominant trait in the first exercise should lead you to identify the purple allele as “P” with the yellow allele as “p”. (j) Fill in the three possibilities for allele combinations that could be present

in kernels below. Then identify whether the appearance of the kernel would be purple or yellow given that genetic content. The first possibility is done for you to serve as a model.

Purple kernel _________

P

P

The pair of alleles that are present at a particular locus is the organism’s genotype (PP, Pp or pp). Its appearance or a functional quality it possesses is its phenotype (purple or white for these pea plants). When the alleles are the same, the genotype is homozygous (PP or pp); when they are different, it is said to be heterozygous (Pp).

The allele combinations you identified on the previous page should be PP, Pp and pp; these are the three possible genotypes that can occur for this trait in corn. Since there are two ways to be homozygous (PP or pp, in this case), the term dominant or recessive is added for clarity. Thus PP is the homozygous dominant genotype, and plants with this genotype have the purple phenotype due to the presence of the dominant allele (i.e., they have the ability to make the enzyme needed to produce the purple pigment). Plants with the heterozygous genotype (Pp) are purple as well, since they also produce the necessary enzyme, but only those with the homozygous recessive genotype (pp) have the yellow phenotype since only these do not have the enzyme and thus retain their “natural” color.

The phenotypes that Mendel described were all physical appearances observable with the naked eye. Our advanced technology allows us to identify phenotypes that are biochemical and cellular in nature, such as whether a person has a disorder such as sickle-cell anemia. In this case, the phenotype is really a set of symptoms that include shortness of breath and joint pain during activity. Since this disorder is controlled by a single gene locus, a person can have the genotypes SS, Ss or ss; only with the ss genotype do the phenotypic symptoms appear. Many of the symptoms of

sickle cell are observable only with a microscope or by using biochemical techniques.

Using your knowledge of meiosis and the vocabulary that you now know, you can effectively and easily predict the genoptypic and phenotypic outcome of genetics crosses by using Punnet squares. First, you must determine whether the trait you are studying adheres to the dominant/recessive patterns described by Mendel. (k)How would you determine whether the height of a corn plant followed

the tall vs. short pattern described by Mendel? If it did, how would you know which trait was dominant?

Once the genetics pattern for a particular trait has been established, and the genotypes of the parents are known, you can create a Punnett square to predict the types of offspring that would be produced from that cross. To complete a Punnett square, first determine the possible alleles that would be present in the sperm or egg produced by each parent. A tall pea plant that has a homozygous genotype (i.e., one that is TT) can only produce eggs or sperm that carry the T allele. Convince yourself that this is true by reviewing how meiosis produces gametes: the available DNA (in this case, containing the T allele) is copied and then sorted over two divisions into gametes. Since only the T allele is present to be copied during DNA replication, only the T allele can be present in the gametes. If the tall parent were heterozygous (Tt), 50% of the gametes would carry the one T allele, while 50% would contain a single t allele for that locus.

The next step is to place the allele possibilities for that parent’s gametes along one axis of the square. Repeat this procedure for the other parent. It is important to note that a Punnet square may not have the same number of

gamete options on the vertical and horizontal axes. (The example below demonstrates this fact.) Complete the square by modeling fertilization events: combine the gametes from each parent in the center boxes. Determine the phenotype of each child using the information regarding dominance that you have already determined. Finally, identify the genotypic and phenotypic ratios in this new generation.

An example of a Punnet square completed for a cross of a heterozygous tall pea plant with a short pea plant is shown on the next page. The logic and steps you would follow to determine the genotypic and phenotypic ratios of the plants produced from this mating follow:1. We first must determine the genotypes of both parents. We already know

from Mendel’s work that tall is the dominant trait, so we assign the letter T for tallness and t for shortness. The heterozygous parent must be Tt. The genotype of the short parent is not given, but to express the recessive genotype, it must be tt.

2. One cell in the tall parent (Tt) undergoing meiosis would produce 4 gametes: two would carry the T allele, while 2 would carry the t allele. In other words, half of the haploid gametes produced by this diploid cell would be T, and half would be t. This information is entered along the vertical axis of the square below.

3. The short parent (tt) can only produce haploid gametes carrying the t allele. This single possibility is listed on the horizontal axis as shown.

