Overview

This lesson examines the principle of segregation versus the principle of independent assortment and their effects on genetic data. It also addresses the concept of dominance. Understanding the inheritance of a single trait which differs between two parents is the goal of a monohybrid cross experiment. In reality though, parents rarely differ by only a single trait so the next logical step in classical genetics would be a dihybrid cross experiment. The goal in this type of experiment is to understand how two traits that vary between parents are inherited. The experiment described in this lesson was a combined effort of Dr. Jim Specht and an undergraduate genetics class.

Authors

• Don Lee, Department of Agronomy and Horticulture, University of Nebraska-Lincoln

Published 2003

Soybean Experiment and Working Hypothesis

Two parents were identified in the field that differed by two traits. One parent had a pale green leaf color and three leaflets per leaf while the other parent had a dark green color and seven leaflets per leaf. Dr. Specht made crosses between the two parents and seven hybrid seeds were produced. He planted these seeds in the field and the following results were obtained.

Parents: pale green, three leaflet crossed with dark green, seven leaflet

Offspring: 4 pale green, three leaflet and 3 dark green, three leaflet

What genetic hypotheses could you pose to explain the results obtained so far? These results can be a little confusing. One way to make the information simpler is to look at one trait at a time.

1. Leaf number trait: the three leaflet x seven leaflet cross gave all progeny with three leaflets
2. Leaf color trait: the pale green x dark green cross gave about [latex]\frac {1}{2}[/latex] pale green + [latex]\frac {1}{2}[/latex] dark green offspring

How could we explain these results? The leaflet number trait hypothesis is easiest to explain. Both parents could be homozygous and the three leaflet trait completely dominant to the seven leaflet trait. Their offspring are heterozygous but express the dominant three leaflet phenotype. Genotypes for this cross could be as follows:

Parents: homozygous dominant LL (3 leaflet) crossed with homozygous recessive ll (7 leaflet) results in heterozygous Ll (3 leaflet)

Now let’s take a look at the leaf color trait, which is a bit more complicated. The results obtained for the leaf color traits could be explained in two ways. All explanations require one parent to be heterozygous.

Parents: heterozygous Yy crossed with homozygous recessive yy results in [latex]\frac {1}{2}[/latex] heterozygous Yy + [latex]\frac {1}{2}[/latex]homozygous recessive yy

The heterozygous Yy genotype could represent the pale green phenotype and homozygous recessive yy dark green, or just as likely, Yy dark green and yy pale green. Either way gives the same resulting progeny from this cross. Therefore, additional experimental evidence must be obtained. What would be the easiest experiment to perform that would test the proposed hypotheses?

Testing the Hypothesis

Soybeans self pollinate when left alone so that is what Dr. Specht did. He then pulled the plants and let the fall semester genetics class continue the experiment. Students carefully removed the pods of seeds from each plant and planted them in trays in the greenhouse. The trays were labeled so that they could be traced back to the pale green or dark green parent plants they came from. Two weeks after planting, the students gathered their data (Fig. 1). The following observations were made as the data was collected, looking at the two traits separately.

Observations:

1. All of the trays had some plants showing the three leaflet and the others showing the seven leaflet trait (Fig. 2).
2. The trays of plants from pale green parents had a mixture of leaf color phenotypes. Yellow, pale green and dark green plants appeared in these trays (Fig. 3).
3. The trays of plants from the dark green parents were all dark green. These results can be compared to the preliminary hypothesis.

Parents: homozygous dominant LL (3 leaflet) crossed with homozygous recessive ll (7 leaflet) results in heterozygous Ll (3 leaflet)

Lack of Dominance

The conflict was that there were three distinct leaf color phenotypes among the selfed progeny. Some had dark green leaves, some had pale green and some had yellow. The plants with yellow leaves were much smaller and many were dying at two weeks of age. How can the pale green plant produce three phenotypes from selfing? Lack of dominance would explain this result. If the two alleles at this leaf color gene pair had a lack of dominance, we could propose the following: First, [latex]Y^1[/latex] is the version of the leaf color gene that controls green color. Plants need two copies of this gene per cell in order to maximize this green color trait. The alternative allele at this gene pair, [latex]Y^2[/latex], is not recessive to the [latex]Y^1[/latex] allele because we can distinguish between plants that are [latex]Y^1[/latex][latex]Y^1[/latex] (dark green) from those that are [latex]Y^1[/latex][latex]Y^2[/latex] (pale green). Selfing a pale green gives [latex]Y^1[/latex][latex]Y^1[/latex] ; [latex]Y^1[/latex][latex]Y^2[/latex]; [latex]Y^2[/latex][latex]Y^2[/latex] progeny as follows (Fig. 4):

