GENETICS, PLANT BREEDING, AND SECTIONS

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Genetics, Plant Breeding, and Selection Hands-on labs, inc. Version 42-0063-00-01

Review the safety materials and wear goggles when working with chemicals. Read the entire exercise before you begin. Take time to organize the materials you will need and set aside a safe work space in which to complete the exercise.

Experiment Summary:

Students will have the opportunity to learn classical Mendelian Genetics and use a Punnett Square to predict the outcome of genetic crosses. Students will perform a monohybrid cross using tobacco mosaic seeds and study a dihybrid cross using corn. They will apply chi square analyses to both crosses to determine the reliability of the data. Students will design an experiment to produce desired traits in a hypothetical crop.

ExpErimEnt

ObjEctivEs ● To predict the genetic frequency of offspring in a monohybrid cross

● To predict the outcomes of genetic crosses using Punnett squares

● To statistically analyze the results of a genetic cross

Time Allocation: Exercise 1 requires 5–10 days of growing the tobacco plants prior to performing the rest of the exercise. Read the exercise and plan accordingly. Two hours of time is required after the seeds have been germinated.

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Experiment Genetics, Plant BreedinG, and selection

matErials

MATERiAlS FRoM:

lABEl oR BoX/ BAg: QTy iTEM DESCRiPTioN:

Student Provides 1 Paper towel 1 Water

1 Warm location for the seeds, either in a warm window or under an incandescent lamp

1 Roll of aluminum foil LabPaq Provides 2 Seed, Tobacco, Seeds-Grn/Alb 50

1 Magnifer, dual

1 Pair of tweezers (may be found in dissection kit)

1 Petri dish, 90 mm

Note: The packaging and/or materials in this LabPaq may differ slightly from that which is listed above. For an exact listing of materials, refer to the Contents List form included in the LabPaq.

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Experiment Genetics, Plant BreedinG, and selection

DiscussiOn anD rEviEw Artificial selection is important in plant production. Classical plant breeding is a process that uses deliberate interbreeding of plants traits. The plants used may be closely or distantly related species, and are interbred to produce new crop varieties. The goal is to produce more desirable traits such as flavor, or a resistance to disease or drought. Artificial selection is also used in breeding of animals. In some instances, there is no advantage to the plant or animal; rather selection is based on human preference. In terms of survivability, it may actually reduce the success of this plant’s survival in a natural environment.

Figure 1: These are different cultivars of the same species, Brassica oleracea, that have been selected for different traits.

In the real world... Protoplast fusion is a technique that removes the cell wall from plants so that cells from two different species can be fused to create a new species. Embryo rescue is an attempt to save a weak or infertile female hybrid by fusing it with more genetically diverse male wild plants. The survival rate in embryo rescue varies with older embryos being more viable. Mutagenesis is the process of changing the genetic material of a plant through DNA insertion, chemical or radiation to obtain desired characteristics.

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Experiment Genetics, Plant BreedinG, and selection

rEviEw Of tErminOlOgy ● Dominance – refers to Mendel’s law that states when two alleles are present in the

heterozygous condition the dominant allele will mask the recessive allele.

● Allele – alternate forms of a gene, for example for the trait freckles the two forms are either present or absent.

● Dominant – when one allele can dominate or mask the other allele.

● Recessive - the term given to the allele that is masked. These are only visible in the phenotype if they are homozygous recessive.

● Heterozygous – the genotypic condition when both forms of the alleles (dominant and recessive) are present in the genotype.

● Homozygous - when the allele pair is the same from the mother and the father (either both are dominant or both are recessive).

● Punnett square – tool used to determine the possible allele combinations that could be present in a zygote.

● genotype – genes present in an organism.

● Phenotype –physical expression of the alleles or what is physically seen.

● Monohybrid – a cross between two individuals that differ at only one trait.

● Dihybrid – a cross between two individuals that differ at two traits.

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Experiment Genetics, Plant BreedinG, and selection

Exercise 1: observing a Monohybrid Cross A monohybrid cross is a simple mixture of traits that uses one pair of alleles. Dominant traits will always be expressed when paired with a recessive trait. Recessive traits are only expressed if they are homozygous (recessive genes from both the mother and father) with no dominant allele present to mask the trait. The dominant trait will be expressed when dominant and recessive genes

When a parent with a homozygous dominant gene for height (TT) is crossed with a parent with a homozygous recessive gene for height (tt), the offspring will be 100% heterozygous (dominant and recessive trait mixture, or Tt). A Punnett square may be helpful in determining all of the possible outcomes in offspring when calculating the expected genotype outcome. A Punnett square is a simple grid that shows the possible combinations of the father’s genes on one side (axis) of the grid and the possible combinations of the mother’s genes on the other. The grid can be completed by filling in the combinations of the gametes possible from the father and the mother. See examples below.