4. The possibilities are combined in the central areas by simply writing both the alleles from the vertical and horizontal axis bordering that box. The order of the alleles is irrelevant, but convention provides for writing the dominant allele, if it exists, first.

5. The phenotype of each offspring is determined: if at least one T is present, the organism is tall, while only those with a homozygous recessive genotype are short.

6. The genotypic and phenotypic ratios are calculated. In this case, they happen to be identical.

Tall, heterozygous parent (Tt) X Short parent (tt)Produces half T and half t gametes produces only gametes with t

Short parent’s gametet

Tall parent’s gametes T Tt – tall offspringt tt – short offspring

The genotypic ratio in the offspring of this cross is 1 Tt : 1tt (50% to 50%)

The phenotypic ratio in the offspring is also 1 tall : 1 short

Note: you could create a 2 X 2 square (the more traditional method) for this cross as shown below. However, since the short parent’s gametes would be alike in both columns, the end result is identical. Use a 2 X 2 box like this one (as the computer does in the exercise below), if this is easier for you.

Short parent’s gametest t

Tall parent’s gametes T Tt – tall offspring Tt – tall offspringt tt – short offspring tt – short

offspringThe genotypic ratio in the offspring of this cross is 1 Tt : 1tt (50% to

50%) The phenotypic ratio in the offspring is also 1 tall : 1 short

EXERCISE B : USING PUNNETT SQUARES TO DETERMINE THE OFFSPRING IN A MONOHYBRID CROSS

7. At this point, examine only one characteristic to complete only monohybrid crosses. (We will examine dihybrid crosses later.) Remember that Mendel always began his work with pure breeding parents, and carried his crosses over two generations. In this case, you can begin with any parents, but the computer only allows you to directly examine the F1 generation. By using the offspring produced in this generation as the parents for the next, however, you can model a two-generation monohybrid cross like those Mendel did.

BACKGROUND : THE TEST CROSS

A test cross is a unique type of cross used for only one purpose: to determine the genotype of an organism that displays the dominant phenotype but whose genotype is unknown. Imagine, for example, that you

found a pea plant that had purple flowers in your garden and that you knew nothing about

its "parents". You know only that purple flowers are dominant to white flowers, and you

would like to know what genotype this plant has.

Mendel devised a simple system for answering this question that works for many organisms. He simply mated this plant with purple flowers with a pea plant that produced only white flowers (i.e., it had the recessive phenotype). He then examined the offspring. If many offspring were produced, and all the offspring peas were purple, the probability that the parent purple plant had the PP genotype was very high. If, however, this mating produced some white flowers, the parent must have been Pp. Consider why this is so, and why we speak of a high probability in the first case and a certainty in the second case by following the logic on the next page:

1. Since "purpleness" is dominant, the purple parent can be either PP or Pp. The parent with white flowers chosen for mating must have the pp (homozygous recessive) genotype (otherwise it wouldn't have white flowers), so this information is known.

2. IF the purple parent was PP, it would produce only P-containing gametes. The white pea plant can only produce p-containing gametes. Fusing these two gametes would result in plants with the Pp genotype, which would be purple.

3. IF the round parent were Pp, half of its gametes would contain the P allele, while the other half would carry the p allele. As the Punnett

square on the previous page shows, half of these offspring would be purple and half white.

4. Thus, it is the offspring that tell us about the parent. IF there are many offspring, and IF all of these offspring have purple flowers, then, as stated above, the probability that the parent was PP is quite high. To understand why we are not certain, consider that this is a case of probability. It is possible, for example, that we could flip a coin 10 times and end up with 10 heads and no tails. This does not mean that the coin does not contain a “tail side”, necessarily; it might have both a head and a tail, but random chance might result in the heads-only result. The greater the number of flips that result in only heads, however, the greater our belief becomes that no tail side is to be found on this coin. 1000 heads in 1000 flips would afford us great confidence that this coin does not have a tail side. Similarly, 1000 round offspring would be much more convincing of the lack of an r allele than 5 would be.

5. Consider why, on the other hand, even a single white-flowered offspring convinces us that, unless a mutation has occurred, the parent must have been Pp. Any white offspring expressing the recessive genotype must have the pp genotype. Since offspring must receive alleles from each parent, the purple parent must have had one p to give its progeny. Since it had purple flowers, it must have had one P allele as well, so its genotype must be Pp.