Thus the green progeny ([latex]Y^1[/latex][latex]Y^1[/latex]) and yellow progeny ([latex]Y^1[/latex][latex]Y^2[/latex]) would be expected to each be about [latex]\frac {1}{4}[/latex] of the progeny and the pale green would be [latex]\frac {1}{2}[/latex]. The frequencies of the leaf color phenotypes obtained in the selfed progeny would be a good test of the hypothesis of lack of dominance. Unfortunately, research is not always as easy as it looks on paper.

Phenotype, Genotype and Environment

The fall semester class encountered a problem when they scored the leaf color phenotypes. The yellow leaf trait was always easy to identify but in many trays the pale green and dark green trait were difficult to distinguish. Because of sunny warm September days, the greenhouse was very hot and the plants were under stress. This limited leaf expansion, making it difficult to quantify the amount of green pigment in the leaf with the naked eye. The best the class could do was group the dark green and pale green plants into the same phenotype group, leaving the yellow in their own group. Environment had influenced the ability to distinguish the two proposed genotypes. However, it is possible to use the data to determine how the leaf color and leaf number traits are inherited together.

Data from the Fall Semester Class

The class recognized that the three dark green plants Dr. Specht had provided them produced similar progeny and all had the same genotype. They combined the data from the progeny of these three plants and got the following results:

The ratio observed among these progeny for the leaflet number trait was about 3:1. Do you remember seeing this ratio in other experiments?

The class also combined the data from the four selfed pale green, three leaflet plants (Table 1).

What data set can we use to study the inheritance of two traits at a time? The dark green parent was passing on the dark green alleles to all of its selfed progeny. Therefore we do not learn anything about the inheritance of that trait. The data set we need to focus on is the one from progeny of the dihybrid pale green, three leaflet parents because all phenotypes for both traits were observed.

The dihybrids Dr. Specht obtained would have the genotype [latex]Y^1[/latex][latex]Y^2[/latex] Ll. The question we have is how are the genes from these two gene pairs getting passed along to the progeny that were grown and characterized by the genetics class? It is easy to get confused by a data set with four phenotype categories. Again, simplifying the data helps us think about trait inheritance.

Leaf color: 200 green and 73 yellow (about [latex]\frac{3}{4}[/latex] to [latex]\frac{1}{4}[/latex])

Leaflet number: 209 three leaflet and 64 seven leaflet (about [latex]\frac{3}{4}[/latex] to [latex]\frac{1}{4}[/latex])

Looking at the data one trait at a time reveals the same 3:1 ratio we observed when segregation at a single gene pair could explain the phenotype variation. It appears that segregation is again happening in this dihybrid cross. If the segregation of the color gene pair was independent of the leaflet number gene pair segregation, than we would make the following phenotypic predictions (Table 2):

The numbers observed obviously coincide with the expected numbers if we propose that the genes controlling each trait are inherited independently from each other (Table 3). The 9:3:3:1 ratio is indicative of the segregation of one gene pair proceeding independently of the segregation at another gene pair. This is the principle of independent assortment.

Mendel’s Dihybrid Cross Experiments

Proper credit must be given to the idea of independent assortment. Gregor Mendel was the first to put this idea down on paper based on what he observed with his pea experiments. Furthermore, Mendel performed additional experiments to back up his ideas. Let’s examine his experiments with peas from the late 1800’s.

The information below and data in Table 4 describes Mendel’s dihybrid cross experiments. The pattern observed in the results should look familiar!

Parents: plant with round, yellow seeds crossed to plant with wrinkled, green seeds

[latex]F_1[/latex] offspring: all round, yellow seeds

Mendel explained his results as follows. The [latex]F_1[/latex] plants have the genotype heterozygous RrYy and can make four kinds of gametes RY, Ry, rY and ry.

