Genetic Inheritance and Sex Linkage in C. elegans: Lab Report

Abstract

The objective of this experiment was to apply Mendelian laws of genetics and inheritance to determine sex linkage. Mendel's three laws of inheritance state that alleles may exist in two forms, dominant or recessive; organisms with at least one dominant allele will display its phenotype; each organism carries two genes for a character, and each gene has variations called alleles; and the alleles of a gene separate during the production of gametes, so each sperm and egg only receive one allele from each parent (Gorp, Lynn Van).

Within a few weeks, we formulated a hypothesis, conducted an experiment, and obtained results to investigate if Mendel's principles and findings about self-fertilization could be applied beyond the category of peas. We successfully determined the occurrence of sex linkage for a recessive mutation by performing a monohybrid cross on the him-8; dpy-3 homozygous hermaphrodites and homozygous him-8 males provided by the lab.

Introduction

During the 1800s, Gregor Mendel conducted studies on the transfer of traits from one generation to the next, using garden peas to elucidate the basic pattern of inheritance that forms the foundation of modern genetics.

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In this lab, we employed Caenorhabditis elegans nematodes or worms as an alternative to garden peas to establish specific trait patterns. Caenorhabditis elegans, also known as C. elegans, are eukaryotic and multicellular nematodes that feed on rotting bacteria found on decaying organic matter, such as fruits (Corsi, 2005).

C. elegans served as the model organism for this experiment due to their rapid life cycle, short lifespan (approximately three to five days from embryo to sexually mature adult), quick generation time, and their ability to produce over three hundred offspring.

These characteristics facilitated the observation of phenotype and genotype ratios, making it easier to complete the necessary procedures for the experiment. Additionally, their transparency allowed for non-invasive analysis (Shrestha et al., 2017).

Worms exist in two sexes: hermaphrodite and male, determined by the ratio of sex chromosomes to autosomes (5 pairs of autosomes and one pair of sex chromosomes). Hermaphrodite worms have XX sex chromosomes (matched pair), while males have XO sex chromosomes (one sex chromosome). Although hermaphrodites produce sperm and oocytes, they are referred to as females for the purposes of this experiment (Shrestha et al., 2017).

The hypothesis we formulated posited that if the him-8 (dpy) gene is recessive and sex-linked, the F2 generation would exhibit patterns of sex-linked inheritance. Throughout the procedures detailed in the methods section, we continually considered whether we would observe sex-linked inheritance and how it might impact our final results. We also questioned whether the him-8 gene would influence the outcomes.

Materials and Methods

The materials utilized in this experiment included:

• Caenorhabditis elegans worms
• Bunsen burners
• Wire picks
• Agar plates
• Experimental plates
• Bacteria
• Dissecting microscope
• Incubator
• Parafilm

We specifically employed two types of C. elegans worms for this experiment: him-8; dpy-3- homozygous hermaphrodites and homozygous dpy-3+; him-8 males.

Prior to commencing the experiment, we conducted preliminary exercises to familiarize ourselves with the materials and procedures. Once prepared, we referred to our provided lab manual and set up our materials. Our objectives were to determine the dominance relationship between the wild type (+) allele and the mutant dumpy (dpy-3) allele, as well as to establish whether dpy-3 is located on the X chromosome, a sex-linked gene. To predict the genotype of the F2 generation resulting from the F1 cross, we employed a Punnett square and performed a Chi-square analysis.

The experiment commenced with rigorous sanitation of hands and surfaces. We conducted a genetic cross between homozygous him-8 males and hermaphrodites that were homozygous for both the him-8 and dpy-3 mutations. Specifically, we focused on the inheritance of the dpy-3 gene, utilizing the him-8 mutant in the background instead of wild type females. A new agar plate was used, and we labeled the bottom of the plate with a Sharpie to facilitate microscope observations without disrupting the worms. Two dpy-3; him-8 homozygous hermaphrodites and 4-6 homozygous him-8 males were transferred to the plate, with an emphasis on transferring L4 worms instead of adults to avoid larvae or embryo transfer. Following observation under a dissecting microscope, we ensured that our experimental plate contained at least 2 hermaphrodites with the dpy phenotype and 4 active and crawling wild type males. We sealed the plates with Parafilm and placed them in an incubator at 15 degrees Celsius, allowing them to incubate for seven days, completing the setup of the parental cross (P₀).