Figure 2a

Father ( T T ) × Mother ( t t )

Father (♂)

gametes T T

Mother (♀) t Tt Tt

t Tt Tt

Figure 2b

Father ( T t ) × Mother ( T t )

Father (♂)

gametes T t

Mother (♀) T TT Tt

t Tt tt

Figure 2: a and b - Parents’ traits shown at top, and their next generation chart shown at bottom.

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Experiment Genetics, Plant BreedinG, and selection

prOcEDurE Before beginning, set up data tables similar to the Data Tables 1 - 8 in the Lab Report Assistant section.

Perform these steps at least ten days before seedling data is required in this laboratory.

1. Cut a piece of paper towel to fit inside a petri dish.

2. Place the paper towel inside the petri dish and moisten with water. Do not oversaturate the paper towel (water should not pool in the petri dish); just moisten the paper towel and squeeze out excess water.

3. Sprinkle all of the seeds onto the paper towel and then cover the petri dish with the petri dish lid.

4. Place seeds near a warm window with indirect light or place seeds under a warm incandescent lamp (with the lamp turned on).

a. Lay a piece of aluminum foil over the top of the petri dish if it is under the light bulb or in direct sunlight to protect it from the direct light. In order to let a little light into the petri dish, just cover the top of the petri dish and allow some light to filter into the sides of the petri dish.

5. Observe seeds for germination each day, and moisten the paper towel as needed. Do not let the paper towel dry out. Also, only add enough water to make the paper towel moist, not oversaturated.

6. There will be two types of F2 seedlings, green and yellow to very pale green/white which are albino (chlorotic). If the petri dishes are kept in filtered light conditions, the green seedlings should quickly develop chlorophyll (green color) after they germinate.

7. Count, classify, and record the color of the seedlings in a data table as they germinate, for the albino seedlings die quickly. Remove the counted seedlings with tweezers to minimize confusion about which seedlings have been counted.

8. Keep the petri dish covered with the plastic lid and limit exposure to room air as much as possible to avoid contamination.

Note: It will take 5–10 days for the seeds in this exercise to germinate.

9. Create a Punnett square (see Data Table 1) that shows the possible outcomes from a cross between a heterozygous male and a heterozygous female.

10. Write a hypothesis, using the Punnett square to determine the approximate number of seedlings (and percentages) that will have a dominant trait and that will have a recessive trait out of the 50 seeds.

11. Once the majority of seeds have germinated, remove the lid of the petri dish and count the number of seedlings that are green versus those that are yellow.

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Experiment Genetics, Plant BreedinG, and selection

12. Record the data into Data Table 2.

13. Compare the expected outcome (Step 7) to actual results found in Data Table 2 (write this information into Data Table 3).

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Experiment Genetics, Plant BreedinG, and selection

Exercise 2: Chi-Square Analysis Statistical testing may be used to determine the accuracy of test results. A chi-square test may determine if the variation between the predicted value and the actual data is an acceptable level of variation (less than is expected by chance). Statistical significance of the chi-square implies that the difference found between the actual data and the expected outcome is not due to chance alone, but instead may be indicative of other variables.

Chi-square analysis uses the following equation, where O = the observed frequency, E = the expected count or frequency, Σ = sum, Χ2 = chi-square test:

Chi-square equation:

After obtaining the chi-square value, you will use a chi-square table to determine significance of the data. To use the chi-square table, you must determine the degrees of freedom (df), which is the number of possible phenotypes [in this exercise, green and yellow (2)] minus 1. The top row of the chi-square data table displays the alpha-level, which is the probability that the two samples are not different (e.g., 0.05 is a probability of 5%). In this Exercise, you will conclude that the samples are different (do not come from the same population) if the alpha-level is below 0.05 (higher than the chi-square distribution value listed in the table).