These gametes are made in equal frequencies in the male and female parts of the plant. The possible gametes from the [latex]F_1[/latex] heterozygous RrYy genotype are:

• Male gametes: [latex]\frac {1}{4}[/latex] RY + [latex]\frac {1}{4}[/latex] Ry + [latex]\frac {1}{4}[/latex] rY + [latex]\frac {1}{4}[/latex] ry
• Female gametes: [latex]\frac {1}{4}[/latex] RY + [latex]\frac {1}{4}[/latex] Ry + [latex]\frac {1}{4}[/latex] rY + [latex]\frac {1}{4}[/latex] ry

Note that with both the Mendel algebra and Punnett square, the RRYY genotype occurs one time and the RrYy genotype occurs four times. Mendel’s algebra and Punnett’s squares can be summarized to give the same results (Fig. 5).

Selfing the [latex]F_2s[/latex] to produce [latex]F_3s[/latex]

The easiest experiment to perform was to let the plants self-pollinate and then keep good records. After scoring his 556 [latex]F_2[/latex] seeds he took the 315 that were round and yellow and planted them in one part of his garden. The plants that grew were allowed to self-pollinate. Of the 315 round and yellow seeds planted, 301 plants matured and produced seed. The seed produced was the [latex]F_3[/latex] generation. At harvest, Mendel needed to exercise the utmost care. Each [latex]F_2[/latex] plant was handled separately. The seeds from the plant were harvested and Mendel then scored the [latex]F_3[/latex] seeds that came from the same [latex]F_2[/latex] plant. This can be referred to as [latex]F_{2.3}[/latex] data and Table 5 summarizes his complete experiment using all of the [latex]F_2[/latex] phenotypes.

Mendel's [latex]F_2[/latex] data supported his principle of independent assortment. There were four different types of round yellow [latex]F_2s[/latex] based on the kinds of progeny they could produce or their breeding behaviors. Based on the [latex]F_3[/latex] progeny produced, the [latex]F_2[/latex] genotype was deduced. For example if a round, yellow seed gave all round progeny it must have the genotype RR. If it gave both round and wrinkled it was Rr. Furthermore, the numbers of [latex]F_2[/latex] plants with each breeding behavior were in agreement with what was expected with independent assortment. There were four times as many round and yellow [latex]F_2s[/latex] that gave all four phenotypes of [latex]F_3[/latex] seeds (138) compared to the round and yellow [latex]F_2s[/latex] that were true breeding (38). Overall, there were nine types of breeding behaviors demonstrated in the [latex]F_2s[/latex] demonstrating that there were nine [latex]F_2[/latex] genotypes. In all cases, the fractions observed in the [latex]F_2s[/latex] were in agreement to the principle of independent assortment. Mendel's well planned experiment provided a convincing demonstration that genes behaved in this predictable manner.

The only thing better than performing an experiment that shows you were right about a new hypothesis is performing two experiments that show that you were right. That is what Gregor Mendel did! In his second experiment he crossed dihybrid [latex]F_1[/latex] plants with homozygous recessive plants in a test cross. This type of cross is named because the geneticist wants to perform a cross that will test or reveal the genotype of an organism. Therefore a test cross is usually made between an organism with a dominant trait and a partner with a recessive version of this trait. Mendel performed the RrYy X rryy testcross and the expected progeny are shown in the Punnett square below (Fig. 6):

The observed result closely matched the expected. The testcross experiment provides additional support for the principle of independent assortment.

Mendel established a rigorous precedent for using carefully planned multi-generation experiments to reveal the principles that governed trait inheritance. The beauty of Mendel's accomplishments is that both the principles and his experimental approach can be applied to understanding the genetic control and inheritance of traits in many kinds of organisms still today.

The Spring Semester Class

Let's return to our genetics class and the soybean experiment. There were seeds remaining on the seven plants that Dr. Specht provided the fall class so the greenhouse generation of the experiment was repeated with the spring semester students. This time, January weather was suitable for non-stressed greenhouse growing conditions and the dark green and pale green phenotypes could be distinguished. The spring semester class collected the following data from selfed progeny of the pale green parent plants (Table 6 and Fig. 7).

Conclusions

Mendel's principles of segregation and independent assortment are valid explanations for genetic variation observed in many organisms. Alleles of a gene pair may interact in a dominant vs. recessive manner or show a lack of dominance. Even so, these principles can be used to predict the future…at least the potential outcome of specific crosses.

Acknowledgements

Development of this lesson was supported in part by Cooperative State Research, Education, & Extension Service, U.S. Department of Agriculture under Agreement Number 98-EATP-1-0403 administered by Cornell University and the American Distance Education Consortium (ADEC). Any opinions, findings, conclusions or recommendations expressed in this publication are those of the author(s) and do not necessarily reflect the view of the U.S. Department of Agriculture.