While awaiting the cross-fertilization between him-8; dpy-3 homozygous hermaphrodites and homozygous him-8 males to generate the first filial generation (F1), we initially predicted that the mutant dpy-3 allele inherited from the hermaphrodite would be dominant to the wild type allele inherited from the male, drawing parallels to human mutation inheritance. After one week of incubation, we retrieved the agar plates and observed the new F1 generation under a dissecting microscope. Based on the observed genotypes, we concluded that the mutant dpy-3 allele inherited from the hermaphrodite was recessive to the wild type allele inherited from the male. Subsequently, we initiated the second part of the experiment by identifying hermaphrodites carrying eggs, indicating readiness for setting up the F2 generation. We repeated the process of labeling a new agar plate on the bottom with a Sharpie, as previously done with the F1 generation. We carefully transferred two F1 L4 stage heterozygous hermaphrodites from the F1 experimental plate to the new agar plate and sealed it with Parafilm. The choice of L4 stage over adult worms was deliberate as they can lay eggs after only eighteen hours.

In the subsequent phase of the lab, we observed the phenotypes of the F2 generation. We sorted and counted the worms based on sex and phenotype, recording the collected data. This data was later utilized to conduct chi-square tests and statistically determine whether we could accept or reject our null hypothesis. When creating the chi-square test for comparing phenotypic ratios, we initiated the process by identifying all possible phenotypes or combinations, observed the numbers of each, and calculated the expected numbers for each using a Punnett square. Subsequently, we computed the chi-square value and consulted the chi-square table to determine the degrees of freedom, the expected chi-square value, and the p-value, which allowed us to either accept or reject our null hypothesis.

Results

Table 1. Sex Linked Punnett Square

This Punnett square was constructed to hypothesize the sex of the C. elegans, indicating that all individuals should be hermaphrodites (females).

Table 2. F2 Punnett Square

This Punnett square demonstrates a phenotype ratio of 3:1 and a genotype ratio of 1:2:1.

Table 3. Phenotype Observation

This table presents recorded data on genetics and phenotypic observations of the F2 cross.

According to Rana, the chi-square test is a nonparametric test used for two specific purposes: to test the hypothesis of no association between two or more groups, populations, or criteria; and to assess how well the observed distribution of data aligns with the expected distribution. This test is designed for the analysis of categorical data and is not meant for parametric or continuous data analysis. To calculate the P value for their experiment, scientists refer to large tables of values typically organized with the vertical axis on the left corresponding to degrees of freedom (df) and the horizontal axis at the top corresponding to P value.

Table 4. First Chi Square Analysis of C. elegans Ratios

This table presents the results of the first Chi-square analysis for C. elegans ratios, focusing on autosomal phenotypes (genotype).

Level of Significance: 0.05

X^2 value: 26.5

Degree of Freedom: 1

We rejected our null hypothesis, which posited that our observed values would match the expected ones, resulting in an F2 generation displaying a 3:1 ratio of wild type to dumpy phenotypes. This rejection was based on the autosomal analysis. The presence of the him-8 gene altered the results, and the F2 generation exhibited patterns of sex-linked inheritance, leading to a discrepancy between our observed and expected values. From the outset of the experiment, we were aware of the existence of sex linkage, prompting us to formulate an alternative hypothesis. According to this alternative hypothesis, our observed values would not match our expected values because the F2 generation would not adhere to a 3:1 ratio of wild type to dumpy phenotypes.

Table 5. Second Chi Square Analysis of C. elegans Ratios

This table presents the results of the second Chi-square analysis for C. elegans ratios, focusing on sex-linked phenotypes (genotype).

Level of Significance: 0.05

X^2 value: 85.23

Degree of Freedom: 1

We rejected our null hypothesis, which assumed that our observed values would match the expected ones, resulting in an expected phenotype ratio of 0.655:0.345 for wild type to dumpy. Our expected values were determined using the provided equation. The presence of the him-8 gene, which enables us to observe sex linkage, led us to reject this hypothesis.

