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LAB 4: GENETIC ANALYSIS OF THE MAIZE PLANT By: Billy Bob Joe Student No. 911911911 Lab Section: 416 T.A: Chic Nextdoor Date: October 13, 2003 1 Introduction: In this experiment, the pattern of inheritance of two genes of a maize were examined. A gene is a fundamental unit of a heritable character, which resides on a locus of a chromosome. Loci (locus - singular) are particular positions of genes on chromosomes. The loci examined in this study were the R locus and the Su locus. A gene in the R locus for the maize can either express the royal purple colour corn kernel or yellow kernel. Similarly a gene in the Su locus can express either a starchy kernel or a sweet kernel. Since there can be two expressions of the same gene, then it can be concluded that there are two versions of the same gene. This is called an allele. An allele can exist as a dominant or recessive form. Both dominant and recessive alleles determine the phenotype (an observable trait or character) of an organism, but recessive alleles determine the phenotype only when it is the only allele present. Alleles occur in like chromosomes that have the same genetic make up, known as homologous chromosomes. This condition of having two collections of chromosomes is known as diploid; in contrast, the condition of having one collection of chromosomes is haploid. During meiosis (the process by which the number of chromosomes in a cell is “halved”), the homologous chromosomes in the diploid cell containing the two possible alleles line up at the metaphase I plate and separate. So by the end of meiosis, the 4 haploid gametes have one of two possible versions of the gene. This explains Mendel’s 1st law, Law of Segregation of alleles, that “when a diploid individual produces gametes, the alleles separate, so that each gamete receives only one member of the pair of alleles” (Purves, 2000).1 2 Independent assortment of alleles is also accounted by chromosomal behaviour and relates to Mendel’s 2nd Law, which states: “alleles of different genes assort independently of one another during gamete formation” (Purves, 2000).1 Alleles assort independently because in metaphase I of meiosis, the pairs of homologous chromosomes line up at the metaphase I plate independent of other pairs of homologous chromosomes. However, homologous chromosomes are dependent of each other during the alignment along the metaphase plate since homologous chromosomes are coupled together by chiasmata (an X-shaped connection, where reciprocal genetic exchange occurs). Nonhomologous chromosomes are not connected in any way like homologous chromosomes, so non-homologous chromosomes do not have any influence on other homologous pairs, thus the alleles sort independent of each other. However, not all alleles sort independent of each other, sometimes two sets of a gene pairs can be inherited together. This is due to the linkage of genes in the chromosomes. During meiosis, if two loci are very close to each other on a chromosome, then it would be exceptionally rare for reciprocal genetic exchange to occur between. Since these genes are close enough to not be separated, they will not sort independently, and therefore will be linked. In the maize experiment, it was hypothesized that “the two genes are sorting independently and therefore expect four meiotic products” (Hasenkampf & Olaveson, 2003).2 Materials and Methods: The dominant alleles in the experiment were the royal purple color kernels, R, and the smooth starchy kernel, Su. The recessive alleles were the yellow corn kernel, r, and the wrinkled sweet kernel, su. A parent that was homozygous dominant for both alleles 3 (R and Su) was crossed with another parent that was also homozygous recessive for both alleles. The parents who were crossed are known as the parental generation or simply P. The offspring of these parents are referred to as the F1 (first filial generation) offspring. These F1 offspring, which were 4 different possible gametes were self-crossed (siblings of F1 mated with each other) to produce a new set of offspring called the F2 (second filial generation) offspring. The phenotypes of the F2 on the maize were then counted and recorded to make comparisons. Results: From the self-crossing of the F1, the phenotypes and the genotypes of the F2 were generated using the Punnett Square (Figure 1). The Punnett Square consists of all the possible male gametes of the F1 on one axis, and all possible female gametes on the other axis. From the Punnett Square (Figure 1), the phenotypes of the kernels were determined. The phenotypes were found to be in the expected ratio of 9:3:3:1. That is to say, out of the total number of 16 possible offspring, 9 were purple and smooth, 3 were purple and wrinkled, another 3 were yellow and smooth, and 1 was yellow and wrinkled. Figure 1. F2 Offspring Produced from Self-crossing of F1 R Su R Su R su r Su r su R su r Su R su RR SuSu RR Susu Rr SuSu Rr Susu (purple & smooth) (purple & smooth) (purple & smooth) (purple & smooth) RR Susu RR susu Rr Susu Rr susu (purple & smooth) (purple & wrinkled) (purple & smooth) (purple & wrinkled) Rr SuSu Rr Susu Rr SuSu rr Susu (purple & smooth) (purple & smooth) (yellow & smooth) (yellow & smooth) Rr Susu Rr susu Rr Susu rr susu (purple & smooth) (purple & wrinkled) (yellow & smooth) (yellow & wrinkled) Phenotypic Ratio: 9 purple & smooth : 3 purple & wrinkled : 3 yellow & smooth :1 yellow & wrinkled NOTE: The dominant allele RR or Rr show royal purple color. The dominant allele SuSu or Susu produce smooth starchy kernel. The recessive allele susu produces wrinkled corn kernel. The recessive allele rr show a yellow corn kernel. 4 In the experiment, a total of 487 kernels were examined, and the number of observed kernels for each phenotype was near the expected number of kernels for that phenotype (Table 1). The expected numbers of kernels were found by using Equation 1. Expected Number of kernels for a phenotype = (Phenotypic ratio for that phenotype) × (Total number of observed kernels) (1) Although the observed number of kernels differed only by very little amounts from the expected number, the differentiation was calculated by measuring a Chi-Square value. The Chi-Square value was used for the purpose of determining “how much the observed results differ from the expected value” (Hasenkampf & Olaveson, 2003).2 The Chi-Squared value was determined by using Equation 2. That is taking the sum of all the values of the 5th column in Table 1. The Chi-Square value for this experiment was determined to be 6.375. Chi - Square value = ∑ (Obs. − Exp.) Exp. 2 (2) Table 1. Phenotypic Classes for F2 Data Classes (description of phenotype) Phenotypic Ratio Observed Number of kernels Expected Number of kernels (Obs.− Exp.) Exp Purple & Smooth 9:16 248 274 2.467 Purple & Wrinkled 3:16 106 91 2.473 Yellow & Smooth 3:16 96 91 0.274 Yellow & Wrinkled 1:16 37 31 1.161 1 2 2 The phenotypic ratio was determined from the Punnett Square (Figure 1). The expected numbers of kernels were rounded off to the nearest whole number. 5 The Chi-Square value was then used to determine a P-value from Table 4.1. (Hasenkampf & Olaveson, 2003).2 The P-value “indicates how often the differentiation would occur from the expected number of kernels due to chance alone (random variation), even when the hypothesis is true “(Hasenkampf & Olaveson, 2003).2 The obtained P-value for this experiment was 0.1, which indicates that 10% of the time, there would be some deviation from the expected values. “In genetics, a P-value of 0.05 is usually a cut off value, that is to say, if there is 5% chance or less of getting a Chi-Square value that large, the hypothesis is rejected” (Hasenkampf & Olaveson, 2003).2 In the experiment, the P-value was 0.1, greater then 0.05, therefore the hypothesis can be accepted. For that reason, it can be said that the alleles of the maize were sorting independently. References Citied: 1. Purves WK, Sadava D, Orians GH and Heller Hc (2000) Life the Science of Biology, 6th Edition, Sinauer Assoc. (Publishers). pp 180 - 183 2. Dr. Clare Hasenkampf and Mary Olaveson (2003) Introductory Biology Part I Laboratory Manual, University of Toronto at Scarborough Department of Life Sciences. Lab 3: pp 57 – 63. 6