Table 1: Chi square table of probabilities

p value (alpha level) 0.1 0.05 0.01

df chi-square distribution values 1 2.71 3.84 6.63 2 4.61 5.99 9.21 3 6.25 7.81 11.34 4 7.78 9.49 13.28 5 9.24 11.07 15.09 6 10.64 12.59 16.81 7 12.02 14.07 18.48 8 14.36 15.51 20.09 9 14.68 16.92 21.67

10 15.99 18.31 23.21

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Experiment Genetics, Plant BreedinG, and selection

prOcEDurE Statistical testing will be used to determine the validity of data collected in Exercise 1.

1. Record the total number of sprouted seedlings from Exercise 1 in Data Table 4 in the “Total” row under the columns entitled “Specimens Observed (O)” and “Specimens Expected (E).”

Note: This must be done after the seedlings are counted. When using a chi-square test, the total values (sum of the expected and the sum of the actual value) must be the same.

2. Determine the “expected values” of the seedlings by multiplying the total number of sprouted seedlings by the percentages (ratios) of green and yellow seedlings that are expected (determined by the Punnett square in Exercise 1).

3. Record the Expected Values in Column B of Data Table 4.

4. Subtract the observed values in Column A from the expected values in Column B of Data Table 4, and record these values in Column C.

5. Perform the equation shown in Column D: Use the value found in Column C, square that value, and divide it by the Expected Values (E).

6. Add the two values in Column D together (green and yellow) and record the final sum in the “Total” row of Column D.

7. To determine the probability that the data values occurred by chance, use the chi-square table; see Table 1.

Experiment Genetics, Plant BreedinG, and selection

Exercise 3: Dihybrid Crossing with Corn This is an example of how plant breeding may be analyzed.

In this exercise, you will observe an ear of corn (using photos) to determine the type of cross (parents’ genes) responsible for the coloration and texture of the corn kernels. There are four grain phenotypes in the ear of corn. The quarter section of corn from the LabPaq is the result of a dihybrid cross. Count the number of purple or yellow kernels and then smooth or wrinkled kernels. The alleles in this case would be P (dominant purple color), p (recessive yellow color), S (smooth shape) and s (wrinkled shape).

Figure 3: Phenotypes in an ear of corn: A) yellow and wrinkled; B) purple and smooth; C) purple and wrinkled; D) yellow and smooth.

prOcEDurE Monohybrid Relationships

1. Count the number of purple and yellow kernels on the ear of corn between the lines in Figures 4–7.

Figure 4: Phenotypes in an ear of corn: A) yellow and wrinkled; B) purple and smooth; C) purple and wrinkled; D) yellow and smooth.

2. Record the data in Data Table 5.

Note: This data represents a cross between a purple kernel corn plant and a yellow kernel corn plant.

Experiment Genetics, Plant BreedinG, and selection

Figure 5: Ear of Corn, Side A

Figure 6: Ear of Corn, Side C

Figure 7: Ear of Corn, Side D

3. Count the number of smooth and wrinkled seeds on the ear of corn.

4. Record the data in Data Table 5.

Note: This data represents a cross between a smooth kernel corn plant and a wrinkled kernel corn plant.

Experiment Genetics, Plant BreedinG, and selection

Dihybrid Cross

5. Construct a Punnett square using Data Table 6.

6. A dihybrid cross involves two alleles, such as PpSs, which represents a heterozygous dihybrid.

7. The ear of corn is a result of a cross between plants that were both heterozygous for color and texture written as PpSs.

8. To create gametes for this type of cross to place on a Punnett square, use the acronym FOIL for first (first of the two types), outside (letters on the very outside), inside (letters on the inside) and last (last of the two types). An example of alleles from PpSs crossing with PpSs would yield:

a. First: PS

b. Outside: Ps

c. Inside: pS

d. Last: ps

9. Place these gametes into the Punnett square in Data Table 6 for both the male and the female.

10. Calculate the phenotypic ratios that are expected to be found for each type of seed.

a. Purple & smooth _______________

b. Purple & wrinkled ______________

c. Yellow & smooth _______________

d. Yellow & wrinkled ______________

11. Count the number of each on the quarter ear of corn and record the numbers in Data Table 7.

12. Calculate the expected number based on total number and assuming the correct ratio. Record the values in Data Table 8.

13. Calculate the individual chi square values for each row.

14. Add them together to determine the overall chi square value.

15. Determine if the chi square value is a good fit with data.

Note: The degrees of freedom or df is the number of possible phenotypes minus 1. In this case, 4 – 1 = 3. Using Table 1, find the number in that row that is closest to the chi square value. Circle that number.

Experiment Genetics, Plant BreedinG, and selection