Discussion

As we meticulously analyzed the worms and conducted the experiment, we gathered substantial data to substantiate our initial hypothesis. Leveraging our understanding of Mendelian genetics, our observations in the F1 generation revealed that the dpy-3 gene was recessive rather than dominant. Instead of a uniform population of dumpy phenotypes, we observed a significant mixture of wild-type and dumpy phenotypes. We employed Punnett squares (Table 1 and Table 2) to organize genotype combinations, delineate phenotype ranges, and establish genotype and phenotype ratios. While Punnett squares indicated the potential for the dumpy gene to be either dominant or recessive, our conclusive determination of its recessiveness was based on direct observations of the worms under a dissecting microscope. Additionally, these observations hinted at the presence of sex linkage on the X chromosomes.

Sex chromosomes play a pivotal role in determining an organism's sex, while autosomes exhibit no sexual dimorphism and are the same in both males and females. Most sex-linked traits are X-linked, and inheritance depends on the sex of the parent carrying the trait. Male C. elegans are considered rare and arise due to errors in chromosomal separation during meiosis. As hermaphrodites possess two X chromosomes and males have only one X chromosome, the X/A ratio for males is 1:0.5. In nature, crosses between hermaphrodites and males result in a 50/50 population. Disregarding sex when studying the dpy gene would result in a uniform dumpy phenotype because it would involve a cross between homozygous wild-type and homozygous dumpy individuals. However, considering sex led to distorted results, as the worms exhibited varying phenotypes. These results demonstrated that the dpy gene, when influenced by him-8, is indeed sex-linked, thereby validating our hypothesis.

In the first Chi-square analysis (Table 4), the P-value was less than 0.05, necessitating the rejection of our null hypothesis, which posited that our observed values would match the expected ones and that the F2 generation would display a 3:1 ratio of wild type to dumpy phenotypes. Conversely, in the second Chi-square analysis (Table 5), the P-value was greater than 0.05, indicating similarity between the expected and observed ratios. Our null hypothesis, which anticipated the observed values aligning with the expected values and resulting in a phenotype ratio of 0.655:0.345 for wild type to dumpy, was accepted. The degree of freedom for the monohybrid cross was calculated as one since there were only two phenotype groups, resulting in 2-1=1.

While the results supported our hypotheses, it is essential to acknowledge potential sources of error that may have affected the accuracy of our findings. For instance, during the second part of the experiment, there may have been instances of accidental transfer of males instead of hermaphrodites due to the exceedingly small size of the worms (1mm). Distinguishing between the two sexes is exceptionally challenging, even under the highest magnification of the microscope, and errors can occur when recording phenotypes. Limited mobility among the worms could potentially hinder mating. However, we maintain that the overall ratios and the failure to reject the null hypothesis would likely have remained consistent even with improved accuracy.

In conclusion, our hypothesis received robust support from the gathered evidence and data, indicating that the dpy-3 gene is a recessive sex-linked gene found in Caenorhabditis elegans. Our initial assumptions about the inheritance of mutations in these nematodes compared to humans were disproven. C. elegans serves as a valuable model organism not only for the study of development but also for understanding various aspects of human biology. Nichols (Year) highlights that "since around 40% of the genes implicated in human disease have equivalents in worms, these tools also make it possible to learn more about the underlying mechanism of many diseases by examining the function of the related gene in the worm."

References

• Corsi, A. K., Wightman, B., & Chalfie, M. (2005). A transparent window into Biology: A primer on Caenorhabditis elegans. Genetics, 200, 387-407.
• Gorp, Lynn Van. (2009). Gregor Mendel: Genetics Pioneer. Compass Point Books.
• Nicholas, H. (2017). Animals In Research: C. Elegans (Roundworm). The Conversation. University of Sydney.
• Rana, R., & Singhal, R. (2015). Chi-square test and its application in hypothesis testing. J Pract Cardiovascular Science, 1, 69-71.
• Shrestha, R., C. McEntee, & Schvarzstein, M. (2017). Understanding Mendelian Genetics using Caenorhabditis elegans. New York: Brooklyn College, CUNY.