EXERCISE C : EXAMINING THE RESULTS OF A TEST CROSS IN CORN

8. The biological supply company has also completed a test cross of a

purple kernel (a corn seed) of unknown origin. As is done in all test

crosses, they mated a plant grown from this kernel, with a plant

expressing the recessive phenotype. The corn plant with the recessive

phenotype would have the genotype and the phenotype

(color) . The progeny of this cross are on cob E. First count

enough kernels to effectively describe the phenotype or phenotypic ratio

on this cob. (You are free to combine your data with other groups as you

did in the first exercise to increase your sample size.) Record your data,

and explain what these observations tell you about the original purple

kernel. Make sure that the logic you present in your conclusion is

thorough and clear.

BACKGROUND : THE DIHYBRID CROSS

In a dihybrid cross, two traits or characteristics of parents and offspring are attended to. In Mendel’s case, he might have attended both to the height of a pea plant and the color of its flowers while ignoring characteristics of its pods and seeds. Since two traits are being examined, the genotypes at two loci must be considered. For example, a tall plant with purple flowers that is homozygous for both traits would have the genotype TTPP, while one that is also homozygous for its height but is heterozygous for its flower color would be TTPp. A short plant with white flowers must be ttpp.

Though the process of creating a Punnett square is the same, determining the alleles in the gametes can be a little more difficult. The important thing to remember is that all gametes must contain one allele for each trait in the organism. In the above example, each gamete should have an allele for height and an allele for flower color. For example, gametes produced from the TTPp parent might be TP or Tp. 9. Why couldn’t this plant produce a gamete with the tp genotype?

As he did with the monohybrid cross, Mendel began with purebred parents and examined the offspring in the F1 generation. He allowed these offspring to self-pollinate, and then recorded the phenotypes in the F2. Our understanding of meiosis and Punnett squares allows us to explain the 9:3:3:1 phenotypic ratio that Mendel observed in these dihybrid crosses. Follow the argument presented in section 9.5 and examine the figure below. In this figure, Mendel attended to the color and the shape of the seeds. The

data demonstrate that the alleles for the two traits (color - Y or y, and shape - R or r) assort independently; in other words, the off spring can have any combination of traits as long as the original alleles were present in their parents. (Note: this is not true if the alleles are close together on the same chromosome.) When you understand the figure below, complete the remaining exercises to become familiar with the dihybrid cross

EXERCISE E : EXAMINING THE RESULTS OF A DIHYBRID CROSS IN CORN

12.Recall that for kernel color, purple is dominant to yellow. The presence

of a gene that codes for an enzyme that catalyzes the synthesis of sugar

molecules into long starch polymers makes the kernels plump with starch

granules. Kernels that lack this gene are sweeter and have a more

shriveled appearance (the recessive phenotype).

10.In a Mendelian dihybrid cross, one parental type is homozygous

dominant at both loci; its genotype at these two loci is

. The other parental type is homozygous recessive; its genotype is

. Explain why all F1 offspring will be heterozygous at

both loci.

11.The genotype of the F1 plants is __________. Each of these F1 plants

can make four types of gametes. They are:

12.Make a Punnett square of an F1 x F1 dihybrid cross. Diagram this

cross here. Once the interior of the square contains the genotypes of each

of these F2 offspring, determine the phenotype of each F2 organism.

Finally, determine the expected phenotypic ratio in the F2 by counting

the number of each phenotype. Record this information and circle it.

13.Take out a cob that is a result of a dihybrid cross from your bag. Score

several rows of kernels on the F2 cob for kernel shape and color. (Sample

at least 100 kernels; remember that it is fine to combine your data with

other groups to increase your end sample size and thus improve your

confidence in your data.) Record your results in the chart below. Also

indicate the relative proportions of each phenotype by dividing the smallest

number into each of the larger ones.

Kernel Shape and Color on F2 Cob.

Phenotype Total Phenotypic Ratio

Starchy, Purple

14.Compare the relative proportions of each of the four phenotypes that you

observed with those that you expected from your Punnett square results.

How similar were your results to those that you had predicted?